Abstract
Disclosed are microalgal cells having an ablated or downregulated fatty acyl-ACP thioesterase (FATA) gene, wherein the cell is modified to express a heterologous lysophosphatidic acid acyltransferase (LPAAT) comprising an amino acid sequence that has at least 80% identity to an acyltransferase encoded by SEQ ID NO: 90, 89, 92, 93 or 95 and wherein the modified microalgal cell produces an oil with an elevated ratio of saturated-unsaturated-saturated triglycerides over trisaturated triglycerides as compared to a corresponding unmodified cell. Also disclosed are microalgal oils comprising at least 60% stearate-oleate-stearate (SOS) triglycerides, less than 5% trisaturates and wherein the fatty acid profile of the oil comprises at least 50% C18:0. Related methods of producing an oil are also disclosed.
Claims
1. A microalgal cell having an ablated or downregulated fatty acyl-ACP thioesterase (FATA) gene, wherein the cell is modified to express a heterologous lysophosphatidic acid acyltransferase (LPAAT) comprising an amino acid sequence that has at least 80% identity to an acyltransferase encoded by SEQ ID NO: 90, 89, 92, 93 or 95 and wherein the modified microalgal cell produces an oil with an elevated ratio of saturated-unsaturated-saturated triglycerides over trisaturated triglycerides as compared to a corresponding unmodified cell.
2. The microalgal cell of claim 1, wherein the cell is modified to coexpress with the heterologous LPAAT at least one exogenous gene that encodes an enzyme selected from the group consisting of invertase, a fatty acyl-ACP thioesterase, a melibiase, a ketoacyl synthase and a THIC.
3. The microalgal cell of claim 1, wherein the cell is modified to ablate or downregulate the expression of at least one endogenous gene selected from the group consisting of: a stearoyl ACP desaturase, a fatty acyl desaturase, a fatty acyl-ACP thioesterase (FATA or FATB), a ketoacyl synthase (KASI, KASII, KASIII or KAS IV) and an acyltransferase (DGAT, GPAT or LPCAT).
4. The microalgal cell of claim 2, wherein the cell is further modified to overexpress a gene encoding a C18:0-specific FATA1 thioesterase.
5. The microalgal cell of claim 4, wherein the C18:0-specific FATA1 thioesterase is a variant Garcinia thioesterase.
6. The microalgal cell of claim 5, wherein the variant Garcinia thioesterase has at least 80% identity to SEQ ID NO: 142.
7. The microalgal cell of claim 6, wherein the variant Garcinia thioesterase comprises one or more of amino acid variants selected from the group consisting of L91F, L91K, L91S, G96A, G96T, G96V, G108A, G108V, S111A, S111V T156F, T156A, T156K, T156V and V193A.
8. The microalgal cell of claim 7, wherein the variant Garcinia thioesterase is a variant comprising the substitutions S111A and V193A, a variant comprising the substitution G96A, or a variant comprising the substitution G108A.
9. The microalgal cell of claim 2, wherein the ketoacyl synthase is a KASII.
10. The microalgal cell of claim 4, wherein the ketoacyl synthase is a KASII.
11. The microalgal cell of claim 3, wherein the cell is modified to ablate or downregulate the expression of an endogenous stearoyl ACP desaturase-2 (SAD2) gene and an endogenous fatty acyl desaturase-2 (FAD2) gene.
12. The microalgal cell of claim 10, wherein the cell is modified to ablate or downregulate the expression of an endogenous stearoyl ACP desaturase-2 (SAD2) gene and an endogenous fatty acyl desaturase-2 (FAD2) gene.
13. The microalgal cell of claim 1, wherein the cell is modified to express a Theobroma cacao diacylglycerol O-acyltransferase.
14. The microalgal cell of claim 13, wherein the Theobroma cacao diacylglycerol O-acyltransferase is a Theobroma cacao diacylglycerol O-acyltransferase-1 or a Theobroma cacao diacylglycerol O-acyltransferase-2.
15. The microalgal cell of claim 12, wherein the Theobroma cacao diacylglycerol O-acyltransferase is a Theobroma cacao diacylglycerol O-acyltransferase-1 or a Theobroma cacao diacylglycerol O-acyltransferase-2.
16. The microalgal cell of claim 1, wherein the cell is of the genus Prototheca or Chlorella.
17. The microalgal cell of claim 16, wherein the cell is a Prototheca moriformis cell.
18. The microalgal cell of claim 15, wherein the cell is a Prototheca moriformis cell.
19. A method of producing an oil comprising: (a) cultivating the microalgal cell of claim 1 under conditions to produce the oil; and (b) extracting the oil from the microalgal cell; wherein the oil comprises at least 50% stearate-oleate-stearate (SOS) triglycerides with an elevated ratio of saturated-unsaturated-saturated triglycerides over trisaturated triglycerides as compared to a corresponding unmodified cell.
20. A method of producing an oil comprising: (a) cultivating the microalgal cell of claim under conditions to produce the oil; and (b) extracting the oil from the microalgal cell; wherein the oil comprises at least 60% stearate-oleate-stearate (SOS) triglycerides, less than 5% trisaturates and wherein the fatty acid profile of the oil comprises at least 50% C18:0.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1. TAG profiles of S7815 versus the S6573 parent. TAGs in brackets co-elute with the peak of the main TAG, but are present in trace amounts, and do not contribute significantly to the area. M=myristate (C14:0), P=palmitate (C16:0), Po=palmitoleate (C16:1), Ma=margaric (C17:0), S=stearate (C18:0), O=oleate (C18:1), L=linoleate (C18:2), Ln=linolenate (C18:3 ), A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0). Sat-Sat-Sat=trisaturates. See Example 5.
[0039] FIG. 2. TAG profiles of lipids from fermentations of S7815 versus S6573. TAGs in brackets co-elute with the peak of the main TAG, but are present in trace amounts, and do not contribute significantly to the area. M=myristate (C14:0), P=palmitate (C16:0), S=stearate (C18:0), O=oleate (C18:1), L=linoleate (C18:2), Ln=linolenate (C18:3 ), A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0). Sat-Sat-Sat=trisaturates. See Example 5.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0040] An allele refers to a copy of a gene where an organism has multiple similar or identical gene copies, even if on the same chromosome. An allele may encode the same or similar protein.
[0041] An oil, cell oil or cell fat shall mean a predominantly triglyceride oil obtained from an organism, where the oil has not undergone blending with another natural or synthetic oil, or fractionation so as to substantially alter the fatty acid profile of the triglyceride. In connection with an oil comprising triglycerides of a particular regiospecificity, the cell oil or cell fat has not been subjected to interesterification or other synthetic process to obtain that regiospecific triglyceride profile, rather the regiospecificity is produced naturally, by a cell or population of cells. For a cell oil produced by a cell, the sterol profile of oil is generally determined by the sterols produced by the cell, not by artificial reconstitution of the oil by adding sterols in order to mimic the cell oil. In connection with a cell oil or cell fat, and as used generally throughout the present disclosure, the terms oil, and fat are used interchangeably, except where otherwise noted. Thus, an oil or a fat can be liquid, solid, or partially solid at room temperature, depending on the makeup of the substance and other conditions. Here, the term fractionation means removing material from the oil in a way that changes its fatty acid profile relative to the profile produced by the organism, however accomplished. The terms oil, cell oil and cell fat encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching, deodorized, and/or degumming, which does not substantially change its triglyceride profile. A cell oil can also be a noninteresterified cell oil, which means that the cell oil has not undergone a process in which fatty acids have been redistributed in their acyl linkages to glycerol and remain essentially in the same configuration as when recovered from the organism.
[0042] As used herein, an oil is said to be enriched in one or more particular fatty acids if there is at least a 10% increase in the mass of that fatty acid in the oil relative to the non-enriched oil. For example, in the case of a cell expressing a heterologous FatB gene described herein, the oil produced by the cell is said to be enriched in, e.g., C8 and C16 fatty acids if the mass of these fatty acids in the oil is at least 10% greater than in oil produced by a cell of the same type that does not express the heterologous FatB gene (e.g., wild type oil).
[0043] Exogenous gene shall mean a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a transgene. A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
[0044] FADc, also referred to as FAD2 or FAD is a gene encoding a delta-12 fatty acid desaturase. SAD is a gene encoding a stearoyl ACP desaturase, a delta-9 fatty acid desaturase. The desaturases desaturates a fatty acyl chain to create a double bond. SAD converts stearic acid, C18:0 to oleic acid, C18:1 and FAD converts oleic acid, C18:1 to linoleic acid, C18:2.
[0045] Fatty acids shall mean free fatty acids, fatty acid salts, or fatty acyl moieties in a glycerolipid. It will be understood that fatty acyl groups of glycerolipids can be described in terms of the carboxylic acid or anion of a carboxylic acid that is produced when the triglyceride is hydrolyzed or saponified.
[0046] Fixed carbon source is a molecule(s) containing carbon, typically an organic molecule that is present at ambient temperature and pressure in solid or liquid form in a culture media that can be utilized by a microorganism cultured therein. Accordingly, carbon dioxide is not a fixed carbon source. Typical fixed carbon source include sucrose, glucose, fructose and other well-known monosaccharides, disaccharides and polysaccharides.
[0047] In operable linkage is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.
[0048] Microalgae are eukaryotic microbial organisms that contain a chloroplast or other plastid, and optionally that is capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae also include mixotrophic organisms that can perform photosynthesis and metabolize one or more fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca.
[0049] As used with respect to nucleic acids, the term isolated refers to a nucleic acid that is free of at least one other component that is typically present with the naturally occurring nucleic acid. Thus, a naturally occurring nucleic acid is isolated if it has been purified away from at least one other component that occurs naturally with the nucleic acid.
[0050] In connection with fatty acid length, mid-chain shall mean C8 to C16 fatty acids.
[0051] In connection with a recombinant cell, the term knockdown refers to a gene that has been partially suppressed (e.g., by about 1-95%) in terms of the production or activity of a protein encoded by the gene. Inhibitory RNA technology to down-regulate or knockdown expression of a gene are well known. These techniques include dsRNA, hairpin RNA, antisense RNA, interfering RNA (RNAi) and others.
[0052] Also, in connection with a recombinant cell, the term knockout refers to a gene that has been completely or nearly completely (e.g., >95%) suppressed in terms of the production or activity of a protein encoded by the gene. Knockouts can be prepared by ablating the gene by homologous recombination of a nucleic acid sequence into a coding sequence, gene deletion, mutation or other method. When homologous recombination is performed, the nucleic acid that is inserted (knocked-in) can be a sequence that encodes an exogenous gene of interest or a sequence that does not encode for a gene of interest. The ablation by homologous recombination can be performed in one, two or more alleles of the gene of interest.
[0053] An oleaginous cell is a cell capable of producing at least 20% lipid by dry cell weight, naturally or through recombinant or classical strain improvement. An oleaginous microbe or oleaginous microorganism is a microbe, including a microalga that is oleaginous (especially eukaryotic microalgae that store lipid). An oleaginous cell also encompasses a cell that has had some or all of its lipid or other content removed, and both live and dead cells.
[0054] An ordered oil or ordered fat is one that forms crystals that are primarily of a given polymorphic structure. For example, an ordered oil or ordered fat can have crystals that are greater than 50%, 60%, 70%, 80%, or 90% of the 13 or 13 polymorphic form.
[0055] In connection with a cell oil, a profile is the distribution of particular species or triglycerides or fatty acyl groups within the oil. A fatty acid profile is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID), as in Example 1. The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME-GC-FID measurement approximate weight percentages of the fatty acids. A sn-2 profile is the distribution of fatty acids found at the sn-2 position of the triacylglycerides in the oil. A regiospecific profile is the distribution of triglycerides with reference to the positioning of acyl group attachment to the glycerol backbone without reference to stereospecificity. In other words, a regiospecific profile describes acyl group attachment at sn-1/3 vs. sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate) and SOP (stearate-oleate-palmitate) are treated identically. A stereospecific profile describes the attachment of acyl groups at sn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such as SOP and POS are to be considered equivalent. A TAG profile is the distribution of fatty acids found in the triglycerides with reference to connection to the glycerol backbone, but without reference to the regiospecific nature of the connections. Thus, in a TAG profile, the percent of SSO in the oil is the sum of SSO and SOS, while in a regiospecific profile, the percent of SSO is calculated without inclusion of SOS species in the oil. In contrast to the weight percentages of the FAME-GC-FID analysis, triglyceride percentages are typically given as mole percentages; that is the percent of a given TAG molecule in a TAG mixture.
[0056] The term percent sequence identity, in the context of two or more amino acid or nucleic acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted using the NCBI BLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. For example, to compare two nucleic acid sequences, one may use blastn with the BLAST 2 Sequences tool Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: 2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For a pairwise comparison of two amino acid sequences, one may use the BLAST 2 Sequences tool Version 2.0.12 (Apr. 21, 2000) with blastp set, for example, at the following default parameters: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap x drop-off 50; Expect: 10; Word Size: 3; Filter: on.
[0057] Recombinant is a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi), hairpin RNA or dsRNA that reduce the levels of active gene product in a cell. A recombinant nucleic acid is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, using chemical synthesis, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a recombinant protein is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid. A recombinant protein will have a different pattern of glycosylation than the protein isolated from the wild-type organism.
[0058] The genes can be used in a variety of genetic constructs including plasmids or other vectors for expression or recombination in a host cell. The genes can be codon optimized for expression in a target host cell. The proteins produced by the genes can be used in vivo or in purified form.
[0059] For example, the gene can be prepared in an expression vector comprising an operably linked promoter and 5UTR. Where a plastidic cell is used as the host, a suitably active plastid targeting peptide can be fused to the FATB gene, as in the examples below. Generally, for the newly identified FATB genes, there are roughly 50 amino acids at the N-terminal that constitute a plastid transit peptide, which are responsible for transporting the enzyme to the chloroplast. In the examples below, this transit peptide is replaced with a 38 amino acid sequence that is effective in the Prototheca moriformis host cell for transporting the enzyme to the plastids of those cells. Thus, the invention contemplates deletions and fusion proteins in order to optimize enzyme activity in a given host cell. For example, a transit peptide from the host or related species may be used instead of that of the newly discovered plant genes described here.
[0060] A selectable marker gene may be included in the vector to assist in isolating a transformed cell. Examples of selectable markers useful in microlagae include sucrose invertase antibiotic resistance genes and other genes useful as selectable markers. The S. carlbergensis MEL1 gene (conferring the ability to grow on melibiose), A. thaliana THIC gene (conferring the ability to grow in media free of thiamine, Saccharomyces sucrose invertase (conferring the ability to grow on sucrose) are disclosed in the Examples. Other known selectable markers are useful and within the ambit of a skilled artisan.
[0061] The terms triglyceride, triacylglyceride and TAG are used interchangeably as is known in the art.
II. Embodiments of the Invention
[0062] Illustrative embodiments of the present invention feature oleaginous cells that produce altered fatty acid profiles and/or altered regiospecific distribution of fatty acids in glycerolipids, and products produced from the cells. Examples of oleaginous cells include microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae and, where applicable, oil producing cells of higher plants including but not limited to commercial oilseed crops such as soy, corn, rapeseed/canola, cotton, flax, sunflower, safflower and peanut. Other specific examples of cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae and methods of cultivation are also provided in co-owned applications WO2008/151149, WO2010/063031, WO2010/063032, WO2011/150410, WO2011/150411, WO2012/061647, WO2012/061647, WO2012/106560, and WO2013/158938, WO2014/120829, WO2014/151904, WO2015/051319, WO2016/007862, WO2016/014968, WO2016/044779, WO2016/164495, all of which are incorporated by reference, including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs. The oleaginous cells can be, for example, capable of producing 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, or about 90% oil by cell weight, 5%. Optionally, the oils produced can be low in highly unsaturated fatty acids such as DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA. The above-mentioned publications also disclose methods for cultivating such cells and extracting oil, especially from microalgal cells; such methods are applicable to the cells disclosed herein and incorporated by reference for these teachings. When microalgal cells are used they can be cultivated autotrophically (unless an obligate heterotroph) or in the dark using a sugar (e.g., glucose, fructose and/or sucrose) In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock. Alternately, or in addition, the cells can metabolize xylose from cellulosic feedstocks. For example, the cells can be genetically engineered to express one or more xylose metabolism genes such as those encoding an active xylose transporter, a xylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase and a xylose reductase. See WO2012/154626, GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE, published Nov. 15, 2012, including disclosure of genetically engineered Prototheca strains that utilize xylose.
[0063] The host cells expressing the acyltransferases or the variant B. napus thioesterases or the variant G. mangostana thioesterase may, optionally, be cultivated in a bioreactor/fermenter. For example, heterotrophic oleaginous microalgal cells can be cultivated on a sugar-containing nutrient broth. Optionally, cultivation can proceed in two stages: a seed stage and a lipid-production stage. In the seed stage, the number of cells is increased from a starter culture. Thus, the seed stage(s) typically includes a nutrient rich, nitrogen replete, media designed to encourage rapid cell division. After the seed stage(s), the cells may be fed sugar under nutrient-limiting (e.g. nitrogen sparse) conditions so that the sugar will be converted into triglycerides. As used herein, standard lipid production conditions are disclosed here. In one embodiment, the culture conditions are nitrogen limiting. Sugar and other nutrients can be added during the fermentation but no additional nitrogen is added. The cells will consume all or nearly all of the nitrogen present, but no additional nitrogen is provided. For example, the rate of cell division in the lipid-production stage can be decreased by 50%, 80%, or more relative to the seed stage. Additionally, variation in the media between the seed stage and the lipid-production stage can induce the recombinant cell to express different lipid-synthesis genes and thereby alter the triglycerides being produced. For example, as discussed below, nitrogen and/or pH sensitive promoters can be placed in front of endogenous or exogenous genes. This is especially useful when an oil is to be produced in the lipid-production phase that does not support optimal growth of the cells in the seed stage.
[0064] The oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes. As a result, some embodiments feature cell oils that were not obtainable from a non-plant or non-seed oil, or not obtainable at all.
[0065] The oleaginous cells, including microalgal cells, can be improved via classical strain improvement techniques such as UV and/or chemical mutagenesis followed by screening or selection under environmental conditions, including selection on a chemical or biochemical toxin. For example the cells can be selected on a fatty acid synthesis inhibitor, a sugar metabolism inhibitor, or an herbicide. As a result of the selection, strains can be obtained with increased yield on sugar, increased oil production (e.g., as a percent of cell volume, dry weight, or liter of cell culture), or improved fatty acid or TAG profile. Co-owned application PCT/US2016/025023 filed on 31 Mar. 2016, herein incorporated by reference, describes methods for classically mutagenizing oleaginous cells.
[0066] The cells can be selected on one or more of 1,2-Cyclohexanedione; 19-Norethindone acetate; 2,2-dichloropropionic acid; 2,4,5-trichlorophenoxyacetic acid; 2,4,5-trichlorophenoxyacetic acid, methyl ester; 2,4-dichlorophenoxyacetic acid; 2,4-dichlorophenoxyacetic acid, butyl ester; 2,4-dichlorophenoxyacetic acid, isooctyl ester; 2,4-dichlorophenoxyacetic acid, methyl ester; 2,4-dichlorophenoxybutyric acid; 2,4-dichlorophenoxybutyric acid, methyl ester; 2,6-dichlorobenzonitrile; 2-deoxyglucose; 5-Tetradecyloxy-w-furoic acid; A-922500; acetochlor; alachlor; ametryn; amphotericin; atrazine; benfluralin; bensulide; bentazon; bromacil; bromoxynil; Cafenstrole; carbonyl cyanide m-chlorophenyl hydrazone (CCCP); carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP); cerulenin; chlorpropham; chlorsulfuron; clofibric acid; clopyralid; colchicine; cycloate; cyclohexamide; C75; DACTHAL (dimethyl tetrachloroterephthalate); dicamba; dichloroprop ((R)-2-(2,4-dichlorophenoxy)propanoic acid); Diflufenican; dihyrojasmonic acid, methyl ester; diquat; diuron; dimethylsulfoxide; Epigallocatechin gallate (EGCG); endothall; ethalfluralin; ethanol; ethofumesate; Fenoxaprop-p-ethyl; Fluazifop-p-Butyl; fluometuron; fomasefen; foramsulfuron; gibberellic acid; glufosinate ammonium; glyphosate; haloxyfop; hexazinone; imazaquin; isoxaben; Lipase inhibitor THL (()-Tetrahydrolipstatin); malonic acid; MCPA (2-methyl-4-chlorophenoxyacetic acid); MCPB (4-(4-chloro-o-tolyloxy)butyric acid); mesotrione; methyl dihydrojasmonate; metolachlor; metribuzin; Mildronate; molinate; naptalam; norharman; orlistat; oxadiazon; oxyfluorfen; paraquat; pendimethalin; pentachlorophenol; PF-04620110; phenethyl alcohol; phenmedipham; picloram; Platencin; Platensimycin; prometon; prometryn; pronamide; propachlor; propanil; propazine; pyrazon; Quizalofop-p-ethyl; s-ethyl dipropylthiocarbamate (EPTC); s,s,s-tributylphosphorotrithioate; salicylhydroxamic acid; sesamol; siduron; sodium methane arsenate; simazine; T-863 (DGAT inhibitor); tebuthiuron; terbacil; thiobencarb; tralkoxydim; triallate; triclopyr; triclosan; trifluralin; and vulpinic acid and others.
[0067] The oleaginous cells produce a storage oil, which is primarily triacylglyceride and may be stored in storage bodies of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. The raw oil may comprise sterols produced by the cells. Patent applications WO2008/151149, WO2010/063031, WO2010/063032, WO2011/150410, WO2011/150411, WO2012/061647, WO2012/061647, WO2012/106560, WO2013/158938, WO2014/120829, WO2014/151904, WO2015/051319, WO2016/007862, WO2016/014968, WO2016/044779, and WO2016/164495 disclose heterotrophic cultivation and oil isolation techniques for oleaginous microalgae. For example, oil may be obtained by providing or cultivating, drying and pressing the cells. The oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939. The raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. Even after such processing, the oil may retain a sterol profile characteristic of the source. Sterol profiles of microalga and the microalgal cell oils are disclosed below. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, drilling fluids, as animal feed, for human nutrition, or for fertilizer.
[0068] In an embodiment of the invention nucleic acids that encode novel acyl transferases are provided. The novel acyltransferases are useful in altering the fatty acid profile and/or altering the regiospecific profile of an oil produced by a host cell. The nucleic acids of the invention may contain control sequences upstream and downstream in operable linkage with the gene of interest. These control sequences include promoters, targeting sequences, untranslated sequences and other control elements. Nucleic acids of the invention encode acyltransferases that function in type II fatty acid synthesis. The acyltransferase genes are isolated from higher plants and can be expressed in a wide variety of host cells. The acyltransferases include lysophosphatidic acid acyltransferase (LPAAT), glycerol phosphate acyltransferase (GPAT), diacyl glycerol acyltransferase (DGAT), lysophosphatidylcholine acyltransferase (LPCAT), or phospholipase A2 (PLA2). and other lipid biosynthetic pathway genes as discussed herein. The acyltransferases of the invention are shown in Table 5. In one embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 96.3%, 98%, or 99% identity to an acyltransferase of clade 1 of Table 5. In another embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 93.9%, 98%, or 99% identity to an acyltransferase of clade 2 of Table 5. In one embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 86.5%, 90%, 95%, 98%, or 99% identity to an acyltransferase of clade 3 of Table 5. In one embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 78.5%, 80%, 85%, 90%, 95%, 98%, or 99% identity to an acyltransferase of clade 4 of Table 5. The acyltransferases when expressed increase the SOS, POP, POS, SLS, PLO, and/or PLO content DCW in host cells and the oils recovered from the host cells. The acyltransferases when expressed in host cells decreases the sat-sat-sat content of the oil by DCW. The acyltransferases when expressed in host cells increases the sat-unsat-sat/sat-sat-sat ratio of the oil by DCW.
[0069] In an embodiment of the invention nucleic acids that encode variant Brassica napus thiosterases (FATA) are provided. The novel thioesterases are useful in altering the fatty acid profile of an oil produced by a host cell. The variant Brassica napus thiosterases prefer to hydrolyze long chain fatty acyl groups from the acyl carrier protein. The nucleic acids of the invention may contain control sequences upstream and downstream in operable linkage with the gene of interest. These control sequences include promoters, targeting sequences, untranslated sequences and other control elements. Nucleic acids of the invention encode thiosterases that function in type II fatty acid synthesis. The thioesterase genes, isolated from higher plants, are altered to create variant thioesterases that have certain amino acids that have been altered from the wild type enzyme. Due to the altered amino acid(s), the substrate specificity of the thioesterase is altered. The variant thioesterases can be expressed in a wide variety of host cells. The nucleic acids encode the variant thioesterases having amino acid sequences that are 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 165, 166, 167, or 198_and comprise one or more of amino acid variants D124A, D209A, D127A or D212A. The variant BnOTE enzymes increased C18:0 content by DCW, decreased C18:1 content by DCW, and decreased C18:2 content by DCW in host cells and the oils recovered from the host cells.
[0070] In an embodiment of the invention nucleic acids that encode variant Garcinia mangostana thiosterases (FATA) are provided. The novel thioesterases are useful in altering the fatty acid profile of an oil produced by a host cell. The variant Garcinia mangostana thiosterases prefer to hydrolyze long chain fatty acyl groups from the acyl carrier protein. The nucleic acids of the invention may contain control sequences upstream and downstream in operable linkage with the gene of interest. These control sequences include promoters, targeting sequences, untranslated sequences and other control elements. Nucleic acids of the invention encode thiosterases that function in type II fatty acid synthesis. The thioesterase genes, isolated from higher plants, are altered to create variant thioesterases that have certain amino acids that have been altered from the wild type enzyme. Due to the altered amino acid(s), the substrate specificity of the thioesterase is altered. The variant thioesterases can be expressed in a wide variety of host cells. The nucleic acids encode the variant thioesterases having amino acid sequences that are 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 and comprise one or more of amino acid variants L91F, L91K, L91S, G96A, G96T, G96V, G108A, G108V, S111A, S111V T156F, T156A, T156K, T156V, or V193A. The variant GmFATA enzymes increased C18:0 content by DCW, decreased C18:1 content by DCW, and decreased C18:2 content by DCW in host cells and the oils recovered from the host cells.
[0071] The nucleic acids of the invention can be codon optimized for expression in a target host cell (e.g., using the codon usage tables of Tables 1a, 1b, 2a, and 2b. For example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codons used can be the most preferred codon according to Tables 1a, 1b, 2a, and 2b. Alternately, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codons used can be the first or second most preferred codon according to Tables 1a, 1b, 2a, and 2b. Preferred codons for Prototheca strains and for Chlorella protothecoides are shown below in Tables 1a and 1b, respectively.
TABLE-US-00001 TABLE 1a Preferred codon usage in Prototheca strains. Ala GCG 345 (0.36) Asn AAT 8 (0.04) GCA 66 (0.07) AAC 201 (0.96) GCT 101 (0.11) GCC 442 (0.46) Pro CCG 161 (0.29) CCA 49 (0.09) Cys TGT 12 (0.10) CCT 71 (0.13) TGC 105 (0.90) CCC 267 (0.49) Asp GAT 43 (0.12) Gln CAG 226 (0.82) GAC 316 (0.88) CAA 48 (0.18) Glu GAG 377 (0.96) Arg AGG 33 (0.06) GAA 14 (0.04) AGA 14 (0.02) CGG 102 (0.18) Phe TTT 89 (0.29) CGA 49 (0.08) TTC 216 (0.71) CGT 51 (0.09) CGC 331 (0.57) Gly GGG 92 (0.12) GGA 56 (0.07) Ser AGT 16 (0.03) GGT 76 (0.10) AGC 123 (0.22) GGC 559 (0.71) TCG 152 (0.28) TCA 31 (0.06) His CAT 42 (0.21) TCT 55 (0.10) CAC 154 (0.79) TCC 173 (0.31) Ile ATA 4 (0.01) Thr ACG 184 (0.38) ATT 30 (0.08) ACA 24 (0.05) ATC 338 (0.91) ACT 21 (0.05) ACC 249 (0.52) Lys AAG 284 (0.98) AAA 7 (0.02) Val GTG 308 (0.50) GTA 9 (0.01) Leu TTG 26 (0.04) GTT 35 (0.06) TTA 3 (0.00) GTC 262 (0.43) CTG 447 (0.61) CTA 20 (0.03) Trp TGG 107 (1.00) CTT 45 (0.06) CTC 190 (0.26) Tyr TAT 10 (0.05) TAC 180 (0.95) Met ATG 191 (1.00) Stop TGA/TAG/TAA
TABLE-US-00002 TABLE 1b Preferred codon usage in Chlorella protothecoides. TTC (Phe) TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met) AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu)
TABLE-US-00003 TABLE 2a Codon usage for Cuphea wrightii UUU F 0.48 19.5 (52) UCU S 0.21 19.5 (52) UAU Y 0.45 6.4 (17) UGU C 0.41 10.5 (28) UUC F 0.52 21.3 (57) UCC S 0.26 23.6 (63) UAC Y 0.55 7.9 (21) UGC C 0.59 15.0 (40) UUA L 0.07 5.2 (14) UCA S 0.18 16.8 (45) UAA * 0.33 0.7 (2) UGA * 0.33 0.7 (2) UUG L 0.19 14.6 (39) UCG S 0.11 9.7 (26) UAG * 0.33 0.7 (2) UGG W 1.00 15.4 (41) CUU L 0.27 21.0 (56) CCU P 0.48 21.7 (58) CAU H 0.60 11.2 (30) CGU R 0.09 5.6 (15) CUC L 0.22 17.2 (46) CCC P 0.16 7.1 (19) CAC H 0.40 7.5 (20) CGC R 0.13 7.9 (21) CUA L 0.13 10.1 (27) CCA P 0.21 9.7 (26) CAA Q 0.31 8.6 (23) CGA R 0.11 6.7 (18) CUG L 0.12 9.7 (26) CCG P 0.16 7.1 (19) CAG Q 0.69 19.5 (52) CGG R 0.16 9.4 (25) AUU I 0.44 22.8 (61) ACU T 0.33 16.8 (45) AAU N 0.66 31.4 (84) AGU S 0.18 16.1 (43) AUC I 0.29 15.4 (41) ACC T 0.27 13.9 (37) AAC N 0.34 16.5 (44) AGC S 0.07 6.0 (16) AUA I 0.27 13.9 (37) ACA T 0.26 13.5 (36) AAA K 0.42 21.0 (56) AGA R 0.24 14.2 (38) AUG M 1.00 28.1 (75) ACG T 0.14 7.1 (19) AAG K 0.58 29.2 (78) AGG R 0.27 16.1 (43) GUU V 0.28 19.8 (53) GCU A 0.35 31.4 (84) GAU D 0.63 35.9 (96) GGU G 0.29 26.6 (71) GUC V 0.21 15.0 (40) GCC A 0.20 18.0 (48) GAC D 0.37 21.0 (56) GGC G 0.20 18.0 (48) GUA V 0.14 10.1 (27) GCA A 0.33 29.6 (79) GAA E 0.41 18.3 (49) GGA G 0.35 31.4 (84) GUG V 0.36 25.1 (67) GCG A 0.11 9.7 (26) GAG E 0.59 26.2 (70) GGG G 0.16 14.2 (38)
TABLE-US-00004 TABLE 2b Codon usage for Arabidopsis UUU F 0.51 21.8 (678320) UCU S 0.28 25.2 (782818) UAU Y 0.52 14.6 (455089) UGU C 0.60 10.5 (327640) UUC F 0.49 20.7 (642407) UCC S 0.13 11.2 (348173) UAC Y 0.48 13.7 (427132) UGC C 0.40 7.2 (222769) UUA L 0.14 12.7 (394867) UCA S 0.20 18.3 (568570) UAA * 0.36 0.9 (29405) UGA * 0.44 1.2 (36260) UUG L 0.22 20.9 (649150) UCG S 0.10 9.3 (290158) UAG * 0.20 0.5 (16417) UGG W 1.00 12.5 (388049) CUU L 0.26 24.1 (750114) CCU P 0.38 18.7 (580962) CAU H 0.61 13.8 (428694) CGU R 0.17 9.0 (280392) CUC L 0.17 16.1 (500524) CCC P 0.11 5.3 (165252) CAC H 0.39 8.7 (271155) CGC R 0.07 3.8 (117543) CUA L 0.11 9.9 (307000) CCA P 0.33 16.1 (502101) CAA Q 0.56 19.4 (604800) CGA R 0.12 6.3 (195736) CUG L 0.11 9.8 (305822) CCG P 0.18 8.6 (268115) CAG Q 0.44 15.2 (473809) CGG R 0.09 4.9 (151572) AUU I 0.41 21.5 (668227) ACU T 0.34 17.5 (544807) AAU N 0.52 22.3 (693344) AGU S 0.16 14.0 (435738) AUC I 0.35 18.5 (576287) ACC T 0.20 10.3 (321640) AAC N 0.48 20.9 (650826) AGC S 0.13 11.3 (352568) AUA I 0.24 12.6 (391867) ACA T 0.31 15.7 (487161) AAA K 0.49 30.8 (957374) AGA R 0.35 19.0 (589788) AUG M 1.00 24.5 (762852) ACG T 0.15 7.7 (240652) AAG K 0.51 32.7 (1016176) AGG R 0.20 11.0 (340922) GUU V 0.40 27.2 (847061) GCU A 0.43 28.3 (880808) GAU D 0.68 36.6 (1139637) GGU G 0.34 22.2 (689891) GUC V 0.19 12.8 (397008) GCC A 0.16 10.3 (321500) GAC D 0.32 17.2 (535668) GGC G 0.14 9.2 (284681) GUA V 0.15 9.9 (308605) GCA A 0.27 17.5 (543180) GAA E 0.52 34.3 (1068012) GGA G 0.37 24.2 (751489) GUG V 0.26 17.4 (539873) GCG A 0.14 9.0 (280804) GAG E 0.48 32.2 (1002594) GGG G 0.16 10.2 (316620)
[0072] The cell oils of this invention can be distinguished from conventional vegetable or animal triacylglycerol sources in that the sterol profile will be indicative of the host organism as distinguishable from the conventional source. Conventional sources of oil include soy, corn, sunflower, safflower, palm, palm kernel, coconut, cottonseed, canola, rape, peanut, olive, flax, tallow, lard, cocoa, shea, mango, sal, illipe, kokum, and allanblackia.
[0073] The oils provided herein are not vegetable oils. Vegetable oils are oils extracted from plants and plant seeds. Vegetable oils can be distinguished from the non-plant oils provided herein on the basis of their oil content. A variety of methods for analyzing the oil content can be employed to determine the source of the oil or whether adulteration of an oil provided herein with an oil of a different (e.g. plant) origin has occurred. The determination can be made on the basis of one or a combination of the analytical methods. These tests include but are not limited to analysis of one or more of free fatty acids, fatty acid profile, total triacylglycerol content, diacylglycerol content, peroxide values, spectroscopic properties (e.g. UV absorption), sterol profile, sterol degradation products, antioxidants (e.g. tocopherols), pigments (e.g. chlorophyll), d13C values and sensory analysis (e.g. taste, odor, and mouth feel). Many such tests have been standardized for commercial oils such as the Codex Alimentarius standards for edible fats and oils.
[0074] Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, b-sitosterol, and stigamsterol are common plant sterols, with b-sitosterol being a principle plant sterol. For example, b-sitosterol was found to be in greatest abundance in an analysis of certain seed oils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74% in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Cell and Molecular Biology 5:71-79, 2006).
[0075] The sterol profile of a microalgal oil is distinct from the sterol profile of oils obtained from higher plants or animals. Oil isolated from Prototheca moriformis strain UTEX1435 were separately clarified (CL), refined and bleached (RB), or refined, bleached and deodorized (RBD) and were tested for sterol content according to the procedure described in JAOCS vol. 60, no. 8, August 1983. Results of the analysis are shown Table 3 below (units in mg/100 g):
TABLE-US-00005 TABLE 3 (units in mg/100 g) Refined, Refined & bleached, & Sterol Crude Clarified bleached deodorized 1 Ergosterol 384 398 293 302 (56%) (55%) (50%) (50%) 2 5,22-cholestadien-24- 14.6 18.8 14 15.2 methyl-3-ol (2.1%) (2.6%) (2.4%) (2.5%) (Brassicasterol) 3 24-methylcholest-5- 10.7 11.9 10.9 10.8 en-3-ol (Campesterol or (1.6%) (1.6%) (1.8%) (1.8%) 22,23- dihydrobrassicasterol) 4 5,22-cholestadien-24- 57.7 59.2 46.8 49.9 ethyl-3-ol (Stigmasterol (8.4%) (8.2%) (7.9%) (8.3%) or poriferasterol) 5 24-ethylcholest-5-en- 9.64 9.92 9.26 10.2 3-ol (-Sitosterol or (1.4%) (1.4%) (1.6%) (1.7%) clionasterol) 6 Other sterols 209 221 216 213 Total sterols 685.64 718.82 589.96 601.1
[0076] These results show three striking features. First, ergosterol was found to be the most abundant of all the sterols, accounting for about 50% or more of the total sterols. The amount of ergosterol is greater than that of campesterol, -sitosterol, and stigmasterol combined. Ergosterol is steroid commonly found in fungus and not commonly found in plants, and its presence particularly in significant amounts serves as a useful marker for non-plant oils. Secondly, the oil was found to contain brassicasterol. With the exception of rapeseed oil, brassicasterol is not commonly found in plant based oils. Thirdly, less than 2% -sitosterol was found to be present. -sitosterol is a prominent plant sterol not commonly found in microalgae, and its presence particularly in significant amounts serves as a useful marker for oils of plant origin. In summary, Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of -sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol:-sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.
[0077] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% -sitosterol. In other embodiments the oil is free from -sitosterol.
[0078] In some embodiments, the oil is free from one or more of -sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from -sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.
[0079] In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-ethylcholest-5-en-3-ol. In some embodiments, the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% clionasterol.
[0080] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-methylcholest-5-en-3-ol. In some embodiments, the 24-methylcholest-5-en-3-ol is 22, 23-dihydrobrassicasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% 22,23-dihydrobrassicasterol.
[0081] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 5,22-cholestadien-24-ethyl-3-ol. In some embodiments, the 5, 22-cholestadien-24-ethyl-3-ol is poriferasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% poriferasterol.
[0082] In some embodiments, the oil content of an oil provided herein contains ergosterol or brassicasterol or a combination of the two. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 40% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of a combination of ergosterol and brassicasterol.
[0083] In some embodiments, the oil content contains, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, or 5% brassicasterol. In some embodiments, the oil content contains, as a percentage of total sterols less than 10%, 9%, 8%, 7%, 6%, or 5% brassicasterol.
[0084] In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.
[0085] In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% -sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% -sitosterol. In some embodiments, the oil content further comprises brassicasterol.
[0086] Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes. Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols. The sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols b-sitosterol and stigmasterol. In contrast, the sterol profiles of non-plant organisms contain greater percentages of C27 and C28 sterols. For example the sterols in fungi and in many microalgae are principally C28 sterols. The sterol profile and particularly the striking predominance of C29 sterols over C28 sterols in plants has been exploited for determining the proportion of plant and marine matter in soil samples (Huang, Wen-Yen, Meinschein W. G., Sterols as ecological indicators; Geochimica et Cosmochimia Acta. Vol 43. pp 739-745).
[0087] In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than b-sitosterol and stigmasterol. In some embodiments of the microalgal oils, C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.
[0088] In some embodiments the microalgal oils provided herein contain C28 sterols in excess of C29 sterols. In some embodiments of the microalgal oils, C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95% by weight of the total sterol content. In some embodiments the C28 sterol is ergosterol. In some embodiments the C28 sterol is brassicasterol.
[0089] Where a fatty acid profile of a triglyceride (also referred to as a triacylglyceride or TAG) cell oil is given here, it will be understood that this refers to a nonfractionated sample of the storage oil extracted from the cell analyzed under conditions in which phospholipids have been removed or with an analysis method that is substantially insensitive to the fatty acids of the phospholipids (e.g. using chromatography and mass spectrometry). The oil may be subjected to an RBD process to remove phospholipids, free fatty acids and odors yet have only minor or negligible changes to the fatty acid profile of the triglycerides in the oil. Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the TAGs in the cell. Examples 1 and 2 below give analytical methods for determining TAG fatty acid composition and regiospecific structure.
[0090] Broadly categorized, certain embodiments of the invention include (i) recombinant oleaginous cells that comprise an ablation of one or two or all alleles of an endogenous polynucleotide, including polynucleotides encoding lysophosphatidic acid acyltransferase (LPAAT) or (ii) cells that produce oils having low concentrations of polyunsaturated fatty acids, including cells that are auxotrophic for unsaturated fatty acids; (iii) cells producing oils having high concentrations of particular fatty acids due to expression of one or more exogenous genes encoding enzymes that transfer fatty acids to glycerol or a glycerol ester; (iv) cells producing regiospecific oils, (v) genetic constructs or cells encoding a an LPAAT, a lysophosphatidylcholine acyltransferase (LPCAT), a phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), diacylglycerol cholinephosphotransferase (DAG-CPT) or fatty acyl elongase (FAE), (vi) cells producing low levels of saturated fatty acids and/or high levels of C18:1, C18:2, C18:3, C20:1 or C22:1, (vii) and other inventions related to producing cell oils with altered profiles. The embodiments also encompass the oils made by such cells, the residual biomass from such cells after oil extraction, oleochemicals, fuels and food products made from the oils and methods of cultivating the cells.
[0091] In any of the embodiments below, the cells used are optionally cells having a type II fatty acid biosynthetic pathway such as plant cells, yeast cells, microalgal cells including heterotrophic or obligate heterotrophic microalgal cells, including cells classified as Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae, or cells engineered to have a type II fatty acid biosynthetic pathway using the tools of synthetic biology (i.e., transplanting the genetic machinery for a type II fatty acid biosynthesis into an organism lacking such a pathway). Use of a host cell with a type II pathway avoids the potential for non-interaction between an exogenous acyl-ACP thioesterase or other ACP-binding enzyme and the multienzyme complex of type I cellular machinery. In specific embodiments, the cell is of the species Prototheca moriformis, Prototheca krugani, Prototheca stagnora or Prototheca zopfii or has a 23S rRNA sequence with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide identity SEQ ID NO: 25. By cultivating in the dark or using an obligate heterotroph, the cell oil produced can be low in chlorophyll or other colorants. For example, the cell oil can have less than 100, 50, 10, 5, 1, 0.0.5 ppm of chlorophyll without substantial purification.
[0092] The stable carbon isotope value 13C is an expression of the ratio of .sup.13C/.sup.12C relative to a standard (e.g. PDB, carbonite of fossil skeleton of Belemnite americana from Peedee formation of South Carolina). The stable carbon isotope value 13C () of the oils can be related to the 13C value of the feedstock used. In some embodiments the oils are derived from oleaginous organisms heterotrophically grown on sugar derived from a C4 plant such as corn or sugarcane. In some embodiments the 13C () of the oil is from 10 to 17 or from 13 to 16.
[0093] In specific embodiments and examples discussed below, one or more fatty acid synthesis genes (e.g., encoding an acyl-ACP thioesterase, a keto-acyl ACP synthase, an LPAAT, an LPCAT, a PDCT, a DAG-CPT, an FAE a stearoyl ACP desaturase, or others described herein) is incorporated into a microalga. It has been found that for certain microalga, a plant fatty acid synthesis gene product is functional in the absence of the corresponding plant acyl carrier protein (ACP), even when the gene product is an enzyme, such as an acyl-ACP thioesterase, that requires binding of ACP to function. Thus, optionally, the microalgal cells can utilize such genes to make a desired oil without co-expression of the plant ACP gene.
[0094] For the various embodiments of recombinant cells comprising exogenous genes or combinations of genes, it is contemplated that substitution of those genes with genes having 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% nucleic acid sequence identity can give similar results, as can substitution of genes encoding proteins having 60%, 70%, 80%, 85%, 90%, 91% 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 100% amino acid sequence identity. Nucleic acids encoding the acyltransferases encode acyltransferases that have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to the acyltransferase disclosed in clade 1, clade 2, clade 3 or clade 4 of Table 5. Likewise, for novel regulatory elements, it is contemplated that substitution of those nucleic acids with nucleic acids having 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid can be efficacious. In the various embodiments, it will be understood that sequences that are not necessary for function (e.g. FLAG tags or inserted restriction sites) can often be omitted in use or ignored in comparing genes, proteins and variants.
[0095] The novel genes and gene combinations reported here can be used in higher plants using techniques that are well known in the art. For example, the use of exogenous lipid metabolism genes in higher plants is described in U.S. Pat. Nos. 6,028,247; 5,850,022; 5,639,790; 5,455,167; 5,512,482; and 5,298,421 disclose higher plants with exogenous acyl-ACP thioesterases. WO2009129582 and WO1995027791 disclose cloning of LPAAT in plants. FAD2 ablation and/or down regulation in higher plants is taught in WO 2013112578, and WO2008/006171. SAD ablation and/or down regulation in higher plants is taught in WO 2013112578, and WO 2008006171.
[0096] The expression of the novel acyltransferases is shown in Examples 4, 5, 6 and 7. The expression of Cuphea paucipetala or Cuphea ignea LPATs markedly increased the C8:0 and C10:0 fraction of the cell oil. Additionally, the expression of Cuphea paucipetala or Cuphea ignea LPAATs markedly increased the incorporation of C8:0 and C10:0 fatty acids in the sn-2 position of the TAG. This is disclosed in Example 4.
[0097] The expression of LPAT genes in host cells increased C18:2 levels and elevated the sat-unsat-sat/sat-sat-sat, (e.g., SOS/SSS) ratio of the cell oil. For example, the expression of Theobroma cacoa LPAT2 drives the transfer of unsaturated fatty acids toward the sn-2 position and reduces the incorporation of saturated fatty acids at sn-2.
[0098] The novel LPAATs, GPATs, DGATs, LPCATs, and PLA2 with specificity for mid-chain fatty acids are disclosed. In Example 7, expression of LPAATs and DGATs are disclosed.
[0099] When an acyltransferase of the invention is expressed in a host cell, one or more additional exogenous genes can concomitantly be expressed. An embodiment of this invention provides host cells that express a recombinant acyltransferase and concomitantly express one or more additional recombinant genes. The one or more additional genes include invertase, fatty acyl-ACP thioesterase (FATA, FATB), melibiase, ketoacyl synthase (KASI, KASII, KASIII, KASIV), antibiotic selective markers, tags such as FLAG, and THIC. In Examples 4, 5, 6, and 7, the co-expression of nucleic acids that encode LPAATs co-expressed with one or more exogenous genes that encode invertase, fatty acyl-ACP thioesterase, melibiase, ketoacyl synthase, THIC are disclosed.
[0100] When an acyltransferase of the invention is expressed in a host cell, an endogenous gene of the host call can concomitantly be ablated or downregulated, thereby eliminating or decreasing the expression of the gene of the host cell. This can be accomplished by using homologous recombination techniques or other RNA inhibitory technologies. The ablated or downregulated gene can be any gene in the host cell. The ablated or downregulated endogenous gene can be stearoyl ACP desaturase, fatty acyl desaturase, fatty acyl-ACP thioesterase (FATA or FATB), ketoacyl synthase (KASI, KASII, KASIII or KAS IV), or an acyltransferase (LPAAT, DGAT, GPAT, LPCAT). When an endogenous is ablated, one, two or more alleles of the endogenous can be ablated. In Example 5, the expression of a Brassica LPAAT, while concomitantly ablating an endogenous stearoyl ACP desaturase is disclosed. In Example 6, LPAATs, GPATs, DGATs, LPCATs and PLA2s with specificity for mid-chain fatty acids were expressed, while ablating a gene encoding stearoyl ACP desaturase. In Example 7 the down regulation of an endogenous FAD2 and a hairpin RNA is disclosed. In co-owned PCT/US2016/026265, applicants disclosed concomitant ablation of an endogenous LPAAT and expression of an exogenous LPAAT.
[0101] In one embodiment, the expression of the acyl transferases alters the fatty acid profile and/or the sn-2 profile of the oil produced by the host organism. The fatty acid profiles and the sn-2 profiles that result from the expression of various acyltransferases are disclosed in Tables 6, 7, 10, 11, 12, 13, 16, 17, 18, 19, 20, 22, 23, and 24. The invention provides host cells with altered fatty acid profiles and altered sn-2 profiles according to Tables 6, 7, 10, 11, 12, 13, 16, 17, 18, 19, 20, 22, 23, and 24.
[0102] As described in PCT/US2016/026265, co-owned by applicant, transcript profiling was used to discover promoters that modulate expression in response to low nitrogen conditions. The promoters are useful to selectively express various genes and to alter the fatty acid composition of microbial oils. In accordance with an embodiment, there are non-natural constructs comprising a heterologous promoter and a gene, wherein the promoter comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any of the promoters of SEQ ID NOs: 1-18 and the gene is differentially expressed under low vs. high nitrogen conditions. In particular, the Prototheca moriformis AMT02 (SEQ ID NO: 18) and AMT03 promoter (SEQ ID NO: 18) are useful promoters for controlling the expression of an exogenous gene. For example, the promoters can be placed in front of a FAD2 gene in a linoleic acid auxotroph to produce an oil with less than 5, 4, 3, 2, or 1% linoleic acid after culturing first under high nitrogen conditions, then next culturing under low nitrogen conditions. Additional promoters, in particulare Prototheca and Chlorella promoters are described in the sequences and descriptions in this application. For example, the Prototheca HXT1, SAD, LDH1 and other Prototheca promoters are described in Examples 6, 7, 8, and 9. Additionally, the Chlorella SAD, ACT and other Chlorella promoters are described in Examples 6, 7, 8, and 9.
[0103] In embodiments of the present invention, oleaginous cells expressing one or more of the genes encoding acyltransferases and/or variant FATA can produce an oil with at least 20, 40, 60 or 70% of C8, C10, C12, C14, C16, or C18 fatty acids.
[0104] The invention also provides host cells expressing one or more of the genes encoding acyltransferases and/or variant FATA can produce an oil enriched is oils that are sat-unsat-sat. Oils of this type include SOS, POP, POS, SLS, PLO, PLO. The sat-unsat-sat oils comprise at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cell oil by dry cell weight.
[0105] The invention also provides host cells expressing one or more of the genes encoding acyltransferases and/or variant FATA can produce an oil that is decreased in tri-saturated oils, sat-sat-sat. Oils of this type include PPP, PSS, PPS, SSS, SPS, and PSP. The sat-sat-sat oils comprise less than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the cell oil by molar fraction or dry cell weight.
[0106] The host cells of the invention can produce 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, or about 90% oil by cell weight, 5%. Optionally, the oils produced can be low in DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA.
[0107] In other embodiments of the invention, there is a process for producing an oil, triglyceride, fatty acid, or derivative of any of these, comprising transforming a cell with any of the nucleic acids discussed herein. In another embodiment, the transformed cell is cultivated to produce an oil and, optionally, the oil is extracted. Oil extracted in this way can be used to produce food, oleochemicals or other products.
[0108] The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, etc). The oils, triglycerides, fatty acids from the oils may be subjected to CH activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation, methylation, dimerization, thiolation, metathesis, hydro-alkylation, lactonization, or other chemical processes.
[0109] After extracting the oil, a residual biomass may be left, which may have use as a fuel, as an animal feed, or as an ingredient in paper, plastic, or other product. For example, residual biomass from heterotrophic algae can be used in such products.
EXAMPLES
Example 1: Fatty Acid Analysis by Fatty Acid Methyl Ester Detection
[0110] Lipid samples were prepared from dried biomass. 20-40 mg of dried biomass was resuspended in 2 mL of 5% H.sub.2SO.sub.4 in MeOH, and 200 ul of toluene containing an appropriate amount of a suitable internal standard (C19:0) was added. The mixture was sonicated briefly to disperse the biomass, then heated at 70-75 C. for 3.5 hours. 2 mL of heptane was added to extract the fatty acid methyl esters, followed by addition of 2 mL of 6% K.sub.2CO.sub.3 (aq) to neutralize the acid. The mixture was agitated vigorously, and a portion of the upper layer was transferred to a vial containing Na.sub.2SO.sub.4 (anhydrous) for gas chromatography analysis using standard FAME GC/FID (fatty acid methyl ester gas chromatography flame ionization detection) methods. Fatty acid profiles reported below were determined by this method.
Example 2: Analysis of Regiospecific Profile
[0111] LC/MS TAG distribution analyses were carried out using a Shimadzu Nexera ultra high performance liquid chromatography system that included a SIL-30AC autosampler, two LC-30AD pumps, a DGU-20A5 in-line degasser, and a CTO-20A column oven, coupled to a Shimadzu LCMS 8030 triple quadrupole mass spectrometer equipped with an APCI source. Data was acquired using a Q3 scan of m/z 350-1050 at a scan speed of 1428 u/sec in positive ion mode with the CID gas (argon) pressure set to 230 kPa. The APCI, desolvation line, and heat block temperatures were set to 300, 250, and 200 C., respectively, the flow rates of the nebulizing and drying gases were 3.0 L/min and 5.0 L/min, respectively, and the interface voltage was 4500 V. Oil samples were dissolved in dichloromethane-methanol (1:1) to a concentration of 5 mg/mL, and 0.8 L of sample was injected onto Shimadzu Shim-pack XR-ODS III (2.2 m, 2.0200 mm) maintained at 30 C. A linear gradient from 30% dichloromethane-2-propanol (1:1)/acetonitrile to 51% dichloromethane-2-propanol (1:1)/acetonitrile over 27 minutes at 0.48 mL/min was used for chromatographic separations.
Example 3: Cultivation of Microalgae
Standard Lipid Production Conditions:
[0112] Cells scraped from a source plate with toothpicks were used to inoculate pre-seed cultures of 0.5 mL EB03, 0.5% glucose, 1DAS2 cultures in 96-well blocks. Pre-seed cultures were grown for 70-75 h at 28 C., 900 rpm in a Multitron shaker. 40 l of pre-seed cultures were used to inoculate seed cultures of 0.46 mL H29, 4% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1DAS2 (8% inoculum), and grown for 24-28 h at 28 C., 900 rpm in a Multitron shaker. 40 L of seed cultures were used to inoculate lipid production cultures of 0.46 mL H43, 6% glucose, 25 mM citrate pH 5, 1DAS2 (8% inoculum), and grown for 70-75 h at 28 C., 900 rpm in a Multitron shaker. Fatty acid profiles and lipid titer analyses were performed as disclosed in Examples 1 and 2.
50 mL Shake Flask Format
[0113] Cells scraped from a source plate with inoculation loops, or cell cultures from cryovials were used to inoculate pre-seed cultures of 10 mL EB03, 0.5% glucose, 1DAS2 cultures in 50 mL bioreactor tubes. Pre-seed cultures were grown for 70-75 h at 28 C., 200 rpm in a Kuhner shaker. 0.8 mL of pre-seed cultures were used to inoculate seed cultures of 10 mL H29, 4% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1DAS2 (8% inoculum), and grown for 24-28 h at 28 C., 200 rpm in a Kuhner shaker. 100 L of seed cultures were used to inoculate lipid production cultures of 49.9 mL H43, 6% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1DAS2 (0.2% inoculum), and grown for 118-122 h at 28 C., 200 rpm in a Kuhner shaker. Fatty acid profiles and lipid titer analyses were performed as disclosed in Examples 1 and 2.
EB03
[0114]
TABLE-US-00006 Dry chemicals Component Concentration (g/L) K2HPO4 3 Sodium Phosphate Dibasic Heptahydrate 5.66 (Na2HPO4 7H2O) citric acid monohydrate 1.2 ammonium sulfate 1 MgSO4 7H2O 0.23 CaCl2 2H2O 0.03 Stock solutions Component Concentration (mL/L) 100X C-Trace (3) 10 Antifoam Sigma 204 0.225
H29
[0115]
TABLE-US-00007 Dry chemicals Final Component Concentration (g/L) K.sub.2HPO.sub.4 (Potassium phosphate 0.25 dibasic anhydrous) NaH.sub.2PO.sub.4 (Sodium phosphate 0.18 monobasic) MgSO.sub.47H.sub.2O (Magnesium 0.24 sulfate heptahydrate) Citric acid monohydrate 0.25 Stock solutions Component Concentration (mL/L) 0.017M stock CaCl.sub.22H.sub.2O 10 0.151M (NH.sub.4).sub.2SO.sub.4 52.2 100X C-Trace (2) 10 Antifoam Sigma 204 0.225
H43
[0116]
TABLE-US-00008 Dry chemicals Final Component Concentration (g/L) K2HPO4 0.25 NaH2PO4 0.18 MgSO4 7H2O 0.24 Citric acid H2O 0.25 Stock solutions Component Concentration (mL/L) 0.017M stock CaCl2 2H2O 10 100X C-Trace (2) 10 Antifoam Sigma 204 0.225 0.151M (NH4)2SO4 12.5
1000DAS2
[0117]
TABLE-US-00009 Dry chemicals Final Component Concentration (g/L) Thiamine-HCl 0.67 d-Biotin 0.010 Cyanocobalimin (vit B-12) 0.008 Calcium Pantothenate 0.02 PABA (p-aminobenzoic acid) 0.04
100C-Trace(2)
[0118]
TABLE-US-00010 Dry chemicals Final Component Concentration (g/L) CuSO45H2O 0.011 CoC126H2O 0.081 H3BO3 0.33 ZnSO47H2O 1.4 MnSO4H2O 0.81 Na2MoO42H2O 0.039 FeSO47H2O 0.11 NiCl26H2O 0.013 Citric Acid Monohydrate 3.0
100C-Trace (3)
[0119]
TABLE-US-00011 Dry chemicals Final Component Concentration (g/L) CuSO45H2O 0.011 H3BO3 0.33 ZnSO47H2O 1.4 MnSO4H2O 0.81 Na2MoO42H2O 0.039 FeSO47H2O 0.11 NiCl26H2O 0.013 Citric Acid Monohydrate 3.0
Example 4: Identification of Novel LPAAT Genes from Sequenced Transcriptomes and Engineering Sn-2 Tag Regiospecificity in UTEX1435 by Expression of Heterologous LPAAT Genes from Cuphea paucipetala, Cuphea ignea, Cuphea painteri, and Cuphea hookeriana
[0120] Lysophosphatidic acyltransferase (LPAAT) genes from plant seeds were cloned and expressed in the transgenic strain, S6511, derived from UTEX 1435 (P. moriformis). Expression of the heterologous LPAATs increases C8:0 and C10:0 fatty acid levels and dramatically increases incorporation of C8:0 and C10:0 fatty acids at the sn-2 position of triacylglycerols (TAGs) in transgenic strains.
[0121] TAGs are synthesized from various chain length acyl-CoAs and glycerol-3-phosphate by consecutive action of three ER-resident enzymes of the Kennedy pathway-glycerol phosphate acyltransferase (GPAT), LPAAT, and diacylglycerol acyltransferase (DGAT). Substrate specificities of these acyltransferases are known to determine the fatty acid composition of the resulting TAGs. LPAAT acylates the sn-2 hydroxyl group of lysophosphatidic acid (LPA) to form phosphatidic acid (PA), a precursor to TAG. In co-owned applications WO2013/158938, WO2015/051139, and PCT/US2016/026265 we demonstrated expression of LPAAT from Cocos nucifera (CnLPAAT, accession no. AAC49119; Knutzon et al., 1995).
[0122] Strain S6511 expresses the acyl-ACP thioesterase (FATB2) gene from Cuphea hookeriana (ChFATB2), leading to C8:0 and C10:0 fatty acid accumulation of ca. 14% and 28%, respectively. Strain S6511 is a strain made according to the methods disclosed in co-owned WO2010/063031 and WO2010/063032, herein incorporated by reference. Briefly, S6511 is a strain that express sucrose invertase and a C. hookeriana FATB2. The construct pSZ3101: 6S::CrTUB2-ScSUC2-CvNR_a:PmAMT03-CpSAD1tp_trimmed:ChFATB2-CvNR_d::6S was engineered into S3150, a strain classically mutagenized to increase lipid yield. We identified novel C8:0- and C10:0-specific LPAATs from seeds exhibiting high levels of C8:0 and C10:0 fatty acids. After we identified and cloned LPAATs we expressed the LPAAT genes in S6511.
Method for Identification of LPAATs
[0123] Seeds were obtained from species exhibiting elevated levels of midchain and other specialized fatty acids (Table 4).
TABLE-US-00012 TABLE 4 Fatty acid profiles of mature seeds. C18:1 C22: C8: C10: C12: C14: C16: C18: C18: (petro- C18: C20: C20: C22: C22: C22: 2n9, C22: 0 0 0 0 0 0 1 selinate) 2 0 1 0 1n17 1n9 17 2n6 S01_Cc Cinnamomum 0.4 54.7 39.0 1.6 0.7 0.1 2.9 0.6 0.0 camphora S02_Uc Umbellularia 0.9 28.8 63.0 2.3 0.4 0.1 3.4 0.6 0.0 californica S03_Ld Limnanthes 0.0 0.0 0.0 0.4 0.7 0.4 2.7 1.5 1.5 59.9 0.3 2.8 17.4 9.3 0.5 douglasii S04_Chs Cuphea 0.2 6.5 83.7 5.1 1.1 0.1 0.0 1.7 0.1 hyssopifolia S05_Ccr Cuphea 1.6 8.1 59.2 15.2 3.9 0.6 0.0 5.4 0.2 carthagenensis S06_Cpr Cuphea 2.0 11.5 61.3 10.8 2.7 0.5 0.0 5.2 0.1 parsonsia S07_Cg Cuphia 7.1 85.1 1.7 0.3 1.0 0.2 0.0 2.1 0.1 glossostoma S08_Cht Cuphea 3.5 44.3 40.0 4.3 1.2 0.3 2.2 3.6 0.1 heterophylla S11_Dc Daucus 0.0 0.0 0.0 0.1 5.9 0.8 11.5 65.9 13.0 0.5 0.3 0.3 carrota S14_Cw Cuphea 0.5 20.2 62.5 5.8 2.2 0.3 2.7 4.7 wrightii S15_Bj Brassica 0.0 0.0 0.0 0.1 3.2 0.7 12.1 19.2 0.5 6.3 0.8 38.9 1.3 juncea S16_Br Brassica 0.0 0.0 0.0 0.1 2.8 1.0 16.0 16.8 0.7 8.3 1.0 40.4 0.8 rapa nipposinica S17_Ca Cuphea 90.8 2.7 0.0 0.1 1.2 0.1 1.8 2.8 avigera var. pulcherrima S18_Ch Cuphea 64.7 29.7 0.1 0.2 1.3 0.1 1.9 2.0 hookeriana S19_Cpal Cuphea 28.9 0.8 1.3 55.1 6.2 0.2 3.0 3.4 palustris S20_Cpai Cuphea 67.0 20.8 0.1 0.2 2.6 0.3 3.1 4.5 painteri S21_Cpau Cuphea 1.5 91.0 1.2 0.7 1.5 0.2 1.1 2.1 paucipetala S22_Chook Cuphea 62.8 31.9 0.2 0.2 1.0 0.1 2.1 1.2 hookeriana S23_Cglut Cuphea 5.2 29.9 46.4 3.9 1.9 0.4 0.0 8.1 glutinosa S24_Caequ Cuphea 27.1 0.0 1.4 57.4 6.0 0.2 3.2 3.8 aequipetala S25_Ccalc Cuphea 8.0 20.4 46.8 7.6 3.2 0.6 3.7 8.5 calcarata S26_Chook Cuphea 70.4 23.1 0.1 0.2 1.5 0.2 2.5 1.8 hookeriana S27_Cproc Cuphea 0.9 86.3 0.0 1.6 2.2 0.4 3.2 3.3 procumbens S28_Cignea Cuphea 3.1 84.9 0.7 0.3 2.6 0.2 2.9 4.4 ignea S35_Ccras Cuphea 1.3 87.7 1.3 0.4 2.0 0.5 3.3 2.7 crassiflora S36_Ckoe Cuphea 0.0 87.4 1.4 0.8 2.2 0.4 2.3 4.5 koehneana S37_Clept Cuphea 1.3 86.1 1.3 0.4 2.2 0.5 3.1 4.1 leptopoda S40_Clop Cuphea 0.5 82.3 2.4 1.6 3.0 0.6 3.9 4.9 lophostoma S41_Sal Sassafras 4.3 65.2 22.8 0.9 0.8 5.1 0.0 0.6 albidum db The percentage of each fatty acid making up the seed oil is shown; abundant and unusual fatty acid species are indicated in bold.
[0124] Briefly, RNA was extracted from dried plant seeds and submitted for paired-end sequencing using the Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using the Trinity software package. LPAAT-containing cDNA contigs were identified by mining transcriptomes for sequences with homology to a known LPAAT that was previously identified in-house, CuPSR23 LPAAT2-1 (see WO2013/158938), using BLAST. For some sequences, a high-confidence, full-length transcript was assembled using Trinity. The resulting amino acid sequences of all new LPAATs were subjected to phylogenetic analyses using previously known, full-length LPAAT sequences (available via NCBI) as well as sequences of previously known LPAATs whose sequences were derived at Solazyme. The analysis showed that the amino acid sequences of the newly discovered LPPAATs were not similar to previously known LPAATs. Table 5 shows the clade analysis in which the novel LPAATs were clustered according to a neighbor joining algorithm. These were found to form 4 clades as listed in Table 5.
TABLE-US-00013 TABLE 5 Clade Analysis of LPAATs Percent amino acid Amino Acid identity Clade SEQ ID Nos. to members No. in Clade Full Genus Species Function of clade 1 S15 BjLPAAT1d Brassica juncea 96.3 S15 BjLPAAT1c Brassica juncea S15 BjLPAAT1a Brassica juncea S15 BjLPAAT1b Brassica juncea 2 CuPSR23LPAAT2-1 Cuphea PSR23 Prefer C8/ 93.9 S40 ClopLPAAT1 Cuphea lophostoma C10 sn-2 S21 CpauLPAAT1 Cuphea paucipetala S37 CleptLPAAT1 Cuphea leptopoda S27 CprocLPAAT1b Cuphea procumbens S27 CprocLPAAT1 Cuphea procumbens S04 ChsLPAAT2 Cuphea hyssopifolia S28 CigneaLPAAT1 Cuphea ignea S05 CcrLPAAT2a Cuphea carthagenensis S06 CprLPAAT1 Cuphea parsonsia S05 CcrLPAAT2b Cuphea carthagenensis S17 CaLPAAT3 Cuphea avigera var. pulcherrima S26 ChookLPAAT1 Cuphea hookeriana S20 CpaiLPAAT1 Cuphea painteri S04 ChsLPAAT1 Cuphea hyssopifolia S25 Ccalc1a Cuphea calcarata S25 Ccalc1b Cuphea calcarata S14 CwLPAAT1 Cuphea wrightii S08 ChtLPAAT1a Cuphea heterophylla S08 ChtLPAAT1b Cuphea heterophylla S36 CkoeLPAAT2 Cuphea koehneana S02 UcLPAAT1b Umbellularia californica S02 UcLPAAT1a Umbellularia californica S01 CcLPAAT1a Cinnamomum camphora S01 CcLPAAT1b Cinnamomum camphora S41 SaILPAAT1 Sassafras albidum db 3 S14 CwLPAAT2a Cuphea wrightii C18:2 86.5 S14 CwLPAAT2b Cuphea wrightii S25 CcalcLPAAT2 Cuphea calcarata S19 CpaILPAAT1 Cuphea palustris S22 ChookLPAAT3b Cuphea hookeriana S17 CaLPAAT1 Cuphea avigera var. pulcherrima S22 ChookLPAAT3a Cuphea hookeriana CuPSR23LPAAT3-1 Cuphea PSR23 S27 CprocLPAAT2b Cuphea procumbens S27 CprocLPAAT2a Cuphea procumbens S18 ChLPAAT2a Cuphea hookeriana S24 CaequLPAAT1d Cuphea aequipetala S24 CaequLPAAT1b Cuphea aequipetala S24 CaequLPAAT1a Cuphea aequipetala S24 CaequLPAAT1c Cuphea aequipetala S23 CglutLPAAT1a Cuphea glutinosa S23 CglutLPAAT1b Cuphea glutinosa S26 ChookLPAAT2b Cuphea hookeriana S07 CgLPAAT1c Cuphia glossostoma S07 CgLPAAT1b Cuphia glossostoma S07 CgLPAAT1a Cuphia glossostoma S28 CigneaLPAAT2 Cuphea ignea S36 CkoeLPAAT1 Cuphea koehneana S35 CcrasLPAAT1a Cuphea crassiflora S35 CcrasLPAAT1c Cuphea crassiflora S35 CcrasLPAAT1b Cuphea crassiflora S35 CcrasLPAAT1d Cuphea crassiflora 4 Gh LPAAT2B Garcinia hombroriana Reduced 78.5 Gi LPAAT2B-1 Garcinia indica trisaturates, Gh LPAAT2A Garcinia hombroriana increase Gi LPAAT2A Garcinia indica unsaturates Gh LPAAT2C Garcinia hombroriana at Sn-2 Gi LPAAT2C-2 Garcinia indica position S03 LdLPAAT1 Limnanthes douglasii S11 DcLPAAT1 Daucus carrota (carrot) S11 DcLPAAT2 Daucus carrota (carrot) S11 DcLPAAT2 Daucus carrota (truncated) (carrot)
Functionality of LPAATs in P. moriformis
[0125] To increase the levels of C8:0 and C10:0 fatty acids in strain S6511, as well as to test the functionality of the newly identified LPAATs, we identified midchain-specific LPAATs from the transcriptomes of species exhibiting high levels of C8:0 and C10:0 fatty acids in their oil seeds and introduced the genes into S6511. LPAATs that co-clustered with CuPSR23 LPAAT2-1, specifically CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1, were selected for synthesis and testing. CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1 were synthesized in a codon-optimized form to reflect UTEX 1435 codon usage. Transgenic strains were generated via transformation of the strain S6511 with a construct encoding one of the four LPAAT genes. The construct pSZ3840 encoding CpauLPAAT1 is shown as an example, but identical methods were used to generate each of the remaining three constructs. Construct pSZ3840 can be written as pLOOP::PmHXT1-ScarMEL1-CvNR:PmAMT3-CpauLPAAT1-CvNR::pLOOP. The sequence of the transforming DNA is provided in FIG. 2 (pSZ3840). The relevant restriction sites in the construct from 5-3, BspQI, KpnI, SpeI, XhoI, EcoRI, SpeI, XhoI, SacI, BspQI, respectively, are indicated in lowercase, bold, and underlined. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Bold lowercase sequences at the 5 and 3 end of the construct represent genomic DNA from UTEX 1435 that target integration to the pLOOP locus via homologous recombination. Proceeding in the 5 to 3 direction, the selection cassette has the P. moriformis HXT1 promoter driving expression of the Saccharomyces carlsbergensis MEL1 (conferring the ability to grow on melibiose) and the Chlorella vulgaris Nitrate reductase (NR) gene 3 UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScarMEL1 are indicated in bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3 UTR is indicated by lowercase underlined text. The second cassette containing the codon optimized CpauLPAAT1 gene from Cuphea paucipetala is driven by the P. moriformis AMT3 promoter and has the Chlorella vulgaris Nitrate reductase (NR) gene 3 UTR. In this cassette, the AMT3 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CpauLPAAT1 gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics. The 3 UTR is indicated by lowercase underlined text. The final construct was sequenced to ensure correct reading frame and targeting sequences.
TABLE-US-00014 SEQIDNO:19 pSZ3840/D2554transformingconstruct(CpauLPAAT1) [00001]![embedded image]()
aatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgt cttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaa ttacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggc ttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagcca ggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggtatgggaaccacgggg [00002]![embedded image]()
[00003]![embedded image]()
[00004]![embedded image]()
[00005]![embedded image]()
[00006]![embedded image]()
[00007]![embedded image]()
[00008]![embedded image]()
[00009]![embedded image]()
[00010]![embedded image]()
[00011]![embedded image]()
[00012]![embedded image]()
gcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggacacggccga ccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctccggccgcgactccga cggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgtt cggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacgcccagttct tcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcccgagatctcctacca ccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccctgtgcaactggggccaggacctga ccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcggagttcacgcgccccgactcccgct gcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctgaacaaggccgccccc atgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaacctgacggacgacga ggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaacaacctgaaggcctcct cctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtctggcgctacta cgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtggc gctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctccaagaa gctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgcaaca agaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttcgg ccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttctaccgcctgcgcccc [00013]![embedded image]()
acacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagtt gctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacg ctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtact [00014]![embedded image]()
[00015]![embedded image]()
[00016]![embedded image]()
[00017]![embedded image]()
[00018]![embedded image]()
[00019]![embedded image]()
[00020]![embedded image]()
[00021]![embedded image]()
[00022]![embedded image]()
[00023]![embedded image]()
[00024]![embedded image]()
[00025]![embedded image]()
[00026]![embedded image]()
[00027]![embedded image]()
atcaacctgttccaggccctgtgcttcgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccg agctgctgctgtccgagctgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttcc gcctgatgggcaaggagcacgccctggtgatcatcaaccacatgaccgagctggactggatgctgggctgggtgatgggcca gcacctgggctgcctgggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttct ccgagtacctgtacatcgagcgctcctgggccaaggaccgcaccaccctgaagtcccacatcgagcgcctgaccgactacccc ctgcccttctggatggtgatcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcct ccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgccc gccgtgtacgacgtgaccgtggccttccccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcgtgc tgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagtt cgtggagaaggacgccctgctggacaagcacaacgccgaggacaccttctccggccaggaggtgcaccgcaccggctcccg ccccatcaagtccctgctggtggtgatctcctgggtggtggtgatcaccttcggcgccctgaagttcctgcagtggtcctcctgga agggcaaggccttctccgtgatcggcctgggcatcgtgaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctc [00028]![embedded image]()
[00029]![embedded image]()
gacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtg ctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatcc ctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaac [00030]![embedded image]()
gtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcct ggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgc cagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactct cgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaag ggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttctt [00031]![embedded image]()
[0126] The sequence for all of the other LPAAT constructs are identical to that of pSZ3840 with the exception of the encoded LPAAT. The LPAAT sequence alone with flanking SpeI and XhoI restriction sites is provided for the remaining LPAAT constructs are shown below. The amino acid sequence of the LPAAT proteins is provided below.
TABLE-US-00015 pSZ3841/D2555(CpaiLPAAT1) SEQIDNO:20 actagt![custom-character]()
gccatcccctccgccgccgtggtgttcctgttcggcctgc tgttcttcacctccggcctgatcatcaacctgttccaggccttctgctt cgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgc gtgacgccgagctgctgcccctggagacctgtggctgttccactggtgc gccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctga tgggcaaggagcacgccctggtgatcatcaaccacaagatcgagctgga ctggatggtgggctgggtgctgggccagcacctgggctgcctgggctcc atcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggct ggtccctgtggttctccggctacctgttcctggagcgctcctgggccaa ggacaagatcaccctgaagtcccacatcgagtccctgaaggactacccc ctgcccttctggctgatcatcttcgtggagggcacccgcttcacccgca ccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgt gccccgcaacgtgctgatcccccacaccaagggcttcgtgtcctccgtg tcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggcct tccccaagacctcccccccccccaccatgctgaagctgacgagggccag tccgtggagctgcacgtgcacatcaagcgccacgccatgaaggacctgc ccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtgga gaaggacgccctgctggacaagcacaactccgaggacaccttctccggc caggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtga tctcctgggtggtggtgatcatcttcggcgccctgaagttcctgctgtg gtcctccctgctgtcctcctggaagggcaaggccactccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcct gtcctcccaggccgagggctccaaccccgtgaaggccgcccccgccaag ctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaac ![custom-character]()
![custom-character]()
ctcgag pSZ3842/D2556(CigneaLPAAT1) SEQIDNO:21 actagt![custom-character]()
gccatcgccgccgccgccgtgatcttcctgttcggcctgc tgttcttcgcctccggcatcatcatcaacctgttccaggccctgtgctt cgtgctgatctggcccctgtccaagaacgtgtaccgccgcatcaaccgc gtgacgccgagctgctgctgatggacctgctgtgcctgttccactggtg ggccggcgccaagatcaagctgacaccgaccccgagaccttccgcctga tgggcatggagcacgccctggtgatcatgaaccacaagaccgacctgga ctggatggtgggctggatcctgggccagcacctgggctgcctgggctcc atcctgtccatcgccaagaagtccaccaagttcatccccgtgctgggct ggtccgtgtggactccgagtacctgttcctggagcgctcctgggccaag gacaagtccaccctgaagtcccacatggagaagctgaaggactaccccc tgcccttctggctggtgatcttcgtggagggcacccgcttcacccgcac caagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtg ccccgcaacgtgctgatcccccacaccaagggcttcgtgtcctgcgtgt ccaacatgcgctccacgtgcccgccgtgtacgacgtgaccgtggccttc cccaagtcctcccccccccccaccatgctgaagctgttcgagggccagt ccatcgtgctgcacgtgcacatcaagcgccacgccctgaaggacctgcc cgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggag aaggacgccctgctggacaagcacaacgccgaggacaccttctccggcc aggaggtgcaccacatcggccgccccatcaagtccctgctggtggtgat cgcctgggtggtggtgatcatcttcggcgccctgaagttcctgcagtgg tcctccctgctgtccacctggaagggcaaggccttctccgtgatcggcc tgggcatcgccaccctgctgatgcacatgctgatcctgtcctcccaggc cgagcgctccaaccccgccaaggtggccaag![custom-character]()
ctcgag pSZ3844/D2557(ChookLPAAT1) SEQIDNO:22 actagt![custom-character]()
gccatcccctccgccgccgtggtgttcctgttcggcctgc tgttcttcacctccggcctgatcatcaacctgttccaggccttctgctt cgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgc gtgacgccgagctgctgcccctggagacctgtggctgttccactggtgc gccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctga tgggcaaggagcacgccctggtgatcatcaaccacaagatcgagctgga ctggatggtgggctgggtgctgggccagcacctgggctgcctgggctcc atcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggct ggtccctgtggttctccgagtacctgttcctggagcgctcctgggccaa ggacaagatcaccctgaagtcccacatcgagtccctgaaggactacccc ctgcccttctggctgatcatcttcgtggagggcacccgcttcacccgca ccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgt gccccgcaacgtgctgatcccccacaccaagggcttcgtgtcctccgtg tcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggcct tccccaagacctcccccccccccaccatgctgaagctgacgagggccag tccgtggagctgcacgtgcacatcaagcgccacgccatgaaggacctgc ccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtgga gaaggacgccctgctggacaagcacaactccgaggacaccttctccggc caggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtga tctcctgggtggtggtgatcatcttcggcgccctgaagttcctgctgtg gtcctccctgctgtcctcctggaagggcaaggccactccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcct gtcctcccaggccgagggctccaaccccgtgaaggccgcccccgccaag ctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaac ![custom-character]()
![custom-character]()
ctcgag
[0127] To determine the impact of the CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1 genes on mid-chain fatty acid accumulation, the above constructs containing the codon optimized CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1 genes were transformed into strain S6511. Primary transformants were clonally purified and grown under standard lipid production conditions at pH7.0 (all the strains require growth at pH 7.0 to allow for maximal expression of the LPAAT gene driven by the pH-regulated AMT3 promoter). The resulting profiles from a set of representative clones arising from these transformations are shown in Table 6.
TABLE-US-00016 TABLE 6 Transformants of pSZ3840 (CpauLPAAT1), pSZ3841 (CpaiLPAAT1), pSZ3842 (CigneaLPAAT1), and pSZ3844 (ChookLPAAT1). The fatty acid profiles for transgenic strains expressing LPAATs derived from C. paucipetala, C. painteri, C. ignea, and C. hookeriana. Sample ID C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 a Parent S6511a 14.4 27.7 0.6 1.3 8.8 1.6 38.2 5.4 0.4 S6511b 14.5 27.7 0.6 1.3 8.6 1.6 38.4 5.3 0.4 pSZ3840 CpauLPAAT1 S6511; T792; D2554-20 16.6 29.9 0.7 1.3 8.0 1.0 35.2 5.2 0.5 S6511; T792; D2554-17 14.6 28.7 0.6 1.3 8.4 1.7 37.1 5.7 0.5 S6511; T792; D2554-41 15.2 28.5 0.7 1.3 8.3 1.4 37.5 5.2 0.4 S6511; T792; D2554-35 14.7 28.4 0.6 1.3 8.6 1.6 37.3 5.6 0.5 S6511; T792; D2554-27 15.2 27.6 0.7 1.3 9.5 1.5 37.1 5.1 0.4 pSZ3841 CpaiLPAAT1 S6511; T792; D2555-34 17.3 29.5 0.7 1.3 7.8 1.2 35.1 5.1 0.4 S6511; T792; D2555-43 17.5 29.1 0.7 1.3 8.0 0.9 35.4 5.0 0.5 S6511; T792; D2555-10 15.7 28.3 0.7 1.3 8.6 1.6 36.2 5.7 0.5 S6511; T792; D2555-22 16.0 27.9 0.7 1.3 8.4 0.9 37.8 5.0 0.4 S6511; T792; D2555-44 15.3 27.5 0.6 1.3 8.1 1.8 38.2 5.4 0.4 pSZ3842 CigneaLPAAT1 S6511; T792; D2556-38 16.2 29.2 0.7 1.3 8.1 1.3 36.1 5.2 0.5 S6511; T792; D2556-22 14.3 28.5 0.7 1.3 8.5 1.6 37.6 5.7 0.5 S6511; T792; D2556-44 13.6 28.4 0.7 1.4 9.0 1.5 36.3 6.7 0.7 S6511; T792; D2556-14 14.1 28.0 0.6 1.3 8.6 1.7 38.0 5.6 0.5 S6511; T792; D2556-36 14.3 28.0 0.6 1.3 8.6 1.7 37.9 5.7 0.5 pSZ3844 ChookLPAAT1 S6511; T792; D2557-47 15.8 29.3 0.7 1.3 8.2 1.2 36.5 5.0 0.5 S6511; T792; D2557-24 16.8 28.8 0.7 1.3 8.1 1.2 35.8 5.4 0.5 S6511; T792; D2557-30 15.2 28.3 0.7 1.3 8.5 1.6 36.8 5.7 0.5 S6511; T792; D2557-39 14.7 28.2 0.7 1.3 8.7 1.5 37.3 5.7 0.5 S6511; T792; D2557-26 15.3 27.7 0.7 1.4 8.7 0.9 37.7 5.4 0.5
[0128] The transformants in Table 6 display a marked increase in the production of C8:0 and C10:0 fatty acids upon expression of the heterologous LPAATs. To determine if expression of the heterologous LPAAT genes affected the regiospecificity of fatty acids at the sn-2 position, we analyzed TAGs from representative D2554 (CpauLPAAT1), D2555 (CpaiLPAAT1), D2556 (CigneaLPAAT1), and D2557 (ChookLPAAT1) strains utilizing the porcine pancreatic lipase method. Cells were grown under conditions to maximize midchain fatty acid levels and to generate sufficient biomass for TAG analysis. TAG and sn-2 profiles are shown in Table 7.
[0129] Table 7:
[0130] Inclusion of C8:0 and C10:0 fatty acids at the sn-2 position of TAGs. Selected transformants were subjected to porcine pancreatic lipase determination of fatty acid inclusion at the sn-2 position. The general fatty acid distribution in triacylglycerols (TAG) is shown to indicate fatty acid abundance for each transformant. In addition, the sn-2-specific distribution is shown. Numbers highlighted in bold and italic reflect significantly increased inclusion of the noted fatty acid compared to the parent S6511.
TABLE-US-00017 TABLE 7 S6511; T792; S6511; T792; S6511; T792; S6511; T792; D2554-20 D2555-34 D2556-38 D2557-24 Strain: S6511 (CpauLPAAT1) (CpaiLPAAT1) (CigneaLPAAT1) (ChookLPAAT1) Analysis TAG sn-2 TAG sn-2 TAG sn-2 TAG sn-2 TAG sn-2 Fatty Acid C8:0 14.4 8.5 16.6 12.8 17.3 22.3 16.2 10.0 16.8 29.1 (area %) C10:0 27.7 26.4 29.9 39.0 29.5 22.2 29.2 36.2 28.8 19.4 C12:0 0.6 0.4 0.7 0.3 0.7 0.4 0.7 0.4 0.7 0.3 C14:0 1.3 1.0 1.3 1.0 1.3 0.9 1.3 1.2 1.3 0.9 C16:0 8.8 0.9 8.0 1.1 7.8 1.1 8.1 1.2 8.1 0.9 C18:0 1.6 0.2 1.0 0.4 1.2 0.5 1.3 0.5 1.2 0.3 C18:1 38.2 52.5 35.2 37.8 35.1 43.6 36.1 42.2 35.8 40.7 C18:2 5.4 8.9 5.2 6.2 5.1 7.9 5.2 7.0 5.4 7.1 C18:3 0.4 0.8 0.5 0.7 0.4 0.9 0.5 0.8 0.5 0.7 C8 + C10 42.2 34.9 46.4 51.8 46.8 44.5 45.5 46.1 45.6 48.5 sum
[0131] As disclosed in Table 7, the CpauLPAAT1 and CigneaLPAAT1 genes show remarkable specificity towards C10:0 fatty acids. D2554-20 exhibits 39.0% of C10:0 in the sn-2 position versus just 26.4% in the S6511 base strain without the heterologous LPAAT, demonstrating a 1.5 fold increase in C10:0 inclusion at the sn-2 position. D2556-38 exhibits 36.2% of C10:0 in the sn-2 position versus 26.4% in the S6511 base strain, demonstrating a 1.4 fold increase in C10:0 inclusion at the sn-2 position. Although there is a small increase in C8:0 levels in the D2554-20 and D2555-34 strains, the vast majority of sn-2 targeting is C10:0-specific. Similarly, CpaiLPAAT1 and ChookLPAAT1 show remarkable specificity towards C8:0 fatty acids. D2555-34 exhibits 22.3% C8:0 in the sn-2 position versus just 8.5% in the S6511 base strain without the heterologous LPAAT, demonstrating a 2.6 fold increase in C8:0 inclusion at the sn-2 position. D2557-24 exhibits 29.1% C8:0 in the sn-2 position versus 8.5%, demonstrating a 3.4 fold increase in C8:0 inclusion at the sn-2 position. We teach that CpauLPAAT1 and CigneaLPAAT1 are C10:0-specific LPAATs and that CpaiLPAAT1 and ChookLPAAT1 are C8:0-specific LPAATs. Knutzon D S, Lardizabal K D, Nelsen J S, Bleibaum J L, Davies H M, Metz J G (1995) Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates. Plant Physiol 109:999-1006
Amino Acid Sequences for Novel LPAAT Genes
[0132]
TABLE-US-00018 CpauLPAAT1 SEQIDNO:23 MAIPAAAVIFLFGLLFFTSGLIINLFQALCFVLVWPLSKNAYRRINRV FAELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTEL DWMLGWVMGQHLGCLGSILSVAKKSTKFLPVLGWSMWFSEYLYIERSW AKDRTTLKSHIERLTDYPLPFWMVIFVEGTRFTRTKLLAAQQYAASSG LPVPRNVLIPRTKGFVSCVSHMRSFVPAVYDVTVAFPKTSPPPTLLNL FEGQSIVLHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAE DTFSGQEVHRTGSRPIKSLLVVISWVVVITFGALKFLQWSSWKGKAFS VIGLGIVTLLMHMLILSSQAERSSNPAKVAQAKLKTELSISKKATDKEN CprocLPAAT1 SEQIDNO:24 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKD KSTLKSHIERLKDYPLPFWLVIFAEGTRFTQTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSCVSHMRSFVPAVYDLTVAFPKTSPPPTLLNLFEGQS VVLHVHIKRHAMKDLPESDDEVAQWCRDKFVEKDALLDKHNAEDTFSGQ ELQHTGRRPIKSLLVVISWVVVIAFGALKFLQWSSWKGKAFSVIGLGIV TLLMHMLILSSQAERSKPAKVAQAKLKTELSISKTVTDKEN CprocLPAAT1b SEQIDNO:25 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKD KSTLKSHIERLKDYPLPFWLVIFAEGTRFTQTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSCVSHMRSFVPAVYDLTVAFPKTSPPPTLLNLFEGQS VVLHVHIKRHAMKDLPESDDEVAQWCRDKFVEK CprocLPAAT2a SEQIDNO:26 IVNLVQAVCFVLVRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIK VFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMK KSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPFWLA LFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSF VPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDA VAQWCRDIFVEKDALLDKHNADDTFSGQELQDTGRPIKSLLVVISWAVL EVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTP AKVAPAKAKIEGESSKTEMEKEK CprocLPAAT2b SEQIDNO:27 IVNLVQAVCFVLVRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIK VFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMK KSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPFWLA LFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSF VPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDA VAQWCRDIFVEKDALLDKHNADDTFSGQELQDTGRPIKSLLV CpaiLPAAT1 SEQIDNO:28 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVF AELLPLEFLWLFHWCAGAKLKLFTDPETFRLMGKEHALVIINHKIELDW MVGWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSGYLFLERSWAKD KITLKSHIESLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSVSHMRSFVPAIYDVTVAFPKTSPPPTMLKLFEGQS VELHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNSEDTFSGQ EVHHVGRPIKALLVVISWVVVIIFGALKFLLWSSLLSSWKGKAFSVIGL GIVAGIVTLLMHILILSSQAEGSNPVKAAPAKLKTELSSSKKVTNKEN ChookLPAAT1 SEQIDNO:29 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVF AELLPLEFLWLFHWCAGAKLKLFTDPETFRLMGKEHALVIINHKIELDW MVGWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSEYLFLERSWAKD KITLKSHIESLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSVSHMRSFVPAIYDVTVAFPKTSPPPTMLKLFEGQS VELHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNSEDTFSGQ EVHHVGRPIKALLVVISWVVVIIFGALKFLLWSSLLSSWKGKAFSVIGL GIVAGIVTLLMHILILSSQAEGSNPVKAAPAKLKTELSSSKKVTNKEN ChookLPAAT2a SEQIDNO:30 LSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWL IDWWAGVKIKVFTDHETFNLMGKEHALVVCNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRL KDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGF VSSVSHMRSFVPAIYDVTVAIPKTSVPPTMLRIFKGQSSVLHVHLKRHL MKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKS LLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILI LFSQSERSTPAKVAPAKPKNEGESSKTEMEKEH ChookLPAAT2b SEQIDNO:31 QIKVFTDHETFNLMGKEHALVVCNHKSDIDWLVGWVLAQWSGCLGSTLA VMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPF WLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHM RSFVPAIYDVTVAIPKTSVPPTMLRIFKGQSSVLHVHLKRHLMKDLPES DDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLLVVISW AVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSER STPAKVAPAKLKKEGESSKPETDKQN ChookLPAAT3a SEQIDNO:32 LSLLFFVSGLIVNLVQAVCFVLIRPLLKNTYRRINRVVAELLWLELVWL IDWWAGIKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRL KDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGF VSSVSQMRSFVPAIYDVTVAIPKTSPPPTLLRMFKGQSSVLHVHLKRHL MNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKS LLVVISWATLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILI LFSQSERSTPAKVAPAKPKNEGESSKTEMEKEH ChookLPAAT3b SEQIDNO:33 LSLLFFVSGLIVNLVQAVCFVLIRPLLKNTYRRINRVVAELLWLELVWL IDWWAGIKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRL KDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGF VSSVSQMRSFVPAIYDVTVAIPKTSPPPTLLRMFKGQSSVLHVHLKRHL MNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKS LLVVISWAVLEIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILI LFSQSERSTPAKVAPAKPKKEGESSKPETDKEN CigneaLPAAT1 SEQIDNO:34 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVF AELLLMDLLCLFHWWAGAKIKLFTDPETFRLMGMEHALVIMNHKTDLDW MVGWILGQHLGCLGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKD KSTLKSHMEKLKDYPLPFWLVIFVEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPHTKGFVSCVSNMRSFVPAVYDVTVAFPKSSPPPTMLKLFEGQS IVLHVHIKRHALKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQ EVHHIGRPIKSLLVVIAWVVVIIFGALKFLQWSSLLSTWKGKAFSVIGL GIATLLMHMLILSSQAERSNPAKVAK CigneaLPAAT2 SEQIDNO:35 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVF AELLLMDLLCLFHWWAGAKIKLFTDPETFRLMGMEHALVIMNHKTDLDW MVGWILGQHLGCLGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKD ESTLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVP KNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSAPPTLLRMFKGQS SVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELHDIGRPVKSLLVVISWAMLVVFGAVKFLQWSSLLSSWKGLAFSGIGL GIITLLMHILILFSQSERSTPAKVAPAKQKNNEGESSKTEMEKEH DeLPAAT1 SEQIDNO:36 SGLVVNLIQAFFFVLVRPFSKNAYRKINRVVAELLWLELIWLIDWWAGV KIQLYTDPETFKLMGKEHALVICNHKSDIDWLVGWILAQRSGCLGSALA VMKKSSKFLPVIGWSMWFSEYLFLERSWAKDENTLKSGFQRLRDFPHAF WLALFVEGTRFTQAKLLAAQEYASSMGLPAPRNVLIPRTKGFVTAVTHM RPFVPAVYDVTLAIPKTSPPPTMLRLFKGQSSVVHIHLKRHLMSDLPKS DDSVAQWCKDAFVVKDNLLDKHKENDSFGDGVLQDTGRPLNSLVVVISW ACLLIFGALKFFQWSSILSSWKGLAFSAVGLGIVTVLMQILIQFSQSER SNRPMPSKHAK DeLPAAT2 SEQIDNO:37 MAIPTAAYVVPLGAIFFFSGLLVNLIQAFFFITVWPLSKKTYIRINKVI VELLWLEFVWLADWWAGLKIEVYADAETFQLMGKEHALVICNHKSDIDW LVGWILAQRAGCLGSSFAVTKKSARYLPVVGWSIWFSGAIFLERSWEKD ENTLKAGFQRLREFPCAFWLGLFVEGTRFTQAKLLAAQEYASTMGLPFP RNVLIPRTKGFIAAVNHMREFVPAIYDLTFAFPKDSPPPTMLRLLKGQP SVVHVHIKRHLMKDLPEKNEAVAQWCKDVFLVKDKLLDKHKDDGSFGDG ELHEIGRPLKSLVVVTTWACLLILGTLKFLLWSSLLSSWKGLIFSATGL AVLTVLMQFLIQSTQSERSNPASLSK CerLPAAT1a SEQIDNO:38 LGLLFFISGLAVNLIQAVCFVFLRPLSKNTYRKINRVLAELLWLQLVWL VDWWAGVKIKVFADRESFNLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSSLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKEGLRRL KDFPRPFWLALFVEGTRFTQAKLLAAQEYATSQGLPVPRNVLIPRTKVH VHVKRHLMKELPETDEAVAQWCKDLFVEKDKLLDKHVAEDTFSDQPLQD IGRPVKPLLVVSSWACLVAYGALKFLQWSSLLSSWKGIAVSAVALAIVT ILMQIMILFSQSERSIPAKVA CerLPAAT1b SEQIDNO:39 LGLLFFISGLAVNLIQAVCFVFLRPLSKNTYRKINRVLAELLWLQLVWL VDWWAGVKIKVFADRESFNLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSSLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKEGLRRL KDFPRPFWLALFVEGTRFTQAKLLAAQEYATSQGLPVPRNVLIPRTKGF VSAVSHMRSFVPAVYDMTVAIPKSSPSPTMLRLFKGQSSVVHVHVKRHL MKELPETDEAVAQWCKDLFVEKDKLLDKHVAEDTFSDQPLQDIGRPVKP LLVVSSWACLVAYGALKFLQWSSLLSSWKGIAVSAVALAIVTILMQIMI LFSQSERSIPTKVA CerLPAAT2a SEQIDNO:40 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVF AELLLLELLWLFHWRAGAKLKLFADPETFRLFGKEHALVICNHRTDLDW MVGWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKD KSTLKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKLHVHIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNA EDTFSGQEVHHIGRPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGK AFSVIGLGIVTLLVNILILSSQAERSNPAKVAPAKLKTELSPSKKVTNK EN CerLPAAT2b SEQIDNO:41 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVF AELLLLELLWLFHWRAGAKLKLFADPETFRLFGKEHALVICNHRTDLDW MVGWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKD KSTLKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSMSHMRSFVPAVYDLTVAFPKTSPPPTLLKLFEGQS VVLHVHIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFSGQ EVHHIGRPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIGL GIVTLLVNILILSSQAERSNPAKVAPAKLKTELSPSKKVTNKEN BrLPAAT1a SEQIDNO:42 AAAVIVPLGILFFISGLVVNLLQAICYVLIRPLSKNTYRKINRVVAETL WLELVWIVDWWAGVKIQVFADNETFNRMGKEHALVVCNHRSDIDWLVGW ILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTL KSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVL IPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVH VHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQN IGRPIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIIT LCMQILIRSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE BrLPAAT1b SEQIDNO:43 AAAVIVPLGILFFISGLVVNLLQAVCYVLVRPMSKNTYRKINRVVAETL WLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHRSDIDWLVGW ILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTL KSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVL IPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVH VHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQN IGRPIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIIT LCMQILIRSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE BrLPAAT1e SEQIDNO:44 MAIAAAVIVPLGLLFFISGLLMNLLQAICYVLVRPLSKNTYRKINRVVA ETLWLELVWIVDWWAGVKIKVFADNETFSRMGKEHALVVCNHRSDIDWL VGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDE STLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPR NVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPS VVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQ EQNIGRPIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLG IITLCMQILIRSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE BjLPAAT1a SEQIDNO:45 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHR SDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASS ELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRL FKGQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAAD TFPGQKEQNIGRPIKSLAVSLIKTFPWLHPHQLTNIFVLFQVVVSWACL LTLGAMKFLHWSNLFSSWKGIALSAFGLGIITLCMQILIRSSQSERSTP AKVAPAKPK BjLPAAT1b SEQIDNO:46 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHR SDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASS ELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRL FKGQPSVVHVHIKCHSMKDLPEPEDEIAQWCRDQFVAKDALLDKHIAAD TFPGQKEQNIGRPIKSLAVVVSWACLLTLGAMKFLHWSNLFSSWKGIAL SAFGLGIITLCMQILIRSSQSERSTPAKVAPAKPK BjLPAAT1e SEQIDNO:47 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHR SDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASS ELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRL FKGQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAAD TFPGQQEQNIGRPIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAF SALGLGIITLCMQILIRSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE BjLPAAT1d SEQIDNO:48 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHR SDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASS ELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRL FKGQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAAD TFPGQQEQNIGRPIKSLAVSLS CeLPAAT1a SEQIDNO:49 MAIGVAAIVVPLGLLFILSGLMVNLIQAICFILVRPLSKNMYRRVNRVV VELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDW LVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKD ESTLKSGLRRLKDFPRPFWLALFVEGTRFTQAKLLAAREYAASTGLPIP RNVLIPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQP SVVHVHIKRHSMNQLPQTDEGVGQWCKDIFVAKDALLDRHLAE CcLPAAT1b SEQIDNO:50 MAIGVAAIVVPLGLLFILSGLMVNLIQAICFILVRPLSKNMYRRVNRVV VELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDW LVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKD ESTLKSGLRRLKDFPRPFWLALFVEGTRFTQAKLLAAREYAASTGLPIP RNVLIPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQP SVVHVHIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEK EFKRIRRPIKSLLVISSWSFLLMFGVFKFLKWSALLSTWKGVAVSTTVL LLVTVVMYMFILFSQSERSSPRKVAPSGPENG UcLPAAT1a SEQIDNO:51 MAIGVAAIVVPLGLLFILSGLIINLIQAICFILVRPLSKNMYRKVNRVV VELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDW LVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKD ESTLKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIP RNVLIPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQP SVVHVHIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEK EFKLIRRPIKSLLVISSWSFLLMFGVFKFLKWSALLSTWKGVAVSTAVL LLVTVVMYMFILFSQSERSSPRKVAPIGPENG UcLPAAT1b SEQIDNO:52 MAIGVAAIVVPLGLLFILSGLIINLIQAICFILVRPLSKNMYRKVNRVV VELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDW LVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKD ESTLKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIP RNVLIPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQP SVVHVHIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAE LdLPAAT1 SEQIDNO:53 SLLFFMSGLVVNFIQAVFYVLVRPISKNTYRRINTLVAELLWLELVWVI DWWAGVKVQLYTDTESFRLMGKEHALLICNHRSDIDWLIGWVLAQRCGC LSSSIAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDENTLKSGLQRLN DFPKPFWLALFVEGTRFTKAKLLAAQEYAASAGLPVPRNVLIPRTKGFV SAVSNMRSFVPAIYDLTVAIPKTTEQPTMLRLFRGKSSVVHVHLKRHLM KDLPKTDDGVAQWCKDQFISKDALLDKHVAEDTFSGLEVQDIGRPMKSL VVVVSWMCLLCLGLVKFLQWSALLSSWKGMMITTFVLGIVTVLMHILIR SSQSEHSTPAK CaequLPAAT1a SEQIDNO:54 QRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSG LKRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPR PTKGFVSSVSHMRSFVAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHL KRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGR PVKSLLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLM HILILFSQSERSTPAKVAPAKPKKEGESSKTETEKEN CaequLPAAT1b SEQIDNO:55 DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGC LGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLK DYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFV SSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLM KDLPESDDAVAQWCRDIFVEKDALLDKHN AEDTFSGQELQDIGRPVKSLLV CaequLPAAT1e SEQIDNO:56 DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGC LGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLK DYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFV SSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLM KDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSL LVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILIL FSQSERSTPAKVAPAKPKKEGESSKTETEKEN CaequLPAAT1d SEQIDNO:57 QRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSG LKRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPR TKGFVSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHL KRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGR PVKSLLV CglutLPAAT1a SEQIDNO:58 LSLLFFVSGLFVNLVQAVCFVLIRPFSKNTYRRINRVVAELLWLELVWL IDWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRL KDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGF VSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHL MKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKS LLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILI LFSQSERSTPAKVAPAKPKKEGESSKTETEKEN CglutLPAAT1b SEQIDNO:59 QAVCFVLIRPFSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDH ETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKF LPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEG TRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIY DVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWC RDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLVVISWAVLVIFGA VKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAP AKPKKEGESSKTETEKEN CprLPAAT1 SEQIDNO:60 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVF AELLLLELLWLFHWRAGAKLKLFADPETFRLFGKEHALVICNHRTDLDW MVGWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKD KSTLKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSMSHMRSFVPAVYDLTVAFPKTSPPPTLLKLFEGQS VVLHVHIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFSGQ EVHHIGRPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIGL GIVTLLVNILILSSQAERSNPAKVVPAKLKTELSPSKKVTNKEN ChsLPAAT1 SEQIDNO:61 MAIPSAAVVFLFGLLFFASGLIINLVQAVCFVLIWPLSKNTCRRINIVF QDMLLSELLWLFHWRAGAKLKFFTDPETYRHMGKEHALVITNHRTDLDW MIGWVLGEHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKD KSTFKSHIERLEDFPQPFWFGIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSVSHMRSFVPAVYETTMTFPKTSPPPTLLKLFEGQP LVLHIHMKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDTFGGL EVHIGRSIKSLMVVICWVVVIIFGALKFLQWSSLLSSWKGIAFIGIGLG IVNLLVHVLILSSQAERSAPTKVAPAKLKTKLLSSKKITNKEN ChsLPAAT2 SEQIDNO:62 MAIPSAAVVFLFGLLFFASGLIINLVQAVCFVLIWPLSKNTCRRINIVF QDMLLSELLWLFHWRAGAKLKFFTDPETYRHMGKEHALVITNHRTDLDW MIGWVLGEHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKD KSTFKSHIERLEDFPQPFWFGIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQS SVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQDIGRPIKSLVVVISWAALVVFGAVKFLQWSSLLSSWKGLAFSGIGL GIITLLMHILILFSQSERSTPAKVAPAKPKREGESSKTEMDKEN CcaleLPAAT1a SEQIDNO:63 MAIPAAAVVFLFGLLFFPSGLIINLFQAVCFVLIWPFSRNTCRRINIVF QEMLLSELLWLFHWRAGAKLKLFADPETYRHMGKEHALLITNHRTDLDW MIGWALGQHLGCLGSILSVVKKSTKFLPSHIERLEDFPQPFWMAIFVEG TRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVY ETTMTFPKTSPPPTLLKLFEGQPIVLHVHMKRHAMKDIPESDEAVAQWC RDKFVEKDSLLDKHNAGDTFSCQEIHIGRPIKSLMVVISWVVVIIFGAL KFLQWSSLLSSWKGIAFSGIGLGIVTLLVHILILSSQAERSTPAKVAPA KLKTELSSSTKVTNKEN CcaleLPAAT1b SEQIDNO:64 MAIPAAAVVFLFGLLFFPSGLIINLFQAVCFVLIWPFSRNTCRRINIVF QEMLLSELLWLFHWRAGAKLKLFADPETYRHMGKEHALLITNHRTDLDW MIGWALGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKD KSTFKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPRTKGFVSCVSHMRSFVPAVYETTMTFPKTSPPPTLLKLFEGQP IVLHVHMKRHAMKDIPESDEAVAQWCRDKFVEKDSLLDKHNAGDTFSCQ EIHIGRPIKSLMVVISWVVVIIFGALKFLQWSSLLSSWKGIAFSGIGLG IVTLLVHILILSSQAERSTPAKVAPAKLKTELSSSTKVTNKEN CcaleLPAAT2 SEQIDNO:65 LSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWL IDWWAGVKIKVFTDHETFRLMGTEHALVISNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRL KDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGF VSSVSHMRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHVHLKRHL MKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKS LVVVISWAALVVFGAVKFLQWSSLLSSWKGLAFSGIALGIITLLMHILI LFSQSERSTPAKVAPAKPKKEGESSKTETDKEN ChtLPAAT1a SEQIDNO:66 MAIPAAAVIFLFSILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVF QEMLLSELLGLFHWRAGAKLKLYTDPETYPLLGKEHALLMINHRTDLDW MIGWVLGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKD KSTFKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKGFVSTVSHMRSFVPAVYDTTLTFPKTSPPPTLLNLFAGQP IVLHIHIKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDAFSDQ EFPISRSIKSLMVVISWVMVIIFGALKFLQWSSLLSSWKGKAFSVIAVG IVTLLMHMSILSSQAERSNPAKVALPKLKTELPSSKKVLNKEN ChtLPAAT1b SEQIDNO:67 MAIPAAAVIFLFSILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVF QEMLLSELLGLFHWRAGAKLKLYTDPETYPLLGKEHALLMINHRTDLDW MIGWVLGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKD KSTFKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKGFVSTVSHMRSFVPAVYDTTLTFPKTSPPPTLLNLFAGQP IVLHIHIKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDAFSDQ EFPISRSIKSLMVVISWVMVIIFGALKFLQWSSLLSSWKGIAFSGIGLG IVTLLMHILILSSQAERSTPAKVAQAKVKTELPSSTKVTNKGN CwLPAAT1 SEQIDNO:68 MAIPAAAVIFLFGILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVF QEMLLSELLWLFHWRAGAELKLFTDPETYRLLGKEHALVMTNHRTDLDW MIGWVTGQHLGCLGSILSIAKKSTKFLPVLGWSMWFSEYLFLERNWAKD KSTFKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSVCHMRSFVPAVYDTTLTFPKNSPPPTLLNLFAGQP IVLHIHIKRHAMKDMPKSDDAVAQWCRDKFVKKDALLDKHNTEDTFSDQ EFPIGRPIKSLMVVISWVVVIIFGTLKFLQWSSLLSSWKGIAFSGIGLG IVTLLVHILILSSQAERSTPPKVAPAKLKTELSSTTKVINKGN CwLPAAT2b SEQIDNO:69 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRLNRVVAELLWLELVWL IDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRL KDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGF RVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMLMFKGQSSVDALLDKHNA DDTFSGQELHDIGRPIKSLLVVISWAVLVVFGAVKFLQWSSLLSSWKGI AFSGIGLGIVTLLVHILILSSQAERSTSAKVAQAKVKTELSSSKKVKNK GN CwLPAAT2a SEQIDNO:70 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRLNRVVAELLWLELVWL IDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRL KDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGF VSSVSHMRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHVHLKRHL MKDLPESDDAVAQWCRDIFVEKDVLLDKHNAEDTFSGQELQDIGRPVKS LLVVISWTLLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILI LFSQSERSTPAKVAPAKPKKEGESSKMETDKEN CgLPAAT1a SEQIDNO:71 LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDE STLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASLGLPVPR NVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSS VLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQE LQDTGRPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLG IITLLMHILILFSQSERSTPAKVAPAKPKNEGESSKAEMEKEK CgLPAAT1b SEQIDNO:72 LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDE STLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASLGLPVPR NVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSS VLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQE LQDTGRPIKSLLVRCFLVLSLIYLNGIMLKLRGPCLQVVISWAVLEVFG AVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVA PAKPKNEGESSKAEMEKEK CgLPAAT1c SEQIDNO:73 LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDE STLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASLGLPVPR NVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSS VLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQE LQDTGRPIKSLLVVTSWAVLVISGAVKFLQWSSLLSSWKGLAFSGIGLG IVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTEKDKEN CpalLPAAT1 SEQIDNO:74 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWL IDWWAGVKIKVFTDHETLSLMGKEHALVICNHKSDIDWLVGWVLAQRSG CLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDENTLKSGLNRL KDYPLPFWLALFVEGTRFTRAKLLAAQQYATSSGLPVPRNVLIPRTKGF VSSVSHMRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHVHLKRHL MKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKS LLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGVGLGIITLLMHILI LFSQSERSTPAKVAPAKPKKDGESSKTEIEKEN CaLPAAT1 SEQIDNO:75 MAIAAAAVIVPVSLLFFVSGLIVNLVQAVCFVLIRPLFKNTYRRINRVV AELLWLELVWLIDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDW LVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKD ESTLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVP RNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLLRMFKGQS SVLHVHLKRHQMNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQDTGRPIKSLLIVISWAVLVVFGAVKFLQWSSLLSSWKGLAFSGIGL GVITLLMHILILFSQSERSTPAKVAPAKPKIEGESSKTEMEKEH CaLPAAT3 SEQIDNO:76 MTIASAAVVFLFGILLFTSGLIINLFQAFCSVLVWPLSKNAYRRINRVF AEFLPLEFLWLFHWWAGAKLKLFTDPETFRLMGKEHALVIINHKIELDW MVGWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSEYLFLERNWAKD KKTLKSHIERLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASAGLPVP TRNVLIPHTKGFVSSVSHMRSFVPAIYDVTVAFPKSPPPTMLKLFEGHF VELHVHIKRHAMKDLPESEDAVAQWCRDKFVEKDALLDKHNAEDTFSGQ EVHHVGRPIKSLLVVISWVVVIIFGALKFLQWSSLLSSWKGIAFSVIGL GTVALLMQILILSSQAERSIPAKETPANLKTELSSSKKVTNKEN SalLPAAT1 SEQIDNO:77 MAIGAAAIVVPLGLLFMLSGLMVNLIQAICFILVRPLSKNMYRRVNRVV VELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHKSDIDW LVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKD ESTLKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIP RNVLIPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQP SVVHVRIKRHSMNQLPPTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEK EFKRIRRPIKSLLVISSWSFLLLFGVFKFLKWSALLSTWKGVAVSTAVL LLVTVVMYMFILFSQSERSSPRKVAPSGPENG CleptLPAAT1 SEQIDNO:78 MAIPAAVVIFLFGLLFFSSGLIINLFQALCFVLIWPLSKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKD KSTLKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSCVNHMRSFVPAVYDLTVAFPKTSPPPTLLNLFEGQS VVLHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSSQ EVHHTGSRPIKSLLVVISWVVVITFGALKFLQWSSWKGKAFSVIGLGIV TLLMHMLILSSQAERSKPAKVTQAKLKTELSISKKVTDKEN ClopLPAAT1 SEQIDNO:79 MAIAAAAVIFLFGLLFFASGLIINLFQALCFVLIRPLSKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETLRLMGKEHALIIINHMTELDW MVGWVMGQHFGCLGSIISVAKKSTKFLPVLGWSMWFSEYLYLERSWAKD KSTLKSHIERLKDYPLPFWLVIFVEGTRFTRTKLLAAQEYAASSGLPVP RNVLIPRTKGFVSCVNHMRSFVPAVYDVTVAFPKTSPQPTLLNLFEGRS IVLHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQ EVHHTGRRPIKSLLVVMSWVVVTTFGALKFLQWSSWKGKAFSVIGLGIV TLLMHVLILSSQAERSNPAKVVQAELNTELSISKKVTNKGN CcrasLPAAT1a SEQIDNO:80 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKD KSTLKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQS SVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQDTGRPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGL GIITLLMHILILFSQSERSTPAKVAPAKAK CcrasLPAAT1b SEQIDNO:81 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKD GKSTLKSHIERLKDYPLPFWLVIFAETRFTRTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQS SVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQDTGRPIKSLLVRCFLVLSLIYLNGIILKLCGLCLQVVISWAVLEVF GAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKV APAKAK CcrasLPAAT1c SEQIDNO:82 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKD KSTLKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQS SVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQDTGRPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGL GIITLLMHILILFSQSERSTPAKVAPAKAKMEGESSKTEMEMEK CcrasLPAAT1d SEQIDNO:83 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDW MVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKD KSTLKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQS SVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQDTGRPIKSLLVRCFLVLSLIYLNGIILKLCGLCLQVVISWAVLEVF GAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKV APAKAKMEGESSKTEMEMEK CkoeLPAAT1 SEQIDNO:84 MAIAAAPVIFLFGLLFFASGLIINLFQAICFVLIWPLSKNAYRRINRVF AELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVITNHKIDLDW MIGWILGQHFGCLGSVISIAKKSTKFLPIFGWSLWFSEYLFLERNWAKD KRTLKSHIERMKDYPLPLWLILFVEGTRFTRTKLLAAQQYAASSGLPVP RNVLIPHTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQS SVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQ ELQETGRPIKSLLVVISWAVLEVYGAVKFLQWSSLLSSWKGLAFSGIGL GLITLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTEMEKEK CkoeLPAAT2 SEQIDNO:85 MHVLLEMVTFRFSSFFVFDNVQALCFVLIWPLSKSAYRKINRVFAELLL SELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVITNHKIDLDWMIGWI LGQHFGCLGSVISIAKKSTKFLPIFGWSLWFSEYLFLERNWAKDKRTLK SHIERMKDYPLPLWLILFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLI PHTKGFVSSVSHMRSFVPAVYDVTVAFPKTSPPPTMLSLFEGQSVVLHV THIKRHAMKDLPDSDDAVAQWCRDKFVEKDALLDKHNAEDFSGQEVHHV GRPIKSLLVVISWMVVIIFGALKFLQWSSLLSSWKGKAFSAIGLGIATL LMHVLVVFSQADRSNPAKVPPAKLNTELSSSKKVTNKEN
Example 5: Expression of LPAATs to Improve Sn-2 Selectivity in Prototheca moriformis
[0133] In the example we disclose genetically engineered Prototheca moriformis strains in which we have modified fatty acid and triacylglycerol biosynthesis to maximize the accumulation of Stearoyl-Oleoyl-Stearoyl (SOS) TAGs, and minimize the production of trisaturated TAGs. Oils from these strains resemble plant seed oils known as structuring fats, which have high proportions of Saturated-Oleate-Saturated TAGs and low levels of trisaturates. These structuring fats (often called butters) are generally solid at room temperature but melt sharply between 35-40 C.
[0134] Strains with high SOS and low trisaturates were obtained by three successive transformations, beginning with S5100, a classically improved derivative of S376 (improved to increase lipid titer), a wild type isolate of Prototheca moriformis. S5100 was transformed with a construct to which increased expression of PmKASII-1 and ablated the SAD2-1 allele. The resultant strain, S5780, produced oil with increased C18:0 and lower C16:0 content relative to S5100. S5780 was prepared according to the methods disclosed in co-owned application WO2013/158938 and as described below. C18:0 levels were increased further by transformation of S5780 with a construct overexpressing the C18:0-specific FATA1 thioesterase gene from Garcinia mangostana (GarmFATA1), generating strain S6573. S6573 was disclosed in co-owned application WO2015/051319. Finally, accumulation of trisaturated TAGS was reduced by expression of genes encoding LPAATs from Brassica napus, Theobroma cacao, Garcinia hombororiana or Garcinia indica in S6573 as described below.
Construct Used for SAD2 Knockout and PmKASII-1 Overexpression in S5100 to Produce S5780
[0135] The sequence of the transforming DNA from the SAD2-1 ablation, PmKASII over-expression construct, pSZ2624, is shown below. The construct is written as: pSZ2624: SAD2-1vD::PmKASII-1tp_PmKASII-1_FLAG-CvNR:CpACT-AtTHIC-CpEF1a::SAD2-1vE Relevant restriction sites are indicated in lowercase, bold, and are from 5-3 PmeI, SpeI, AscI, ClaI, SacI, AvrII, EcoRV, AflII, KpnI, XbaI, MfeI, BamHI, BspQI and PmeI. Underlined sequences at the 5 and 3 flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. The SAD2-1 5 integration flank contained the endogeneous SAD2-1 promoter, enabling the in situ activation of the PmKASII gene. Proceeding in the 5 to 3 direction, the region encoding the PmKASII plastid targeting sequence is indicated by lowercase, underlined italics. The sequence that encodes the mature PmKASII polypeptide is indicated with lowercase italics, while a 3FLAG epitope encoding sequence is in bold italics. The initiator ATG and terminator TGA for PmKASII-FLAG are indicated by uppercase italics. The 3 UTR of the Chlorella vulgaris nitrate reductase (CvNR) gene is indicated by small capitals. Two spacer regions are represented by lowercase text. The CpACT promoter driving the expression of the AtTHIC gene (encoding 4-amino-5-hydroxymethyl-2-methylpyrimidine synthase activity, thereby permitting the strain to grow in the absence of exogeneous thiamine) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3 UTR of the Chlorella protothecoides EF1a (CpEF1a) gene is indicated by small capitals. The use of THIC as a selection marker was described in co-owned applications WO2011/150410 and WO2013/150411.
TABLE-US-00019 pSZ2624Nucleotidesequenceofthetransforming DNA SEQIDNO:86 gtttaaacGCCGGTCACCACCCGCATGCTCGTACTACAGCGCACGCACC GCTTCGTGATCCACCGGGTGAACGTAGTCCTCGACGGAAACATCTGGTT CGGGCCTCCTGCTTGCACTCCCGCCCATGCCGACAACCTTTCTGCTGTT ACCACGACCCACAATGCAACGCGACACGACCGTGTGGGACTGATCGGTT CACTGCACCTGCATGCAATTGTCACAAGCGCTTACTCCAATTGTATTCG TTTGTTTTCTGGGAGCAGTTGCTCGACCGCCCGCGTCCCGCAGGCAGCG ATGACGTGTGCGTGGCCTGGGTGTTTCGTCGAAAGGCCAGCAACCCTAA ATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGTTTGGACCAGATC CGCCCCGATGCGGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCT TTCGTAAATGCCAGATTGGTGTCCGATACCTGGATTTGCCATCAGCGAA ACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGCTACCAGGGTTGC ATACATTGCCCATTTCTGTCTGGACCGCTTTACTGGCGCAGAGGGTGAG TTGATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGCGTGTCT GTTTTCGGCTGCACGAATTCAATAGTCGGATGGGCGACGGTAGAATTGG GTGTGGCGCTCGCGTGCATGCCTCGCCCCGTCGGGTGTCATGACCGGGA CTGGAATCCCCCCTCGCGACCATCTTGCTAACGCTCCCGACTCTCCCGA CCGCGCGCAGGATAGACTCTTGTTCAACCAATCGACAactagtATGcag accgcccaccagcgcccccccaccgagggccactgatcggcgcccgcct gcccaccgcctcccgccgcgccgtgcgccgcgcctggtcccgcatcgcc cgcgggcgcgccgccgccgccgccgacgccaaccccgcccgccccgagc gccgcgtggtgatcaccggccagggcgtggtgacctccctgggccagac catcgagcagactactcctccctgctggagggcgtgtccggcatctccc agatccagaagacgacaccaccggctacaccaccaccatcgccggcgag atcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagc gcgtggacgacgtgatcaagtacgtgtacatcgccggcaagcaggccct ggagtccgccggcctgcccatcgaggccgccggcctggccggcgccggc ctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggca tgacctccacgccgccggcgtggaggccctgacccgcggcggcgtgcgc aagatgaaccccactgcatccccactccatctccaacatgggcggcgcc atgctggccatggacatcggatcatgggccccaactactccatctccac cgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatc cgccgcggcgacgccaacgtgatgctggccggcggcgccgacgccgcca tcatcccctccggcatcggcggcttcatcgcctgcaaggccctgtccaa gcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgc gacggatcgtgatgggcgagggcgccggcgtgctggtgctggaggagct ggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggc ggcgccgccacctccgacgcccaccacatgaccgagcccgacccccagg gccgcggcgtgcgcctgtgcctggagcgcgccctggagcgcgcccgcct ggcccccgagcgcgtgggctacgtgaacgcccacggcacctccaccccc gccggcgacgtggccgagtaccgcgccatccgcgccgtgatcccccagg actccctgcgcatcaactccaccaagtccatgatcggccacctgctggg cggcgccggcgccgtggaggccgtggccgccatccaggccctgcgcacc ggctggctgcaccccaacctgaacctggagaaccccgcccccggcgtgg accccgtggtgctggtgggcccccgcaaggagcgcgccgaggacctgga cgtggtgctgtccaactccttcggcttcggcggccacaactcctgcgtg atcttccgcaagtacgacgag![custom-character]()
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TGAatcgatAGATCTCTTAAGGCAGCAGCAGCTCGGATAGTATCGACA CACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGC CTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGT TTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTAT TTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGC ATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCT CCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCT GTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGC ACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTAATTAAgagct ccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctg tcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgct tggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgtt ggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgtt cacagcctaggtgatatccatcttaaggatctaagtaagattcgaagcg ctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaat gtaagtgggctggcgtaccctgtacgtgagtcaacgtcactgcacgcgc accaccctctcgaccggcaggaccaggcatcgcgagatacagcgcgagc cagacacggagtgccgagctatgcgcacgctccaactaggtaccagttt aggtccagcgtccgtggggggggacgggctgggagcttgggccgggaag ggcaagacgatgcagtccctctggggagtcacagccgactgtglgtgtt gcactgtgcggcccgcagcactcacacgcaaaatgcctggccgacaggc aggccctgtccagtgcaacatccacggtccctctcatcaggctcacctt gctcattgacataacggaatgcgtaccgctctttcagatctgtccatcc agagaggggagcaggctccccaccgacgctgtcaaacttgcttcctgcc caaccgaaaacattattgtttgagggggggggggggggggcagattgca tggcgggatatctcgtgaggaacatcactgggacactgtggaacacagt gagtgcagtatgcagagcatgtatgctaggggtcagcgcaggaaggggg cctttcccagtctcccatgccactgcaccgtatccacgactcaccagga ccagcttcttgatcggcttccgctcccgtggacaccagtglgtagcctc tggactccaggtatgcgtgcaccgcaaaggccagccgatcgtgccgatt cctgggtggaggatatgagtcagccaacttggggctcagagtgcacact ggggcacgatacgaaacaacatctacaccgtgtcctccatgctgacaca ccacagcttcgctccacctgaatgtgggcgcatgggcccgaatcacagc caatgtcgctgctgccataatgtgatccagaccctctccgcccagatgc cgagcggatcgtgggcgctgaatagattcctgtttcgatcactgtttgg gtcctttccttttcgtctcggatgcgcgtctcgaaacaggctgcgtcgg gctttcggatcccttttgctccctccgtcaccatcctgcgcgcgggcaa gttgcttgaccctgggctgataccagggttggagggtattaccgcgtca ggccattcccagcccggattcaattcaaagtctgggccaccaccctccg ccgctctgtctgatcactccacattcgtgcatacactacgttcaagtcc tgatccaggcgtgtctcgggacaaggtgtgcttgagtttgaatctcaag gacccactccagcacagctgctggttgaccccgccctcgcaatctagaA TGgccgcgtccgtccactgcaccctgatgtccgtggtctgcaacaacaa gaaccactccgcccgccccaagctgcccaactcctccctgctgcccgga tcgacgtggtggtccaggccgcggccacccgatcaagaaggagacgacg accacccgcgccacgctgacgacgacccccccacgaccaactccgagcg cgccaagcagcgcaagcacaccatcgacccctcctcccccgacaccagc ccatcccctccacgaggagtgatccccaagtccacgaaggagcacaagg aggtggtgcacgaggagtccggccacgtcctgaaggtgcccaccgccgc gtgcacctgtccggcggcgagcccgccacgacaactacgacacgtccgg cccccagaacgtcaacgcccacatcggcctggcgaagctgcgcaaggag tggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgt actacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgac gcgcgagaagctggaccccgagacgtccgctccgaggtcgcgcggggcc gcgccatcatcccctccaacaagaagcacctggagctggagcccatgat cgtgggccgcaagacctggtgaaggtgaacgcgaacatcggcaactccg ccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccac catgtggggcgccgacaccatcatggacctgtccacgggccgccacatc cacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggca ccgtccccatctaccaggcgctggagaaggtggacggcatcgcggagaa cctgaactgggaggtgaccgcgagacgctgatcgagcaggccgagcagg gcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccc cctgaccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatc cacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagc actgggacgacatcctggacatctgcaaccagtacgacgtcgccctgtc catcggcgacggcctgcgccccggctccatctacgacgccaacgacacg gcccagacgccgagctgctgacccagggcgagctgacgcgccgcgcgtg ggagaaggacgtgcaggtgatgaacgagggccccggccacgtgcccatg cacaagatccccgagaacatgcagaagcagctggagtggtgcaacgagg cgcccactacaccctgggccccctgacgaccgacatcgcgcccggctac gaccacatcacctccgccatcggcgcggccaacatcggcgccctgggca ccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaa ccgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccac gcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacg cgctgtccaaggcgcgatcgagaccgctggatggaccagacgcgctgtc cctggaccccatgacggcgatgtccaccacgacgagacgctgcccgcgg acggcgcgaaggtcgcccacactgctccatgtgcggccccaagactgct ccatgaagatcacggaggacatccgcaagtacgccgaggagaacggcta cggctccgccgaggaggccatccgccagggcatggacgccatgtccgag gagacaacatcgccaagaagacgatctccggcgagcagcacggcgaggt cggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaag TGAcaattgACGGAGCGTCGTGCGGGAGGGAGTGTGCCGAGCGGGGAGT CCCGGTCTGTGCGAGGCCCGGCAGCTGACGCTGGCGAGCCGTACGCCCC GAGGGTCCCCCTCCCCTGCACCCTCTTCCCCTTCCCTCTGACGGCCGCG CCTGTTCTTGCATGTTCAGCGACggatccTAGGGAGCGACGAGTGTGCG TGCGGGGCTGGCGGGAGTGGGACGCCCTCCTCGCTCCTCTCTGTTCTGA ACGGAACAATCGGCCACCCCGCGCTACGCGCCACGCATCGAGCAACGAA GAAAACCCCCCGATGATAGGTTGCGGTGGCTGCCGGGATATAGATCCGG CCGCACATCAAAGGGCCCCTCCGCCAGAGAAGAAGCTCCTTTCCCAGCA GACTCCTTCTGCTGCCAAAACACTTCTCTGTCCACAGCAACACCAAAGG ATGAACAGATCAACTTGCGTCTCCGCGTAGCTTCCTCGGCTAGCGTGCT TGCAACAGGTCCCTGCACTATTATCTTCCTGCTTTCCTCTGAATTATGC GGCAGGCGAGCGCTCGCTCTGGCGAGCGCTCCTTCGCGCCGCCCTCGCT GATCGAGTGTACAGTCAATGAATGGTCCTGGGCGAAGAACGAGGGAATT TGTGGGTAAAACAAGCATCGTCTCTCAGGCCCCGGCGCAGTGGCCGTTA AAGTCCAAGACCGTGACCAGGCAGCGCAGCGCGTCCGTGTGCGGGCCCT GCCTGGCGGCTCGGCGTGCCAGGCTCGAGAGCAGCTCCCTCAGGTCGCC TTGGACGGCCTCTGCGAGGCCGGTGAGGGCCTGCAGGAGCGCCTCGAGC GTGGCAGTGGCGGTCGTATCCGGGTCGCCGGTCACCGCCTGCGACTCGC CATCCgaagagcgtttaaac
[0136] Construct D1683 (pSZ2624), was transformed into S5100. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. Integration of pSZ2624 at the SAD2-1 locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 8). Simultaneous ablation of SAD2-1 and over-expression of PmKASII (driven in situ by the SAD2-1 promoter) resulted in C18:0 levels up to 26.1%. C16:0 accumulation was reduced from 15.3% in S5100 to <6% the strains derived from D1683, demonstrating that PmKASII-1 over-expression promoted the elongation of C16:0 to C18:0. S5780 was chosen for further development as it had the highest lipid titer relative to the S5100 parent.
TABLE-US-00020 TABLE 8 Fatty acid profiles of SAD2-1 ablation, PmKASII-1 overexpression strains derived from D1683-1, compared to the S5100 parent. Primary S5100; T531; D1683.1 Strain S5100 S5780 S5781 S5782 S5783 S5784 Fatty Acid C14:0 0.7 0.7 0.8 0.7 0.7 0.7 Area % C16:0 15.3 5.9 6.0 6.0 5.8 5.8 C16:1 0.5 0.1 0.0 0.1 0.0 0.0 C18:0 4.0 25.6 26.1 26.0 25.0 25.3 C18:1 71.0 55.7 54.5 54.6 56.3 55.6 C18:2 7.3 8.0 8.5 8.5 8.1 8.4 C18:3 0.5 0.7 0.8 0.8 0.7 0.7 C20:0 0.3 1.8 1.9 1.8 1.8 1.8 C20:1 0.2 0.6 0.6 0.6 0.7 0.7 C22:0 0.1 0.2 0.3 0.3 0.3 0.2 C24:0 0.1 0.4 0.4 0.4 0.4 0.4 saturates 20.6 34.7 35.6 35.4 34.1 34.5
[0137] We disclose additional methods of elevating C18:0 levels that can be used in conjunction with SAD2 knockout and KASII over-expression. Previously we described acyl-ACP thioesterases from Brassica napus (BnFATA) (Co-owned application WO2012/106560), Garcinia mangostana (GarmFATA1) (Co-owned application WO2015/051319) and Theobroma cacao (TcFATA) (Co-owned application WO2013/158938) with specificity towards cleavage of C18:0-ACP, and we observed that average C18:0 levels were higher in strains in which we replaced the native BnFATA transit peptide with the Chlorella protothecoides SAD1 transit peptide (CpSAD1tp). A DNA construct was made for expression of a chimeric gene encoding CpSAD1tp fused to the predicted GarmFATA1 mature polypeptide and a FLAG tag sequence.
[0138] The sequence of the transforming DNA from the GarmFATA1 expression construct pSZ3204 is shown below. The construct is written as pSZ3204: 6SA::CrTUB2-ScSUC2-CvNR:PmSAD2-2-CpSAD1tp_GarmFATA1_FLAG-CvNR::6SB. Relevant restriction sites are indicated in lowercase, bold, and are from 5-3 BspQI, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, SpeI, AscI, ClaI, AflII, SacI and BspQI. Underlined sequences at the 5 and 3 flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the 6S locus. Proceeding in the 5 to 3 direction, the CrTUB2 promoter driving the expression of Saccharomyces cerevisiae SUC2 (ScSUC2) gene, enabling strains to utilize exogeneous sucrose, is indicated by lowercase, boxed text. The initiator ATG and terminator TGA of ScSUC2 are indicated by uppercase italics, while the coding region is represented by lowercase italics. The 3 UTR of the CvNR gene is indicated by small capitals. A spacer region is represented by lowercase text. The P. moriformis SAD2-2 (PmSAD2-2) promoter driving the expression of the chimeric CpSAD1tp_GarmFATA1_FLAG gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding CpSAD1tp is represented by lowercase, underlined italics; the sequence encoding the GarmFATA1 mature polypeptide is indicated by lowercase italics; and the 3FLAG epitope tag is represented by uppercase, bold italics. A second CvNR 3 UTR is indicated by small capitals.
TABLE-US-00021 pSZ3204 SEQIDNO:87 gctcttcGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGC CAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATC ACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCC GGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGAC GTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACC GAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCT GGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACG AGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGAC GGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGC ACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGC GCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAA ACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCG ACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGT CACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGT GCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAggtaccattcttgcg ctatgacacttccagcaaaaggtagggcgggctgcgagacggcttcccg gcgctgcatgcaacaccgatgatgcttcgaccccccgaagctccttcgg ggctgcatgggcgctccgatgccgctccagggcgagcgctgtttaaata gccaggcccccgattgcaaagacattatagcgagctaccaaagccatat tcaaacacctagatcactaccacttctacacaggccactcgagcttgtg atcgcactccgctaagggggcgcctcttcctcttcgtttcagtcacaac ccgcaaactctagaatatcaATGctgctgcaggccttcctgttcctgct ggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtcc gaccgccccctggtgcacttcacccccaacaagggctggatgaacgacc ccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtactt ccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggc cacgccacgtccgacgacctgaccaactgggaggaccagcccatcgcca tcgccccgaagcgcaacgactccggcgccttctccggctccatggtggt ggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgc cagcgctgcgtggccatctggacctacaacaccccggagtccgaggagc agtacatctcctacagcctggacggcggctacaccttcaccgagtacca gaagaaccccgtgctggccgccaactccacccagttccgcgacccgaag gtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagt cccaggactacaagatcgagatctactcctccgacgacctgaagtcctg gaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtac gagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagt cctactgggtgatgttcatctccatcaaccccggcgccccggccggcgg ctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgag gccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacg ccctgcagaccttcttcaacaccgacccgacctacgggagcgccctggg catcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaac ccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccg agtaccaggccaacccggagacggagctgatcaacctgaaggccgagcc gatcctgaacatcagcaacgccggcccctggagccggttcgccaccaac accacgttgacgaaggccaacagctacaacgtcgacctgtccaacagca ccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagac gatctccaagtccgtgttcgcggacctctccctctggttcaagggcctg gaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcct ccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaa cccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagc gagaacgacctgtcctactacaaggtgtacggcttgctggaccagaaca tcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacaccta cttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggg gtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagT GAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTG GTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAAT ATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGT ACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACC CCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAAC TTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCA CTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTA CTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGG GATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagag gaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccac aataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgt ccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcg gtggagctgatggtcgaaacgttcacagcctagggatatcctgaagaat gggaggcaggtgttgttgattatgagtgtgtaaaagaaaggggtagaga gccgtcctcagatccgactactatgcaggtagccgctcgcccatgcccg cctggctgaatattgatgcatgcccatcaaggcaggcaggcatactgtg cacgcaccaagcccacaatcttccacaacacacagcatgtaccaacgca cgcgtaaaagttggggtgctgccagtgcgtcatgccaggcatgatgtgc tcctgcacatccgccatgatctcctccatcgtctcgggtgtttccggcg cctggtccgggagccgttccgccagatacccagacgccacctccgacct cacggggtacttttcgagcgtctgccggtagtcgacgatcgcgtccacc atggagtagccgaggcgccggaactggcgtgacggagggaggagaggga ggagagagaggggggggggggggggggatgattacacgccagtctcaca acgcatgcaagacccgtttgattatgagtacaatcatgcactactagat ggatgagcgccaggcataaggcacaccgacgttgatggcatgagcaact cccgcatcatatttcctattgtcctcacgccaagccggtcaccatccgc atgctcatattacagcgcacgcaccgcttcgtgatccaccgggtgaacg tagtcctcgacggaaacatctggctcgggcctcgtgctggcactccctc ccatgccgacaacctttctgctgtcaccacgacccacgatgcaacgcga cacgacccggtgggactgatcggttcactgcacctgcatgcaattgtca caagcgcatactccaatcgtatccgtttgatttctgtgaaaactcgctc gaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtgacctgggtgt ttcgtcgaaaggccagcaaccccaaatcgcaggcgatccggagattggg atctgatccgagcttggaccagatcccccacgatgcggcacgggaactg catcgactcggcgcggaacccagcMcgtaaatgccagattggtgtccga taccttgatttgccatcagcgaaacaagacttcagcagcgagcgtattt ggcgggcgtgctaccagggttgcatacattgcccatttctgtctggacc gctttaccggcgcagagggtgagttgatggggttggcaggcatcgaaac gcgcgtgcatggtgtgtgtgtctgttttcggctgcacaatttcaatagt cggatgggcgacggtagaattgggtgttgcgctcgcgtgcatgcctcgc cccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccctcct gctaacgctcccgactctcccgcccgcgcgcaggatagactctagttca accaatcgacaactagtATGgccaccgcatccactactcggcgacaatg cccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgccc agcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatc gtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccg tggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccga ggacggcctgtcctacaaggagaagacatcgtgcgctgctacgaggtgg gcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcagga ggtgggctgcaaccacgcccagtccgtgggctactccaccggcggatac caccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgc atgcacatcgagatctacaagtaccccgcctggtccgacgtggtggaga tcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactg gatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctcc aagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacg tggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcct ggccaccccgaggagaacaactcctccctgaagaagatctccaagctgg aggacccctcccagtactccaagctgggcctggtgccccgccgcgccga cctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtg ctggagtccatgccccaggagatcatcgacacccacgagctgcagacca tcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactc cctgacctcccccgagccctccgaggacgccgaggccgtgacaaccaca acggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccg caacacctgcacctgctgcgcctgtccggcaacggcctggagatcaacc gcggccgcaccgagtggcgcaagaagcccacccgc![custom-character]()
![custom-character]()
![custom-character]()
![custom-character]()
![custom-character]()
![custom-character]()
TGAatcgatagatctcttaagGCAGCAGCAGCTCGGATA GTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCAC ACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCC TCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGC TTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCAT ATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTC AGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGG GCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAA TGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagcttaa ttaagagctcTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTC TCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAA TTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTT GTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAA CACCGCGTACCTCTGCTTTGCGCAATCTGCCCTGTTGAAATCGCCACCA CATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGA ATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGA CACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCAT AACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCT GAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGC ATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGA AGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCC CAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTG AATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGT TGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTT GTTCGAGCTTgaagagc
[0139] Construct D1940 (pSZ3204), was transformed into the S5780 parent strain. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. Integration of pSZ3204 at the 6S locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 9). Over-expression of GarmFATA1 (driven by the SAD2-2 promoter) resulted in C18:0 levels up to 54.3%. C16:0 levels were comparable in strains derived from D1940 and the S5780 parent. S6573 was chosen for further development as it had the highest lipid titer of the strains with >50% C18:0.
TABLE-US-00022 TABLE 9 Fatty acid profiles of GarmFATA1 overexpressing stable strains derived from D1940 primary transformants. Primary D1683.1 D1940.19 D1940.20 D1940.23 D1940.46 D1940.5 Strain S5100 S5780 S6571 S6572 S6573 S6574 S6575 S6578 S6580 Fatty Acid Area % C14:0 0.7 0.0 0.8 0.0 0.8 0.7 0.7 0.0 0.0 C16:0 18.0 5.9 6.3 6.6 6.3 5.0 5.1 5.0 5.3 C16:1 0.5 0.0 0.1 0.1 0.1 0.0 0.1 0.1 0.1 C18:0 3.9 29.0 52.7 54.3 53.7 43.1 46.0 45.4 47.9 C18:1 69.8 54.3 31.4 30.1 30.5 41.5 38.5 40.0 37.2 C18:2 5.9 6.4 5.7 5.8 5.6 6.3 6.2 6.1 6.2 C18:3 0.5 0.7 0.6 0.6 0.6 0.6 0.5 0.6 0.5 C20:0 0.3 2.4 1.8 1.6 1.7 2.1 2.0 2.0 2.0 C20:1 0.1 0.6 0.1 0.1 0.1 0.2 0.1 0.1 0.1 C22:0 0.1 0.3 0.2 0.2 0.2 0.3 0.3 0.2 0.2 C24:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 saturates 23.1 37.7 61.9 62.8 62.8 51.2 54.2 52.7 55.5
[0140] Lysophosphatidic acid acetyltransferase (LPAAT) enzymes are responsible for the transfer of acyl groups to the sn-2 position on the glycerol backbone. We disclose here that we can reduce the accumulation of excessive amounts of trisaturates in our high SOS strains by expressing heterologous LPAAT genes which were better than the endogenous acyltransferases at discriminating against saturated fatty acids. Expression of LPAT2 homologs from B. napus, T cacao, Garcinia hombroriana and Garcinia indica and their effect on the formation of trisaturated TAGs in the high-C18:0 S6573 strain is disclosed below.
[0141] The sequence of the transforming DNA from the BnLPAT2(Bn1.13) expression construct pSZ4198 is shown below The construct is written as pSZ4198: PLOOP::PmHXT1-ScarMEL1-CvNR:PmSAD2-2v2-BnLPAT2(Bn1.13)-CvNR::PLOOP. Relevant restriction sites are indicated in lowercase, bold, and are from 5-3 BspQI, KpnI, SpeI, SnaBI, EcoRI, SpeI, ClaI, BglII, AflII, HindIII, SacI and BspQI. Underlined sequences at the 5 and 3 flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the PLOOP locus. Proceeding in the 5 to 3 direction, the PmHXT1 promoter driving the expression of S. carlbergensis MEL1 (ScarMEL1) gene, enabling strains to utilize exogeneous melibiose, is indicated by lowercase, boxed text. The initiator ATG and terminator TGA of ScarMEL1 are indicated by uppercase italics, while the coding region is represented by lowercase italics. The 3 UTR of the CvNR gene is indicated by small capitals. The P. moriformis SAD2-2v2 promoter driving the expression of the BnLPAT2(Bn1.13) gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding BnLPAT2(Bn1.13) is represented by lowercase, underlined italics. A second CvNR 3 UTR is indicated by small capitals. The Brassica napus LPAAT2(BN1.13) sequence is from Genbank accession GU045434.
TABLE-US-00023 SEQIDNO:88:NucleotidesequenceofthetransformingDNAfrompSZ4198 gctcttccgctAACGGAGGTCTGTCACCAAATGGACCCCGTCTATTGCGGGAAACCACG GCGATGGCACGTTTCAAAACTTGATGAAATACAATATTCAGTATGTCGCGGGCGG CGACGGCGGGGAGCTGATGTCGCGCTGGGTATTGCTTAATCGCCAGCTTCGCCCC CGTCTTGGCGCGAGGCGTGAACAAGCCGACCGATGTGCACGAGCAAATCCTGAC ACTAGAAGGGCTGACTCGCCCGGCACGGCTGAATTACACAGGCTTGCAAAAATA CCAGAATTTGCACGCACCGTATTCGCGGTATTTTGTTGGACAGTGAATAGCGATG CGGCAATGGCTTGTGGCGTTAGAAGGTGCGACGAAGGTGGTGCCACCACTGTGC CAGCCAGTCCTGGCGGCTCCCAGGGCCCCGATCAAGAGCCAGGACATCCAAACT ACCCACAGCATCAACGCCCCGGCCTATACTCGAACCCCACTTGCACTCTGCAATG GTATGGGAACCACGGGGCAGTCTTGTGTGGGTCGCGCCTATCGCGGTCGGCGAA GACCGGGAAggtaccgcggtgagaatcgaaaatgcatcgtttctaggttcggagacggtcaattccctgctccggcgaatctg tcggtcaagctggccagtggacaatgttgctatggcagcccgcgcacatgggcctcccgacgcggccatcaggagcccaaacagc gtgtcagggtatgtgaaactcaagaggtccctgctgggcactccggccccactccgggggcgggacgccaggcattcgcggtcggt cccgcgcgacgagcgaaatgatgattcggttacgagaccaggacgtcgtcgaggtcgagaggcagcctcggacacgtctcgctag ggcaacgccccgagtccccgcgagggccgtaaacattgtttctgggtgtcggagtgggcattttgggcccgatccaatcgcctcatgc cgctctcgtctggtcctcacgttcgcgtacggcctggatcccggaaagggcggatgcacgtggtgttgccccgccattggcgcccac gtttcaaagtccccggccagaaatgcacaggaccggcccggctcgcacaggccatgctgaacgcccagatttcgacagcaacacca tctagaataatcgcaaccatccgcgttttgaacgaaacgaaacggcgctgtttagcatgtttccgacatcgtgggggccgaagcatgct ccggggggaggaaagcgtggcacagcggtagcccattctgtgccacacgccgacgaggaccaatccccggcatcagccttcatcg acggctgcgccgcacatataaagccggacgcctaaccggtttcgtggttatgactagtATGttcgcgttctacttcctgacggcctgc atctccctgaagggcgtgacggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaaca cgacgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacatgggctaca agtacatcatcctggacgactgctggtcctccggccgcgactccgacggcacctggtcgccgacgagcagaagaccccaacgg catgggccacgtcgccgaccacctgcacaacaactccacctgacggcatgtactcctccgcgggcgagtacacgtgcgccggct accccggctccctgggccgcgaggaggaggacgcccagacttcgcgaacaaccgcgtggactacctgaagtacgacaactgc tacaacaagggccagacggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccg ccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccgg cgacgtcacggcggagacacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcacc actgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaa cctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttaccatgtgggccatggtgaagtcccccctgat catcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactcc aacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtc cggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggagg agatcacttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaa ctccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacg gcctgtccaagaacgacacccgcctgacggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccg cccacggcatcgcgactaccgcctgcgcccctcctccTGAtacgtactcgagGCAGCAGCAGCTCGGATAGT ATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTG CCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATC TTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCAC CCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCT ACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCAC AGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGC ACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAagctgtag aattcctggctcgggcctcgtgctggcactccctcccatgccgacaacctttctgctgtcaccacgacccacgatgcaacgcgacacg acccggtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcatactccaatcgtatccgtttgatttctgtgaaaactcg ctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtgacctgggtgtttcgtcgaaaggccagcaaccccaaatcgcaggc gatccggagattgggatctgatccgagcttggaccagatcccccacgatgcggcacgggaactgcatcgactcggcgcggaaccca gctttcgtaaatgccagattggtgtccgataccttgatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgct accagggttgcatacattgcccatttctgtctggaccgctttaccggcgcagagggtgagttgatggggttggcaggcatcgaaacgc gcgtgcatggtgtgtgtgtctgttttcggctgcacaatttcaatagtcggatgggcgacggtagaattgggtgttgcgctcgcgtgcatgc ctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccctcctgctaacgctcccgactctcccgcccgcgcgcag gatagactctagttcaaccaatcgacaactagtATGgccatggccgccgccgtgatcgtgcccctgggcatcctgacttcatctcc ggcctggtggtgaacctgctgcaggccatctgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcg tggtggccgagaccctgtggctggagctggtgtggatcgtggactggtgggccggcgtgaagatccaggtgacgccgacaacg agaccacaaccgcatgggcaaggagcacgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcc tggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaagacctgcccgtgatcggctggtccatgt ggactccgagtacctgacctggagcgcaactgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgactt cccccgcccataggctggccctgacgtggagggcacccgatcaccgaggccaagctgaaggccgcccaggagtacgccgc ctcctccgagctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgctccttcgt gcccgccatctacgacatgaccgtggccatccccaagacctcccccccccccaccatgctgcgcctgacaagggccagccctcc gtggtgcacgtgcacatcaagtgccactccatgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcgacca gacgtggccaaggacgccctgctggacaagcacatcgccgccgacaccaccccggccagcaggagcagaacatcggccgc cccatcaagtccctggccgtggtgctgtcctggtcctgcctgctgatcctgggcgccatgaagacctgcactggtccaacctgactc ctcctggaagggcatcgccactccgccctgggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcctcccagtccga gcgctccacccccgccaaggtggtgcccgccaagcccaaggacaaccacaacgactccggctcctcctcccagaccgaggtg gagaagcagaagTGAatcgatagatctcttaagGCAGCAGCAGCTCGGATAGTATCGACACACT CTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGT GAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACG CGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCC CCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTG CTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTT GGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCT GATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagcttaattaagagctcAGCGG CGACGGTCCTGCTACCGTACGACGTTGGGCACGCCCATGAAAGTTTGTATACCGA GCTTGTTGAGCGAACTGCAAGCGCGGCTCAAGGATACTTGAACTCCTGGATTGAT ATCGGTCCAATAATGGATGGAAAATCCGAACCTCGTGCAAGAACTGAGCAAACC TCGTTACATGGATGCACAGTCGCCAGTCCAATGAACATTGAAGTGAGCGAACTGT TCGCTTCGGTGGCAGTACTACTCAAAGAATGAGCTGCTGTTAAAAATGCACTCTC GTTCTCTCAAGTGAGTGGCAGATGAGTGCTCACGCCTTGCACTTCGCTGCCCGTG TCATGCCCTGCGCCCCAAAATTTGAAAAAAGGGATGAGATTATTGGGCAATGGA CGACGTCGTCGCTCCGGGAGTCAGGACCGGCGGAAAATAAGAGGCAACACACTC CGCTTCTTAgctcttc
[0142] Additional transforming constructs to test the activity of LPAATs from B. napus, T cacao, G. hombroriana and G. indica contained the same selectable marker, restriction sites, promoters and 3 UTR elements as pSZ4198. The coding sequences of BnLPAT2(Bn1.5), TcLPAT2, GhomLPAT2A, GhomLPAT2B, GhomLPAT2C, GindLPAT2A, GindLPAT2B and GindLPAT2C are shown in below. In each case the initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding the LPAT2 homolog is represented by lowercase italics. The Brassica napus LPAAT2(BN1.13) sequence is from Genbank accession GU045435. The Theobroma cacao LPAAT2 sequence is from the cocoaGenDB database.
TABLE-US-00024 SEQIDNO:89NucleotidesequenceoftheBnLPAT2(1.5)codingsequence, usedinthetransformingDNAfrompSZ4202 ATGgccatggccgccgccgccgtgatcgtgcccctgggcatcctgacttcatctccggcctggtggtgaacctgctgcaggccgt gtgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagaccctgtggctggagctg gtgtggatcgtggactggtgggccggcgtgaagatccaggtgacgccgacgacgagaccacaaccgcatgggcaaggagca cgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctcc gccctggccgtgatgaagaagtcctccaagacctgcccgtgatcggctggtccatgtggactccgagtacctgacctggagcgca actgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgacacccccgccccactggctggccctgacgtg gagggcacccgatcaccgaggccaagctgaaggccgcccaggagtacgccgcctcctcccagctgcccgtgccccgcaacgt gctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgctccttcgtgcccgccatctacgacatgaccgtggccat ccccaagacctcccccccccccaccatgctgcgcctgacaagggccagccctccgtggtgcacgtgcacatcaagtgccactcc atgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcgaccagacgtggccaaggacgccctgctggacaa gcacatcgccgccgacaccaccccggccagaaggagcacaacatcggccgccccatcaagtccctggccgtggtggtgtcctg ggcctgcctgctgaccctgggcgccatgaagacctgcactggtccaacctgactcctccctgaagggcatcgccctgtccgccctg ggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcctcccagtccgagcgctccacccccgccaaggtggcccccg ccaagcccaaggacaagcaccagtccggctcctcctcccagaccgaggtggaggagaagcagaagTGA SEQIDNO:90NucleotidesequenceoftheTcLPAT2codingsequence,used inthetransformingDNAfrompSZ4206 ATGgccatcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttc gtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggctggtgg actggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccctggtggtggccaacc accgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctcc aagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagaacaccctgaaggc cggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtggagggcacccgcttcacccaggccaagttcctggccgc ccaggagtacgccgcctcccagggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgc gctccttcgtgcccgccatctacgacatgaccgtggccatccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctc cgtggtgcacgtgcacatcaagcgctgcctgatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgttcg tggagaaggacaagctgctggacaagcacatcgccgaggacaccttctccgaccagcccatgcaggacctgggccgccccatcaagtcc ctgctggtggtggcctcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcg ccttcttcctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggc ccccggcaagcccaagaacgacggcgagacctccgaggcccgccgcgacaagcagcagTGA SEQIDNO:91NucleotidesequenceoftheGhomLPAT2Acodingsequence, usedinthetransformingDNAfrompSZ4412. ATGgccatccccgccgccatcgtgatcgtgcccgtgggcctgctgttcttcatctccggcctgatcgtgaacctgctgcaggccctgtgcttcg tgctgatccgccccctgtccaagtccgcctaccgcaccatcaaccgccagctggtggagctgctgtggctggagctggtgtgcatcgtggac tggtgggcccgcgtgaagatccagctgttcaccgacaaggagaccctgaactccatgggcaaggagcacgccctggtgatgtgcaacca ccgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccaccgtggccgtgatgaagaagtcctcca aggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagtcc ggcctgcagcgcctgcgcgacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgcc caggagtacgccgcctccaccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccatcacccgc tccttcgtgcccgtgatctacgacatcaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctccg tggtgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgacgtggcccagtggtgccgcgaccagttcgtgg tgaaggactccctgctggacaagcacatcgccgaggacaccttctccgaccaggagctgcaggacatcggccgccccatcaagtccctgg tggtgttcacctcctgggtgtgcatcatcaccttcggcgccctgaagttcctgcagtggtcctccctgctgcactcctggaagggcatcgccat ctccgcctccggcctggccatcgtgaccgtgctgatgcacatcctgatccgcttctcccagtccgagcactccacctccgccaagatcgccgcc gagaagcacaagaacggcggcgtgtcccaggagatgggccgcgagaagcagcacTGA SEQIDNO:92NucleotidesequenceoftheGhomLPAT2Bcodingsequence, usedinthetransformingDNAfrompSZ4413. ATGgagatccccgccgtggccgtgatcgtgcccatcggcatcctgttcttcatctccggcctgatcgtgaacctgatgcaggccatctgcttc ttcctgatccgccccctgtccaagaacacccaccgcatcgtgaaccgccagctggccgagctgctgtggctggagctgatctggatcgtgga ctggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcacctgatgggcaaggagcacgccctggtgatctgcaacc actcctccgacatcgactggctggtgggctggctgctgtgccagcgctccggctgcctgggctccgccctggccgtgatgaagtcctcctcca aggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagtccaccctgaagtcc ggcctgcagcgcctgaaggacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccaggccaagctgctggccgc ccaggagtacgccatgtccgccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgc gctccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccgtgcagcccaccatgctgcgcctgttcaagggccagtcctc cgtggtgcaggtgcacctgaagcgccactccatgaaggacctgcccgagtccgaggacgacgtggcccagtggtgccgcgaccgcttcgt ggtgaaggactccctgctggacaagcacaaggtggaggacaccttcaccgaccaggagctgcaggacctgggccgccccatcaagtccc tggtggtggtgacctgctgggcctgcatcatcatcttcggcatcctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatggc catctccgcctccggcctggccgtggtgaccttcctgatgcagatcctgatccgcttctcccagtccgagcgctccacccccgccaagatcgcc cccgccaagcccaacaaggccggcaactcctccgagaccgtgcgcgacaagcaccagTGA SEQIDNO:93NucleotidesequenceoftheGhomLPAT2Ccodingsequence, usedinthetransformingDNAfrompSZ4414. ATGgccatccccgccgccatcatcatcgtgcccctgggcctgatcttcttcacctccggcctgatcatcaacctgatccaggccgtgtgctacg tgctgatccgccccctgtccaagtccaccttccgccgcatcaaccgcgagctggccgagctgctgtggctggagctggtgtgggtggtggac tggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcactccatgggcaaggagcacgccctggtgatctgcaaccac cgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacttcttcctggagcgcaactgggccatggacgagtccaccctgaagtccg gcctgcagcgcctgaaggacttcccccagcccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgccc aggagtacgccgcctccgccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgaacatcatgcgc tccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctccg tggtgcacgtgcacctgaagcgccacctgatggaggacctgcccgagaccgacgacgacgtggcccagtggtgccgcgaccgcttcgtgg tgaaggactccctgctggacaagtacgtggccgaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccctgg tggtggtgacctcctgggtgtgcatcatcgccttcggctccctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgtgat ctccgccgcctccctggccgtggtgaccgtgctgatgcagatcctgatccgcttctcccagtccgagcgctccacctccgccaagatcgccgcc gccaagcgcaagaacgtgggcgagcacTGA SEQIDNO:94NucleotidesequenceoftheGindPAT2Acodingsequence, usedinthetransformingDNAfrompSZ4415. ATGgccatccccgtggtggtggtgatcgtgcccgtgggcctgctgttcttcatctccggcctgatcgtgaacctgctgcaggccctgtgcttc gtgctgatccgccccctgtccaagtccgcctaccgcaccatcaaccgccagctggtggagctgctgtggctggagctggtgtgcatcgtgga ctggtgggcccgcgtgaagatccagctgttcatcgacaaggagaccctgaactccatgggcaaggagcacgccctggtgatgtgcaacc accgctcctacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccaccgtggccgtgatgaagaagtcctcc aaggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagt ccggcctgcagcgcctgcgcgacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccg cccaggagtacgccgcctccaccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccatcaccc gctccttcgtgcccgtgatctacgacatcaccgtggccatccccaagtcctcctcccagcccaccatgctgaagctgttcaagggccagtcctc cgtggtgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgacgtggcccagtggtgccgcgcccagttcgt ggtgaaggactccctgctggacaagcacatcgccgaggacaccttctccgaccaggagctgcaggacatcggccgccccatcaagtccct ggtggtgttcacctcctgggtgtgcatcatcaccttcggcgccctgaagttcctgcagtggtcctccctgctgcactcctggaagggcatcgcc atctccgcctccggcctggccatcgtgaccgtgctgatgcacatcctgatccgcttctcccagtccgagcactccacctccgccaagatcgccg ccgagaagcacaagaacggcggcgtgtcccaggagatgggccgcgagaagcagcacTGA SEQIDNO:95NucleotidesequenceoftheGindPAT2Bcodingsequence, usedinthetransformingDNAfrompSZ4416. ATGggcatccccgccgtggccgtgatcgtgcccatcggcatcctgttcttcatctccggcttcatcgtgaacctgatgcaggccatctgcttcg tgctgatccgccccctgtccaagaacacctaccgcatcgtgaaccgccagctggccgagttcctgtggctggagctgatctgggtggtggac tggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcacctgatgggcaaggagcacgccctggtgatctgcaacca ccgctccgacatcgactggctggtgggctggctgctgtgccagcgctccggctgcctgggctccgccctggccgtgatgaagtcctcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagtccaccctgaagctgg gcctgcagcgcctgaaggacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccaggccaagctgctggccgccc aggagtacgccatgtccgccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgc tccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccgtgcagcccaccatgctgggcctgttcaagggccagtcctgc gtggtgcaggtgcacctgaagcgccacctgatgaaggacctgcccgagtccgaggacgacgtggcccagtggtgccgcgagcgcttcgt ggtgaaggactccctgctggacaagcacaaggtggaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccct ggtggtggtgatctcctgggcctgcatcctgatcttctggatcctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgcc atctccgcctgcgccatggccgtgatcgccttcctgatgcagatcctgctgcgcttctcccagtccgagcgctccacccccgccaagatcgccc ccgccaagcccaacaacgcccgcaactcctccgagaccgtgcgcgacaagcaccagTGA SEQIDNO:96NucleotidesequenceoftheGindPAT2Ccodingsequence, usedinthetransformingDNAfrompSZ4417. ATGgccatccccgccgccatcatcatcgtgcccctgggcctgatcttcttcacctccggcttcatcatcaacctgatccaggccgtgtgctacg tgctgatccgccccctgtccaagtccaccttccgccgcatcaaccgccagctggccgagctgctgtggctggagctggtgtgggtggtggac tggtgggccggcgtgaagatccagctgttcaccaacaaggagaccctgcactccatcggcaaggagcacgccctggtgatctgcaaccag cgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccatggacgagtccaccctgaagtccg gcctgcagtggctgaaggacttcccccagcccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgcc caggagtacgccgcctccgccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgaacatcatgcg ctccttcgtgcccgccgtgtacgacgtgaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctcc gtggtgcacgtgcacctgaagcgccacctgatggaggacctgcccgagaccgacgacgacgtggcccagtggtgccgcgaccgcttcgtg gtgaaggactccctgctggacaagcacctggccgaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccctg gtggtggtgacctcctgggtgtgcatcatcgccttcggcgccctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgtg atctccgccgcctccctggccgtggtgaccgtgctgatgcagatcctgatccgcttctcccagtccgagcgctccacctccgccaaggtggtg gccgagaagcgcaagaacgtgggcgagcacTGA
[0143] Constructs D2971, D2973, D2975, D3219, D3221, D3223, D3225, D3227 and D3229, derived from pSZ4198, pSZ4202, pSZ4206, pSZ4412, pSZ4413, pSZ4414, pSZ4415, pSZ4416 and pSZ4417, respectively, were transformed into the S6573 parent strain. The fatty acid profiles of primary transformants are shown in Table 10. Also shown are the SOS/SSS ratios determined by LC/MS multiple response measurements. Expression of LPAT2 genes had no discernable effect on C16:0 or C18:0 accumulation, but C18:2 levels increased by 1-2% compared to the S6573 parent in strains when expressing the D2971, D2973, D2975, D3221, D3223, and D3227 constructs. Expression of LPAT2 genes increased C18:2 and also elevated ratios of SOS/SSS, showing reduced accumulation of trisaturated TAGs.
TABLE-US-00025 TABLE 10 Fatty acid profiles and SOS/SSS ratios of D2971, D2973, D2975, D3219, D3221, D3223, D3225, D3227 and D3229 primary transformants. Strain LPAAT gene SOS/SSS C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 saturates S5100 0.7 17.7 4.1 68.5 6.8 0.6 0.4 23.3 S6573.1 15 0.8 6.2 50.7 33.7 5.6 0.7 1.5 59.8 D2971.1 BnLPAT2(1.13) 23 0.8 6.1 51.4 30.5 8.6 0.6 1.4 60.2 D2971.2 16 0.8 6.1 54.3 28.9 7.0 0.6 1.5 63.3 D2971.4 16 0.8 6.4 53.3 29.5 7.3 0.6 1.4 62.6 S6573.2 14 0.8 6.6 52.8 31.7 5.2 0.6 1.5 62.3 D2973.2 BnLPAT2(1.5) 22 0.8 6.2 53.4 28.3 6.4 0.6 1.7 62.7 D2973.38 23 0.9 7.5 51.2 29.1 6.5 0.5 1.4 61.7 D2973.24 24 0.9 6.8 51.7 29.2 6.3 0.5 1.6 61.5 S6573.3 14 0.8 6.6 52.8 31.7 5.2 0.6 1.5 62.3 D2975.33 TcLPAT2 27 0.8 6.6 52.7 29.7 7.1 0.6 1.5 62.3 D2975.13 32 0.8 6.5 52.4 30.2 7.3 0.6 1.4 61.7 D2975.35 27 0.8 6.5 52.8 29.6 7.3 0.6 1.5 62.2 S6573.4 12 0.9 6.4 54.9 28.9 5.7 0.6 1.7 64.5 D3219.19 GhomLPAT2A 12 0.9 7.1 52.4 31.2 4.8 0.5 2.0 63.1 D3219.20 14 0.9 6.6 53.2 30.6 5.5 0.6 1.7 63.0 D3219.32 15 0.8 6.4 53.1 29.8 6.5 0.6 1.5 62.6 S6573.5 12 0.9 6.4 53.7 30.3 5.5 0.6 1.6 63.3 D3220.1 GhomLPAT2B 27 0.9 6.6 52.2 30.0 7.0 0.7 1.4 61.9 D3221.39 20 0.9 6.7 53.9 28.7 6.7 0.6 1.5 63.7 D3221.40 22 0.8 6.5 53.7 29.1 6.8 0.6 1.4 63.2 S6573.6 14 0.8 6.3 54.0 30.2 5.5 0.6 1.6 63.4 D3223.2 GhomLPAT2C 20 0.8 6.5 53.0 29.3 7.3 0.6 1.5 62.4 D3223.6 21 0.8 6.5 53.5 29.3 7.0 0.6 1.4 62.7 D3223.7 21 0.8 6.4 52.5 30.7 6.6 0.5 1.5 61.8 D3225.5 GindLPAT2A 13 0.9 6.6 53.5 30.2 5.6 0.6 1.6 63.2 S6573.7 12 0.9 6.5 53.5 29.9 5.7 0.6 1.8 63.3 D3227.6 GindLPAT2B 23 0.8 6.4 54.1 28.8 6.8 0.6 1.6 63.5 D3227.3 21 0.8 6.5 53.9 29.0 6.7 0.6 1.5 63.4 D3227.17 22 0.8 6.6 53.8 28.8 7.0 0.6 1.4 63.3 S6573.8 11 0.8 6.4 54.3 30.1 5.4 0.6 1.7 63.8 D3229.41 GindLPAT2C 11 0.9 6.6 54.2 29.7 5.6 0.6 1.7 63.9 D3229.27 13 0.8 6.4 54.1 30.0 5.6 0.6 1.7 63.6 D3229.33 12 0.8 6.4 54.0 30.2 5.5 0.6 1.7 63.5
[0144] Table 11 presents the TAG composition of the lipids produced by D2971, D2973, D2975, D3221, D3223, and D3227 primary transformants relative to the S6573 parent. SOS levels in the LPAT2-expressing strains were equivalent or slightly higher than in the S6573 controls. Trisaturates declined by up to 53%, and total Sat-Unsat-Sat levels improved in all of the strains expressing heterologous LPAT2 genes. Among the LPAT2 genes, the strains expressing the T. cacao LPAT2 homolog showed the greatest improvements in their TAG profiles).
TABLE-US-00026 TABLE 11 TAG composition of D2971, D2973, D2975, D3221, D3223, and D3227 primary transformants relative to the S6573 parent. LPAAT gene BnLPAT2 BnLPAT2 Ghom Ghom Gind (1.13) (1.5) TcLPAT2 LPAT2B LPAT2C LPAT2B Strain D2971.1 D2973.38 D2975.33 D2975.13 D3221.39 D3221.40 D3223.6 D3227.3 D3227.6 % S6573 SOS 100 100 110 104 107 107 108 103 105 TAG Sat-Sat-Sat 57 63 48 47 74 62 68 62 70 Sat-U-Sat 109 107 113 110 112 112 109 108 107 Sat-O-Sat 97 100 105 102 106 105 102 104 104 Sat-L-Sat 174 147 155 155 139 143 141 130 125 U-U-U/Sat 85 86 72 83 64 69 78 82 79
[0145] We analyzed the fatty acid profiles, TAG profiles and lipid titers from 50 mL shake flask cultures of stable lines generated from D2975-33. C18:0 and C16:0 levels were comparable between the strains and the S6573 control, and lipid titers ranged from 75-105% of the parent strain titer (Table 12). C18:2 levels increased by more than 2% in the TcLPAT2-expressing strains.
TABLE-US-00027 TABLE 12 Fatty acid profiles of TcLPAT2-expressing stable lines made from D2975-33. Primary D1940.19 D2975.33 Strain S6573 S7813 S7815 S7816 S7817 S7819 Fatty Acid C12:0 0.2 0.2 0.2 0.2 0.2 0.2 Area % C14:0 0.9 0.7 0.8 0.8 0.7 0.7 C16:0 6.5 5.9 6.1 5.9 6.1 6.0 C16:1 0.1 0.1 0.1 0.1 0.1 0.1 cis-9 C17:0 0.2 0.2 0.2 0.2 0.2 0.2 C18:0 56.1 55.6 55.9 56.2 53.9 53.9 C18:1 28.1 26.8 26.6 26.5 28.8 28.4 C18:2 5.5 8.1 7.7 7.9 7.7 7.8 C18:3 0.6 0.5 0.6 0.5 0.6 0.7 C20:0 1.5 1.5 1.4 1.3 1.3 1.5 C22:0 0.2 0.2 0.1 0.1 0.1 0.2 C24:0 0.1 0.1 0.1 0.1 0.1 0.1 saturates 65.7 64.4 65.0 64.9 62.8 62.9
[0146] The TAG profiles of S6573 and S7815 are compared in FIG. 1. SOS levels in the LPAT2-expressing strains were higher than in the S6573 control. Trisaturates were reduced from 10.2% in S6573 to 5.6% in S7815. Much of the improvement in total sat-unsat-sat levels in S7815 came from a 4% increase in stearate-linoleate-stearate (SLS) and a 1.5% increase in palmitate-linoleate-stearate (PLS), consistent with the enhanced C18:2 content of that strain. These results indicate that the T. cacoa LPAT2 reduces the incorporation of saturated fatty acids at the sn-2 position.
[0147] The performance of S7815 versus the S6573 parent strain was compared in high-density fermentations. The fatty acid profile of each strain at the two time points of the fermentations are shown in Table 13. The strains had very similar composition, with 5.5-5.7% C16:0, 56.4-56.8% C18:0, and 27.2-28.6% C18:1 as the major fatty acids. As was observed in the shake flask assays, (see Table 12), C18:2 levels increased from 5.5% in S6573 to 7.7% in S7815(Table 13). Normalized lipid titers and yields were comparable between the two strains, indicating that expression of the TcLPAT2 gene in S7815 did not have deleterious effects on growth or lipid accumulation.
TABLE-US-00028 TABLE 13 Fatty acid profiles of S7815 versus S6573 fermentations. Strain S6573 S7815 Fermentation 140207F25 140208F26 Fatty Acid C12:0 0.19 0.20 0.20 0.21 Area % C14:0 0.71 0.72 0.66 0.66 C16:0 5.69 5.73 5.57 5.54 C16:1 cis-7 0.05 0.05 0.05 0.06 C16:1 cis-9 0.07 0.06 0.05 0.05 C17:0 0.11 0.11 0.12 0.11 C8:0 56.01 56.78 55.50 56.37 C8:1 29.31 28.58 27.92 27.19 C8:2 5.56 5.51 7.75 7.70 C8:3 0.34 0.32 0.40 0.37 C20:0 1.51 1.50 1.35 1.34 C22:0 0.16 0.16 0.14 0.14 C24:0 0.10 0.09 0.09 0.08 sum C18 91.22 91.19 91.57 91.63 saturates 64.54 65.34 63.69 64.51 unsaturates 35.46 34.64 36.30 35.49
[0148] Table 13 compares the TAG profiles of the lipids produced during high-density fermentation of S7815 versus S6573. SOS and Sat-Oleate-Sat levels were almost identical between S7815 and the S6573 control. However, Sat-Linoleate-Sat levels increased by more than 7%, and di-unsaturated and tri-unsaturated TAGs (UU-U/Sat) declined by more than 3% in S7815 compared to S6573. Trisaturates at the end points of the fermentations were reduced from 10.1% in S6573 to 6.1% in S7815. These results indicate that the activity of T. cacoa LPAT2 drives the transfer of unsaturated fatty acids towards the sn-2 position and discriminates against the incorporation of saturated fatty acids at sn-2.
Example 6: Identification and Expression of Novel LPAAT, GPAT, DGAT, LPCAT and PLA2 with Specificity for Mid-Chain Fatty Acids
[0149] In this example, we demonstrate the effect of expression of LPAAT, GPAT, DGAT, LPCAT and PLA2 enzymes involved in triacylglycerol biosynthesis (in previously described P. moriformis (UTEX 1435) transgenic strains, S7858 and S8174. S7858 and S8174 were prepared according to co-owned WO2015/051319, herein incorporated by reference. In addition co-owned WO2010/063031 and WO2010/063032 teach the expression Cuphea hookerianas FATB2. Briefly, strain S7858 is a strain that express sucrose invertase and a Cuphea. hookeriana FATB2. To make S7858, the construct pSZ4329 (SEQ ID NO: 197) was engineered into S3150, a strain classically mutagenized to increase lipid yield. The plasmid, pSZ4329 is written as THI4::CrTUB2-ScSUC2-PmPGH:PmAcp-P1p-CpSAD1tp_trimmed_ChFATB2_FLAG-CvNR::THI4a The annotation of the coding portions of pSZ4329 is shown in the Table A below.
TABLE-US-00029 TABLE A Nucleotide Nucleotide Nucleotide pSZ4329 Identity Number Number Length THI4a 3 flank 3 flanking sequences 5,692 6,394 703 of endogenous THI4 CvNR 3UTR 5,278 5,679 402 ChFATB2 CDS 4,105 5,271 1,167 CpSAD1tp-trimmed CDS 3,991 4,104 114 PmACP-P1 promoter promoter 3,411 3,981 571 Buffer DNA 3,199 3,404 206 UTR04424=PmPGH 3UTR 2,749 3,192 444 UTR ScSUC2(o) CDS 1,144 2,742 1,599 CrTUB2 promoter promoter THI4a 5 flank 5 flanking sequences 820 1,131 312 of endogenous THI4 27 813 787
[0150] Strain S7858, accumulates C8:0 fatty acids to about 12% and C10:0 fatty acids to about 22-24%. Briefly, strain S8174 is a strain that express sucrose invertase and a Cuphea. Avigera var. pulcherrima FATB2. To make S8174, the construct pSZ5078 (SEQ ID NO: 198) was engineered into S3150, a strain classically mutagenized to increase lipid yield. pSZ5078 is written as THI4a5::CrTUB2_ScSUC2_PmPGH:PmAMT3_CpSAD1tp_trimmed-CaFATB1_Flag_CvNR::THI4a3. Strain S8174 accumulates C8:0 fatty acids to about 24% and C10:0 fatty acids to about 10%. The annotation of the coding portions of pSZ5078 is shown in the Table B below.
TABLE-US-00030 TABLE B Nucleotide Nucleotide Nucleotide pSZ5078 Identity Number Number Length THI4a 3 3 flanking sequences 6,200 6,902 703 flank of endogenous THI4 CvNR 3UTR 5,786 6,187 402 CaFATB1 CDS 4,602 5,771 1,170 wild-type CpSAD1tp CDS 4,488 4,601 114 AMT3 promoter eukaryotic 3,411 4,481 1,071 Buffer DNA misc_feature 3,199 3,404 206 PmPGH 3UTR 2,749 3,192 444 ScSUC2(o) CDS 1,144 2,742 1,599 CrTUB2 promoter 820 1,131 312 promoter THI4a 5 5 flanking sequences 27 813 787 flank of endogenous THI4
[0151] The pool of acyl-CoAs in the ER can be utilized for the synthesis of TAGs as well as phospholipids and long chain fatty acids. The enzymes involved in the synthesis of TAGS and phospholids actively compete against each other for the same substrates. Acyl-CoAs can associate with lysophosphatidate to form phosphatidate which is converted to phosphatidylcholine (PC) and other phospholipid species. PC can be desaturated by FAD2 and FAD3 enzymes to generate polyunsaturated fatty acids, which can be cleaved by phosphotransferases and reenter the acyl-CoA pool. Acyl-CoAs can also be generated from PC directly by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT). LPCAT can also catalyze the reverse reaction to consume acyl-CoA. Removal of fatty acids from PC to form acyl-CoAs can also be catalyzed by phospholipase A2 (PLA2). TAG formation in the ER from acyl-CoAs requires action of glycerol phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT) and diacyl glycerol acyltransferase (DGAT).
[0152] The endogenous P. moriformis TAG biosynthesis machinery has evolved to function with the longer chain fatty acids that the strain normally makes. We introduced heterologous acyltransferases and phospholipases from species that naturally accumulate high levels of short chain fatty acids into Prototheca to increase accumulation of C8:0 fatty acids. We identified the following plant enzymes in NCBI as shown in Table 14 below.
TABLE-US-00031 TABLE 14 Genes representing target enzymes identified from higher plants that produce high amounts of C8:0 and C10:0. All these genes were synthesized with codon usage optimized for expression in Prototheca. Species Gene Enzyme ![text missing or illegible when filed]()
cnLPAAT1 LPAAT Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea avigera var. ![text missing or illegible when filed]()
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LPAAT1 Cuphea avigera var. ![text missing or illegible when filed]()
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LPAAT2 Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea ![text missing or illegible when filed]()
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LPAAT1 Cuphea ![text missing or illegible when filed]()
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LPAAT2 Cuphea ![text missing or illegible when filed]()
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LPAAT2 Cuphea ![text missing or illegible when filed]()
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LPAAT2 Cuphea avigera var. ![text missing or illegible when filed]()
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GPAT9![text missing or illegible when filed]()
GPAT Cuphea ![text missing or illegible when filed]()
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GPAT9![text missing or illegible when filed]()
1 Cuphea ![text missing or illegible when filed]()
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GPAT9![text missing or illegible when filed]()
2 Cuphea ![text missing or illegible when filed]()
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GPAT9![text missing or illegible when filed]()
2 Cuphea ![text missing or illegible when filed]()
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GPAT9![text missing or illegible when filed]()
2 Cuphea ![text missing or illegible when filed]()
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GPAT9![text missing or illegible when filed]()
2 Cuphea avigera var. ![text missing or illegible when filed]()
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DGAT1 DGAT Cuphea ![text missing or illegible when filed]()
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DGAT1![text missing or illegible when filed]()
1 Cuphea avigera var. ![text missing or illegible when filed]()
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LPCAT LPCAT Cuphea ![text missing or illegible when filed]()
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LPCAT Cuphea ![text missing or illegible when filed]()
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LPCAT Cuphea ![text missing or illegible when filed]()
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LPCAT1 Cuphea avigera var. ![text missing or illegible when filed]()
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PLA2![text missing or illegible when filed]()
1 PLA2 Cuphea ![text missing or illegible when filed]()
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PLA2![text missing or illegible when filed]()
1 Cuphea ![text missing or illegible when filed]()
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PLA2![text missing or illegible when filed]()
2 Cuphea ![text missing or illegible when filed]()
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PLA2![text missing or illegible when filed]()
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[0153] We made a set of constructs expressing heterologous short chain specific acyltransferases and PLA2s as shown in Table 15. The genes were codon optimized to reflect UTEX 1435 codon usage.
TABLE-US-00032 TABLE 15 List of constructs transformed into S7858 or S8174 D# Strain Construct D4289 S7858 SAD2-1vD::CpauLPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4290 S7858 SAD2-1vD::![text missing or illegible when filed]()
LPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4291 S7858 SAD2-1vD::CigneaLPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4292 S7858 SAD2-1vD::![text missing or illegible when filed]()
LPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4293 S7858 SAD2-1vD::ChookLPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4404 S7858 SAD2-1vD::CnLPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4517 S8174 SAD2-1vD::CavigLPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4518 S8174 SAD2-1vD::CavigLPAAT2![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4519 S8174 SAD2-1vD::CavigLPAAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4690 S8174 SAD2-1vD::CuPSR23 LPAAT2![text missing or illegible when filed]()
1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4728 S8174 SAD2-1vD::CkoeLPAAT![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4729 S8174 SAD2-1vD::CkoeLPAAT2![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4730 S8174 SAD2-1vD::CprocLPAAT2![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4551/D5683 S8174 SAD2-1vD::CavigGPAT9![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4552/D4684 S8174 SAD2-1vD::ChookGPAT9-1-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4553/D4685 S8174 SAD2-1vD::CignGPAT9-1-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4554/D4686 S8174 SAD2-1vD::CignGPAT9-2-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4724 S8174 SAD2-1vD::![text missing or illegible when filed]()
GPAT9-1-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4725 S8174 SAD2-1vD::![text missing or illegible when filed]()
GPAT9-2-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4549 S8174 SAD2-1vD::CavigDGAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4681 S8174 SAD2-1vD::CavigDGAT1![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4556/D4688 S8174 SAD2-1vD::CavigLPCAT![text missing or illegible when filed]()
PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4726 S8174 SAD2-1vD::![text missing or illegible when filed]()
LPCAT-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4556/D4689 S8174 SAD2-1vD::CpauLPCAT-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4727 S8174 SAD2-1vD::CschuLPCAT-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4732 S8174 SAD2-1vD::CavigPLA2-1-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4734 S8174 SAD2-1vD::CignPLA2-1-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4735 S8174 SAD2-1vD::CuPSR23PLA2-2-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex D4736 S8174 SAD2-1vD::CprocPLA2-2-PmATP:PmHXT1![text missing or illegible when filed]()
ScarMEL-PmPGK::SAD2Bex ![text missing or illegible when filed]()
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[0154] All the constructs shown in Table 15 can be written as SAD2-1vD::gene of interest-PmATP-PmHXT1-ScarMEL-PmPGK::SAD2B, and were made to target the transforming DNA to the SAD2 locus on the genome, thereby disrupting the expression of at least one allele of the endogenous stearoyl ACP desaturase. Sequences of all the transforming DNAs are provided below. The relevant restriction sites in the construct from 5-3 are- Pme I, BspQ I, Kpn I, Xho I, Avr II, Spe I, SnaB I, EcoR V, Sac I, BspQ I, Pme I respectively are indicated in lowercase, bold, and underlined. Pme I sites delimit the 5 and 3 ends of the transforming DNA. Bold, lowercase sequences at the 5 and 3 end of the construct represent genomic DNA from UTEX 1435 that target integration to the SAD2 locus via homologous recombination, wherein the SAD2 5 flank provides the promoter for the gene of interest downstream. The primary construct was made with the previously characterized CnLPAAT gene as shown below and all other constructs were made by replacing the CnLPAAT gene with other genes of interest using the restriction sites, Kpn I and Xho I that span the gene on either side. Proceeding in the 5 to 3 direction, the first cassette has the codon optimized Cocos nucifera LPAAT and the Prototheca moriformis ATP synthase (PmATP) gene 3 UTR. The initiator ATG and terminator TGA for cDNAs are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3 UTR is indicated by lowercase underlined text. The second cassette containing the selection gene melibiose from Saccharomyces carlsbergensis (ScarMEL1) is driven by the endogenous HXT1 promoter, and has the endogenous phosphoglycerate kinase (PmPGK) gene 3 UTR. In this cassette, the PmHXT1 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the ScarMEL1 gene are indicated in uppercase italics, while the coding region is indicated by lowercase italics. The 3 UTR is indicated by lowercase underlined text. All the final constructs were sequenced to ensure correct reading frames and targeting sequences.
TABLE-US-00033 pSZX61SequenceofthetransformingDNAexpressing CnLPAATdownstreamoftheSAD2promoterinthecassettefollowedbytheScarMEL1 geneforselectiondownstreamofthePmHXT1promoterinthesecondcassette. SEQIDNO:97 gtttaaacgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacgg aaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgaca cgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagc agttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcg caggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcgg aacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttgg cgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcagg catcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtg gcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactc tcccgaccgcgcgcaggatagactcttgttcaaccaatcgacaggtaccATGgacgcctccggcgcctcctccttcctgcgcggccgct gcctggagtcctgcttcaaggcctccttcggctacgtaatgtcccagcccaaggacgccgccggccagccctcccgccgccccgccgacgcc gacgacttcgtggacgacgaccgctggatcaccgtgatcctgtccgtggtgcgcatcgccgcctgcttcctgtccatgatggtgaccaccatc gtgtggaacatgatcatgctgatcctgctgccctggccctacgcccgcatccgccagggcaacctgtacggccacgtgaccggccgcatgct gatgtggattctgggcaaccccatcaccatcgagggctccgagttctccaacacccgcgccatctacatctgcaaccacgcctccctggtgg acatcttcctgatcatgtggctgatccccaagggcaccgtgaccatcgccaagaaggagatcatctggtatcccctgttcggccagctgtac gtgctggccaaccaccagcgcatcgaccgctccaacccctccgccgccatcgagtccatcaaggaggtggcccgcgccgtggtgaagaag aacctgtccctgatcatcttccccgagggcacccgctccaagaccggccgcctgctgcccttcaagaagggcttcatccacatcgccctccag acccgcctgcccatcgtgccgatggtgctgaccggcacccacctggcctggcgcaagaactccctgcgcgtgcgccccgcccccatcaccgt gaagtacttctcccccatcaagaccgacgactgggaggaggagaagatcaaccactacgtggagatgatccacgccctgtacgtggacc acctgcccgagtcccagaagcccctggtgtccaagggccgcgacgcctccggccgctccaactccTGAttaattaactcgagatgtggaga tgtagggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttccca acgccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcg tctggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggaga gcgtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcg [00032]![embedded image]()
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cgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccga gcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgact gctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagaccccaacggcatgggccacgtcgccgac cacctgcacaacaactccttcctgacggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggcc gcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagt tcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccct gtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcgg agttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatga acatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcg tcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaa cgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatcccc gccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctgg acaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttc gactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggc gtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtcca agaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggc atcgcgttctaccgcctgcgcccctcctccTGAtacaacttattacgtattctgaccggcgctgatgtggcgcggacgccgtcgtac tctttcagactttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgcaattaattgtgtgatgaagaaaggg tggcacaagatggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctggctcaatcttgtcgcatgt ccggcgcaatgtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactgatcgcattgcc atcccgtcaactcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaagcgt caggaaatcgtctcggcagctggaagcgcatggaatgcggagcggagatcgaatcagatatcAAGCTCCATCgagctccagc cacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgaggg ccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctacgccaacatga tgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaacctcttcgccga cttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaag gtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgcc gagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagg gagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccg cgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcaca tcaaagggcccctccgccagagaagaagctcctttcccagcagactcctgaagagcgtttaaac.
[0155] The sequence for all of the other acyltransferase constructs are identical to that of pSZEX61 with the exception of the encoded acyltransferase. The acyltransferase sequence alone is provided below for the remaining acyltransferase constructs.
TABLE-US-00034 SEQIDNO:98CpauLPAAT1 ggtaccATGgccatccccgccgccgccgtgatcttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccagg ccctgtgcttcgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgagc tgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagca cgccctggtgatcatcaaccacatgaccgagctggactggatgctgggctgggtgatgggccagcacctgggctgcctgggctcc atcctgtccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacatcgagcgct cctgggccaaggaccgcaccaccctgaagtcccacatcgagcgcctgaccgactaccccctgcccttctggatggtgatcttcgtg gagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtg ctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggccttcc ccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcgtgctgcacgtgcacatcaagcgccacgccat gaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaag cacaacgccgaggacaccttctccggccaggaggtgcaccgcaccggctcccgccccatcaagtccctgctggtggtgatctcct gggtggtggtgatcaccttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggccttctccgtgatcggcctgggc atcgtgaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctcctccaaccccgccaaggtggcccaggccaagc tgaagaccgagctgtccatctccaagaaggccaccgacaaggagaacTGActcgag SEQIDNO:99CprocLPAAT1 ggtacc![custom-character]()
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ctcgag SEQIDNO:100CpaiLPAAT1 ggtaccATGgccatcccctccgccgccgtggtgacctgacggcctgctgacttcacctccggcctgatcatcaacctgaccagg ccactgcacgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgacgccgagctgctgcccctggagtt cctgtggctgaccactggtgcgccggcgccaagctgaagctgacaccgaccccgagaccaccgcctgatgggcaaggagcac gccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctcca tcctgtccgtggccaagaagtccaccaagacctgcccgtgacggctggtccctgtggactccggctacctgacctggagcgctcc tgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgccataggctgatcatcacgtgga gggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgct gatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccttcccc aagacctcccccccccccaccatgctgaagctgacgagggccagtccgtggagctgcacgtgcacatcaagcgccacgccatg aaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaagc acaactccgaggacaccactccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtgatctcctgggt ggtggtgatcatcacggcgccctgaagacctgctgtggtcctccctgctgtcctcctggaagggcaaggccactccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgagggctccaaccccgtgaaggc cgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:101ChookLPAAT1 ggtaccATGgccatcccctccgccgccgtggtgacctgacggcctgctgacttcacctccggcctgatcatcaacctgaccagg ccactgatcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgacgccgagctgctgcccctggagtt cctgtggctgaccactggtgcgccggcgccaagctgaagctgacaccgaccccgagaccaccgcctgatgggcaaggagcac gccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctcca tcctgtccgtggccaagaagtccaccaagacctgcccgtgacggctggtccctgtggactccgagtacctgacctggagcgctcc tgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgccataggctgatcatcacgtgga gggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgct gatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccttcccc aagacctcccccccccccaccatgctgaagctgacgagggccagtccgtggagctgcacgtgcacatcaagcgccacgccatg aaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaagc acaactccgaggacaccactccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtgatctcctgggt ggtggtgatcatcacggcgccctgaagacctgctgtggtcctccctgctgtcctcctggaagggcaaggccactccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgagggctccaaccccgtgaaggc cgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:102CignLPAAT1 ggtaccATGgccatcgccgccgccgccgtgatcacctgacggcctgctgacttcgcctccggcatcatcatcaacctgaccag gccctgtgcttcgtgctgatctggcccctgtccaagaacgtgtaccgccgcatcaaccgcgtgttcgccgagctgctgctgatggac ctgctgtgcctgaccactggtgggccggcgccaagatcaagctgacaccgaccccgagaccaccgcctgatgggcatggagca cgccctggtgatcatgaaccacaagaccgacctggactggatggtgggctggatcctgggccagcacctgggctgcctgggctc catcctgtccatcgccaagaagtccaccaagacatccccgtgctgggctggtccgtgtggactccgagtacctgacctggagcgc tcctgggccaaggacaagtccaccctgaagtcccacatggagaagctgaaggactaccccctgccataggctggtgatcacgt ggagggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgt gctgatcccccacaccaagggcttcgtgtcctgcgtgtccaacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggcctt ccccaagtcctcccccccccccaccatgctgaagctgacgagggccagtccatcgtgctgcacgtgcacatcaagcgccacgcc ctgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaa gcacaacgccgaggacaccactccggccaggaggtgcaccacatcggccgccccatcaagtccctgctggtggtgatcgcctg ggtggtggtgatcatcacggcgccctgaagacctgcagtggtcctccctgctgtccacctggaagggcaaggccactccgtgatc ggcctgggcatcgccaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctccaaccccgccaaggtggccaag TGActcgag SEQIDNO:103CavigLPAAT1 ggtaccATGaccatcgcctccgccgccgtggtgttcctgttcggcatcctgctgttcacctccggcctgatcatcaacctgttccag gccttctgctccgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagttcctgcccctggag ttcctgtggctgttccactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagc acgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctc catcctgtccgtggccaagaagtccaccaagacctgcccgtgttcggctggtccctgtggttctccgagtacctgttcctggagcgc aactgggccaaggacaagaagaccctgaagtcccacatcgagcgcctgaaggactaccccctgcccttctggctgatcatcttcg tggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctccgccggcctgcccgtgccccgcaac gtgctgatcccccacaccaagggatcgtgtcctccgtgtcccacatgcgctccacgtgcccgccatctacgacgtgaccgtggcct tccccaagacctcccccccccccaccatgctgaagctgttcgagggccacttcgtggagctgcacgtgcacatcaagcgccacgc catgaaggacctgcccgagtccgaggacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggac aagcacaacgccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaagtccctgctggtggtgatctcc tgggtggtggtgatcatcttcggcgccctgaagttcctgcagtggtcctccctgctgtcctcctggaagggcatcgccttctccgtgat cggcctgggcaccgtggccctgctgatgcagatcctgatcctgtcctcccaggccgagcgctccatccccgccaaggagaccccc gccaacctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:104CavigLPAAT2 ggtaccATGgccatcgccgccgccgccgtgatcgtgcccgtgtccctgctgttcttcgtgtccggcctgatcgtgaacctggtgca ggccgtgtgcttcgtgctgatccgccccctgttcaagaacacctaccgccgcatcaaccgcgtggtggccgagctgctgtggctgg agctggtgtggctgatcgactggtgggccggcgtgaagatcaaggtgttcaccgaccacgagaccttccacctgatgggcaagg agcacgccctggtgatctgcaaccacaagtccgacatcgactggctggtgggctgggtgctggcccagcgctccggctgcctggg ctccaccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggactccgagtacctgttcctggag cgcaactgggccaaggacgagtccaccctgaagtccggcctgaaccgcctgaaggactaccccctgcccttctggctggccctgt tcgtggagggcacccgcttcacccgcgccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgca acgtgctgatcccccgcaccaagggatcgtgtcctccgtgtcccacatgcgctcatcgtgcccgccatctacgacgtgaccgtgg ccatccccaagacctcccccccccccaccctgctgcgcatgttcaagggccagtcctccgtgctgcacgtgcacctgaagcgcca ccagatgaacgacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacatcttcgtggagaaggacgccctgctgg acaagcacaacgccgaggacaccttctccggccaggagctgcaggacaccggccgccccatcaagtccctgctgatcgtgatct cctgggccgtgctggtggtgttcggcgccgtgaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggccttctccgg catcggcctgggcgtgatcaccctgctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggccc ccgccaagcccaagatcgagggcgagtcctccaagaccgagatggagaaggagcacTGActcgag SEQIDNO:105CpalLPAAT1 ggtaccATGgccatcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcgtgtccggcctgatcgtgaacctggtgca ggccgtgtgcttcgtgctgatccgccccctgtccaagaacacctaccgccgcatcaaccgcgtggtggccgagctgctgtggctgg agctggtgtggctgatcgactggtgggccggcgtgaagatcaaggtgttcaccgaccacgagaccctgtccctgatgggcaagg agcacgccctggtgatctgcaaccacaagtccgacatcgactggctggtgggctgggtgctggcccagcgctccggctgcctggg ctccaccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgcccgagtcc gacgacgccgtggcccagtggtgccgcgacatcttcgtggagaaggacgccctgctggacaagcacaacgccgaggacacctt ctccggccaggagctgcaggacaccggccgccccatcaagtccctgctggtggtgatctcctgggccgtgctggtgatcttcggcg ccgtgaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggccttctccggcgtgggcctgggcatcatcaccctgct gatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggcccccgccaagcccaagaaggacggcga gtcctccaagaccgagatcgagaaggagaacgttcctggagcgctcctgggccaaggacgagaacaccctgaagtccggcct gaaccgcctgaaggactaccccctgcccttctggctggccctgttcgtggagggcacccgcttcacccgcgccaagctgctggcc gcccagcagtacgccacctcctccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctccgtgtc ccacatgcgctcatcgtgcccgccatctacgacgtgaccgtggccatccccaagacctcccccccccccaccatgctgcgcatgtt caagggccagtcctccgtgctgcacgtgcacctgaagcgccacctgatgaaggacctTGActcgag SEQIDNO:106CuPSR23LPAAT2 ggtaccATGgccatcgccgccgccgccgtgatcacctgttcggcctgatatatcgcctccggcctgatcatcaacctgttccag gccctgtgcttcgtgctgatccgccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgag ctgctgtgcctgacgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccaccgcctgatgggcaaggagc acgccctggtgatcatcaaccacatgaccgagctggactggatggtgggctgggtgatgggccagcacttcggctgcctgggctc catcatctccgtggccaagaagtccaccaagacctgcccgtgctgggctggtccatgtggactccgagtacctgtacctggagcg ctcctgggccaaggacaagtccaccctgaagtcccacatcgagcgcctgatcgactaccccctgcccactggctggtgatcacgt ggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgtgtcctccggcctgcccgtgccccgcaacgt gctgatcccccgcaccaagggcacgtgtcctgcgtgtcccacatgcgctccacgtgcccgccgtgtacgacgtgaccgtggccac cccaagacctcccccccccccaccctgctgaacctgacgagggccagtccatcatgctgcacgtgcacatcaagcgccacgcca tgaaggacctgcccgagtccgacgacgccgtggccgagtggtgccgcgacaagacgtggagaaggacgccctgctggacaa gcacaacgccgaggacaccactccggccaggaggtgtgccactccggctcccgccagctgaagtccctgctggtggtgatctcc tgggtggtggtgaccaccttcggcgccctgaagacctgcagtggtcctcctggaagggcaaggccactccgccatcggcctggg catcgtgaccctgctgatgcacgtgctgatcctgtcctcccaggccgagcgctccaaccccgccgaggtggcccaggccaagctg aagaccggcctgtccatctccaagaaggtgaccgacaaggagaacTGActcgag SEQIDNO:107CkoeLPAAT1 ggtaccATGgccatccccgccgccgtggccgtgatccccatcggcctgctgacatcatctccggcctgatcgtgaacctgatcca ggccgtggtgtacgtgctgatccgccccctgtccaagaacctgcaccgcaagatcaacaagcccatcgccgagctgctgtggctg gagctgatctggctggtggactggtgggccggcatcaaggtggaggtgtacgccgactcccagaccctggagctgatgggcaag gagcacgccctgctgatctgcaaccaccgctccgacatcgactggctggtgggctgggtgctggcccagcgcgcccgctgcctgg gctccgccctggccatcatgaagaagtccgccaagttcctgcccgtgatcggctggtccatgtggactccgactacatcacctgga ccgcacctgggccaaggacgagaagaccctgaagtccggatcgagcgcctggccgacttccccatgccatctggctggccctg acgtggagggcacccgatcaccaaggccaagctgctggccgcccaggagtacgccgcctcccgcggcctgcccgtgccccag aacgtgctgatcccccgcaccaagggatcgtgaccgccgtgacccacatgcgctcctacgtgcccgccatctacgactgcaccg tggacatctccaaggcccaccccgccccctccatcctgcgcctgatccgcggccagtcctccgtggtgaaggtgcagatcacccg ccactccatgcaggagctgcccgagaccgccgacggcatctcccagtggtgcatggacctgttcgtgaccaaggacggcacctg gagaagtaccactccaaggacatcacggctccctgcccgtgcagaacatcggccgccccgtgaagtccctgatcgtggtgctgtg ctggtactgcctgatggccacggcctgacaagacttcatgtggtcctccctgctgtcctcctgggagggcatcctgtccctgggcctg atcctgctggccgtggccatcgtgatgcagatcctgatccagtccaccgagtccgagcgctccacccccgtgaagtccatccaga aggacccctccaaggagaccctgctgcagaacTGActcgag SEQIDNO:108CkoeLPAAT2 ggtaccATGcacgtgctgctggagatggtgaccaccgcactcctccacttcgtgacgacaacgtgcaggccctgtgatcgtgct gatctggcccctgtccaagtccgcctaccgcaagatcaaccgcgtgacgccgagctgctgctgtccgagctgctgtgcctgacga ctggtgggccggcgccaagctgaagctgacaccgaccccgagaccaccgcctgatgggcaaggagcacgccctggtgatcac caaccacaagatcgacctggactggatgatcggctggatcctgggccagcacttcggctgcctgggctccgtgatctccatcgcca agaagtccaccaagacctgcccatcacggctggtccctgtggactccgagtacctgacctggagcgcaactgggccaaggaca agcgcaccctgaagtcccacatcgagcgcatgaaggactaccccctgcccctgtggctgatcctgacgtggagggcacccgat cacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgctgatcccccacac caagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggccttccccaagacctcccc cccccccaccatgctgtccctgacgagggccagtccgtggtgctgcacgtgcacatcaagcgccacgccatgaaggacctgccc gactccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaagcacaacgccgagg acaccactccggccaggaggtgcaccacgtgggccgccccatcaagtccctgctggtggtgatctcctggatggtggtgatcatct tcggcgccctgaagacctgcagtggtcctccctgctgtcctcctggaagggcaaggccactccgccatcggcctgggcatcgcca ccctgctgatgcacgtgctggtggtgactcccaggccgaccgctccaaccccgccaaggtgccccccgccaagctgaacaccga gctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:109CprocLPAAT2 ggtaccATGgccatccccgccgccgtggccgtgatccccatcggcctgctgacatcatctccggcctgatcgtgaacctgatcca ggccgtggtgtacgtgctgatccgccccctgtccaagaacctgtaccgcaagatcaacaagcccatcgccgagctgctgtggctg gagctgatctggctggtggactggtgggccggcatcaaggtggaggtgtacgccgactccgagaccctggagtccatgggcaag gagcacgccctgctgatctgcaaccaccgctccgacatcgactggctggtgggctgggtgctggcccagcgcgcccgctgcctgg gctccgccctggccatcatgaagaagtccgccaagttcctgcccgtgatcggctggtccatgtggttctccgactacatcttcctgga ccgcacctgggagaaggacgagaagaccctgaagtccggcttcgagcgcctggccgacttccccatgcccttctggctggccct gttcgtggagggcacccgcttcaccaaggccaagctgctggccgcccaggagttcgccgcctcccgcggcctgcccgtgcccca gaacgtgctgatcccccgcaccaagggcttcgtgaccgccgtgacccacatgcgctcctacgtgcccgccatctacgactgcacc gtggacatctccaaggcccaccccgccccctccatcctgcgcctgatccgcggccagtcctccgtggtgaaggtgcagatcaccc gccactccatgcaggagctgcccgagacccccgacggcatctcccagtggtgcatggacctgttcgtgaccaaggacgccttcct ggagaagtaccactccaaggacatcttcggctccctgcccgtgcacgacatcggccgccccgtgaagtccctgatcgtggtgctgt gctggtactccctgatggccttcggcactacaagttcttcatgtggtcctccctgctgtcctcctgggagggcatcctgtccctgggcct ggtgctgatcgtgatcgccatcgtgatgcagatcctgatccagtcctccgagtccgagcgctccacccccgtgaagtccgtgcaga aggacccctccaaggagaccctgctgcagaacTGActcgag SEQIDNO:110CavigGPAT9 ggtaccATGgccaccggcggctccctgaagccctcctcctccgacctggacctggaccaccccaacatcgaggactacctgcc ctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccgc cggcgccatcgtggacgactccttcacccgctgatcaagtccatcccccgcgagccctggaactggaacctgtacctgttccccct gtggtgcatcggcgtgctgatccgctacttcatcctgttccccggccgcgtgatcgtgctgaccatgggctggatcaccgtgatctcct catcatcgccgtgcgcgtgctgctgaagggccacgacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tcatcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttcttcatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctgc tgcagtccaccctgctggagtccgtgggctgcatctggacgaccgcgccgaggccaaggaccgcggcatcgtggccaagaagc tgtgggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaacaactactccgtga tgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgccttctgg aactccaagaagcagtccttcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagcc ccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgcccgcgccggcctgaaga aggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaccttcgccgagtcc gtgctgcagcgcctggaggagTGActcgag SEQIDNO:111ChookGPAT9-1 ggtaccATGgccaccgccggctccctgaagccctcccgctccgagctggacttcgaccgccccaacatcgaggactacctgcc ctccggctcctccatcatcgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccgcc ggcgccatcgtggacgactccttcacccgctgatcaagtccaacccccccgagccctggaactggaacatctacctgttccccct gtggtgcttcggcgtgctgatccgctacctgatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcatcttcctgtcctc cttcatccccgtgcacctgctgctgaagggccacgacgccctgcgcatcaagctggagcgcctgctggtggagctgatctgctcat cttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaaccac acctccatgatcgacttcttcatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctgctg cagtccaccctgctggagtccgtgggctgcatctggttcgaccgcgccgaggccaaggaccgcggcatcgtggccaagaagctg tgggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaacaactactccgtgatg ttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgccttctggaa ctccaagaagcagtccttcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagcccc agaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctgaagaag gtgccctgggacggctacctgaagtactcccgcccctcccccaagcacaccgagcgcaagcagcagaacttcgccgagtccgt gctgcagcgcctggagaagaagTGActcgag SEQIDNO:112CignGPAT9-1 ggtaccATGgccaccggcggccgcctgaagccctcctcctccgagctggacctggaccgcgccaacaccgaggactacctgc cctccggctcctccatcaacgagcccgtgggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccg ccggcgccatcgtggacgactccttcacccgctgcttcaagtccatcccccccgagccctggaactggaacatctacctgttccccc tgtggtgatcggcgtgctgatccgctacttcatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcaccgtgatctcct catcaccgccgtgcgcacctgctgaagggccacaacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tcatcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacacctgatcctggaccagatgaccgtgactccgtgatcatgcagaagcaccccggctgggtgggcctg ctgcagtccaccctgctggagtccgtgggctgcatctggacaaccgcgccgaggccaaggaccgcgagatcgtggccaagaag ctgtgggaccacgtgcacggcgagggcaacaaccccctgctgatcaccccgagggcacctgcgtgaacaaccactactccgtg atgacaagaagggcgccacgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcacgtggacgccactg gaactcccgcaagcagtccacaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacaggagc cccagaccctgaagcccggcgagaccgccatcgagacgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctgaag aaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagtccaagcagcagtccacgccgagtcc gtgctgcgccgcctggaggagaagTGActcgag SEQIDNO:113CignGPAT9-2 ggtaccATGgccaccggcggccgcctgaagccctcctcctccgagctggacctggaccgcgccaacaccgaggactacctgc cctccggctcctccatcaacgagcccgtgggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccg ccggcgccatcgtggacgactccacacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgaccccc tgtggtgatcggcgtgctgatccgctacttcatcctgaccccgcccgcgtgatcgtgctgaccatcggctggatcaccgtgatctcct catcaccgccgtgcgcacctgctgaagggccacaacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tccacgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacacctgatcctggaccagatgaccgtgactccgtgatcatgcagaagcaccccggctgggtgggcctg ctgcagtccaccctgctggagtccgtgggctgcatctggacaaccgcgccgaggccaaggaccgcgagatcgtggccaagaag ctgtgggaccacgtgcacggcgagggcaacaaccccctgctgatcaccccgagggcacctgcgtgaacaaccactactccgtg atgacaagaagggcgccacgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcacgtggacgccactg gaactccaagaagcactccacacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacaggagc cccagaccctgaagcccggcgagacccccatcgagacgccgagcgcgtgcgcgacatcatctccgtgcgcgccgacctgaag aaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaagacgccgagtc cgtgctgcgccgcctggaggagaagTGActcgag SEQIDNO:114CpalGPAT9-1 ggtaccATGgccaccgccggccgcctgaagccctcctcctccgagctggagctggacctggaccgccccaacatcgaggact acctgccctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccatgctgaccga ggccgccggcgccatcgtggacgactccacacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgt tccccctgtggtgatcggcgtgctgatccgctacctgatcctgaccccgcccgcgtgatcgtgctgaccgtgggctggatcaccgtg atctcctccacatcaccgtgcgcacctgctgaagggccacgactccctgcgcatcaagctggagcgcctgatcgtgcagctgttct gctcctccacgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgcccccagcaggtgtacgtggcc aaccacacctccatgatcgacttcatcatcctgaaccagatgaccgtgactccgccatcatgcagaagcaccccggctgggtggg cctgatccagtccaccatcctggagtccgtgggctgcatctggacaaccgcgccgaggccaaggaccgcgagatcgtggccaa gaagctgctggaccacgtgcacggcgagggcaacaaccccctgctgatcaccccgagggcacctgcgtgaacaaccactactc cgtgatgacaagaagggcgccacgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcacgtggacgcct tctggaactccaagaagcagtccacaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacagg agccccagaccctgaagcccggcgagacccccatcgagacgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctg aagaaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagtccacgccga gtccgtgctgcgccgcctggagaagcgcTGActcgag SEQIDNO:115CpalGPATt9-2 ggtaccATGgccaccgccggccgcctgaagccctcctcctccgagctggagctggacctggaccgccccaacatcgaggact acctgccctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccatgctgaccga ggccgccggcgccatcgtggacgactccacacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgt tccccctgtggtgatcggcgtgctgatccgctacctgatcctgaccccgcccgcgtgatcgtgctgaccgtgggctggatcaccgtg atctcctccacatcaccgtgcgcacctgctgaagggccacgactccctgcgcatcaagctggagcgcctgatcgtgcagctgttct gctcctccacgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgcccccagcaggtgtacgtggcc aaccacacctccatgatcgacttcatcatcctgaaccagatgaccgtgactccgccatcatgcagaagcaccccggctgggtggg cctgatccagtccaccatcctggagtccgtgggctgcatctggacaaccgcgccgaggccaaggaccgcgagatcgtggccaa gaagctgctggaccacgtgcacggcgagggcaacaaccccctgctgatcaccccgagggcacctgcgtgaacaaccactactc cgtgatgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgcct tctggaactccaagaagctgtcatcaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttgg agccccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctg aagaaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaccttcgccg agtccgtgctgcgccgcctggaggagaagggcaacgtggtgcccaccgtgaacTGActcgag SEQIDNO:116CavigDGAT1 ggtaccATGgccatcgccgacggcggcatcatcggcgccgccggctccatctccgccctgaccgccgacaccgaccccccct ccctgcgccgccgcaacgtgcccgccggccaggcctccgccgtgtccgccttctccaccgagtccatggccaagcacctgtgcga cccctcccgcgagccctccccctcccccaagtcctccgacgacggcaaggaccccgacatcggctccgtggactccctgaacga gaagccctcctcccccgccgccggcaagggccgcctgcagcacgacctgcgatcacctaccgcgcctcctcccccgcccaccg caaggtgaaggagtcccccctgtcctcctccaacatcttcaagcagtcccacgccggcctgttcaacctgtgcgtggtggtgctggt ggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggcctgctgatcaagaccggcttctggttctcctcccgctccct gcgcgactggcccctgttcatgtgctgcctgtccctgcccatcttccccctggccgccttcctggtggagaagctggcccagaagaa ccgcctgcaggagcccaccgtggtgtgctgccacgtgctgatcacctccgtgtccatcctgtaccccgtgctggtgatcctgcgctg cgactccgccgtgctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtcctacgcccactccaactac gacatgcgctacgtggccaagtccctggacaagggcgagcccgtggtggactccgtgatcgccgaccacccctaccgcgtgga ctacaaggacctggtgtacttcatggtggcccccaccctgtgctaccagctgtcctaccccctgaccccctgcgtgcgcaagtcctg gatcgcccgccaggtgatgaagctggtgctgttcaccggcgtgatgggcttcatcgtggagcagtacatcaaccccatcgtgcag aactccaagcaccccctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaacctgtacgtgtggc tgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgatctgcttcggcgaccgcgagttctacaaggactgg tggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgccacatctacttcccct gcctgcgcaacggcatcccccgcggcgtggccgtgctgatcgccttcctggtgtccgccgtgttccacgagctgtgcatcgccgtgc cctgccacgtgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctggtgtccaactgcctgcagaagaagtt ccagtcctccatggccggcaacatgttatctggttcatcttctgcatcttcggccagcccatgtgcgtgctgctgtactaccacgacct gatgaaccgcaagggctcccgcatcgacTGActcgag SEQIDNO:117ChookDGAT1-1 ggtaccATGgccatcgccgacggcggctccgccggcgccgccggctccatctccggctccgacccctccccctccaccgcccc ctccctgcgccgccgcaacgcctccgccggccaggccttctccaccgagtccatggcccgcgacctgtgcgacccctcccgcga gccctccctgtcccccaagtcctccgacgacggcaaggaccccgccgacgacatcggcgccgccgactccgtggactccggcg gcgtgaaggacgagaagccctcctcccaggccgccgccaaggcccgcctggagcacgacctgcgatcacctaccgcgcctcc tcccccgcccaccgcaaggtgaaggagtcccccctgtcctcctccaacatcttcaagcagtcccacgccggcctgttcaacctgtg cgtggtggtgctggtggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggcctgctgatcaagaccggcttctggtt ctcctcccgctccctgcgcgactggcccctgttcatgtgctgcctgtccctgcccatcaccccctggccgccttcctggtggagaagc tggcccagaagaaccgcctgcaggagcccaccgtggtgtgctgccacgtgatcatcacctccgtgtccatcctgtaccccgtgctg gtgatcctgcgctgcgactccgccgtgctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtcctacg cccacgccaactacgacatgcgctccgtggccaagtccctggacaagggcgagaccgtggccgactccgtgatcgtggaccac ccctaccgcgtggactacaaggacctggtgtacttcatggtggcccccaccctgtgctaccagctgtcctaccccctgaccccctac gtgcgcaagtcctgggtggcccgccaggtgatgaagctggtgctgttcaccggcgtgatgggcttcatcgtggagcagtacatcaa ccccatcgtgcagaactccaagcaccccctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaa cctgtacgtgtggctgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgacctgcttcggcgaccgcgagt tctacaaggactggtggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgc cacatctacttcccctgcctgcgcaacggcatcccccgcggcgtggccgtgctgatcgccttcctggtgtccgccgtgttccacgag ctgtgcatcgccgtgccctgccacgtgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctggtgtccaactg cctgcagaagaagttccagtcctccatggccggcaacatgttatctggttcatcttctgcatcttcggccagcccatgtgcgtgctgct gtactaccacgacctgatgaaccgcaagggctcccgcatcgacTGActcgag SEQIDNO:118CavigLPCAT ggtaccATGggcctggtgtccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctggccaccat ccccgtgtccttcctgtggcgcctggtgcccggccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgtcctacct gtcatcggcgcctcctccaacctgcacttcatcgtgcccatgaccctgggctacctgtccatgctgttcttccgcccatctccggcct gctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcggcatcg acgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatgaactacaacgacggcctgctgaaggaggagg gcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacttcggctactgcctgtgctgcggctc ccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcatctggtcccgctcccagaagg agcccaagccctccccatcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgtacctgtacctggtgccc caccaccccctgacccgcttcaccgagcccgtgtactacgagtggggatcttccgccgcctgtcctaccagtacatggccgccctg accgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggaccgagt cctccccccccaagccccgctgggaccgcgccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgcagctgc ccctggtgtggaacatccaggtgtccatctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccccggctt caccagctgctggccacccagaccgtgtccgccgtgtggcacggcctgtaccccggctacatcatatatcgtgcagtccgccctg atgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccccaagatgggcctggtgaagaacatcttcgtgttctt caacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcctacgg ctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccgcccgctccaa ggcccacaaggagcagTGActcgag SEQIDNO:119CpalLPCAT ggtaccATGgagctgggctccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctggccaccat ccccgtgtccttcctgtggcgcctggtgcccggccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgtcctacct gtcatcggcccctcctccaacctgcacttcatcgtgcccatgaccctgggctacctgtccatgctgttcttccgcccatctccggcct gctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcggcatcg acgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggaggagg gcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacatcggctactgcctgtgctgcggctc ccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcgtgtggtcccactccgagaagg agcccaagccctccccatcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgtacatgtacctggtgccc caccaccccctgtcccgatcaccgagcccgtgtactacgagtggggcacttccgccgcctgtcctaccagtacatggccggcctg accgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggaccgagt cctccccccccaagccccgctgggaccgcgccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgcagctgc ccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccccggctt caccagctgctggccacccagaccgtgtccgccatctggcacggcctgtaccccggctacatcatatatcgtgcagtccgccctg atgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccccaagatgggcctggtgaagaacatcttcgtgttctt caacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcctacgg ctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccgcccgctccaa ggcccacaaggagcagTGActcgag SEQIDNO:120CpauLPCAT ggtaccATGgagctggagatcggctccgtggccgccgccatcggcgtgtccgtgcccgtggcccgatcctgctgtgatcagg ccaccatccccgtgtccttcctgtgccgcctgctgcccgcccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgt cctacctgtcatcggcccctcctccaacctgcacttcatcgtgcccatgtccctgggctacctgtccatgctgttcttccgccccttctcc ggcctgctgaccttcttcagggatcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcgg catcgacgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggag gagggcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacttcggctactgcctgtgctgcg gctcccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcatctggtcccgctccgaga aggaccccaagccctccccatcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgcacatgtacctggt gccccaccaccccctgacccgcttcaccgagcccgtgtactacgagtggggatcttccgccgcctgtcctaccagtacatggccg cccagaccgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggac cgagtcctccccccccaagccccgctgggacaaggccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgca gctgcccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccc cggcttcttccagctgctggccacccagaccgtgtccgccgtgtggcacggcctgtaccccggctacatcatcttcttcgtgcagtcc gccctgatgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccagaagatgggcctggtgaagaacatcttcg tgttcttcaacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcc tacggctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccacccg ctccaaggtgcacaaggagcagTGActcgag SEQIDNO:121CschuLPCAT ggtaccATGgagctggagatggagcccctggccgccgccatcggcgtgtccgtggccgtgttccgcttcctggtgtgcttcatcg ccaccatccccgtgtccttcatctgccgcctggtgcccggcggcctgccccgccacctgttctccgccgcctccggcgccgtgctgtc ctacctgtcatcggatctcctccaacctgcacttcctggtgcccatgaccctgggctacctgtccatgatcctgttccgccgatctgc ggcatcctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcgg catcgacgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggag gagggcctgcgcgagtcccagaagaagaaccgcctgatccgcctgccctccctgatcgagtacttcggctactgcctgtgctgcg gctcccacttcgccggccccgtgtacgagatgaaggactacctggactggaccgagggcaagggcatctggtcccactccgaga agggccccaagccctcccccctgcgcgccgccctgcgcgccatcatccaggccggcttctgcatggccatgtacctgtacctggtg ccccactaccccctgacccgcttcaccgaccccgtgtactacgagtggggcatcctgcgccgcctgtcctaccagtacatggcctc cttcaccgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggacc gagtcctccccccccaagccccgctgggaccgcgccaagaacgtggacatcctgggcgtggagctggccaagtcctccgtgca gatccccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccc cggatcctgcagctgctggccacccagaccgtgtccgccatctggcacggcgtgtaccccggctacctgatcacttcgtgcagtcc gccctgatgatcgccggctcccgcgccatctaccgctggcagcaggccgtgccccccaagatgtccctggtgaagaacaccctg gtgttcttcaacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctc ctacggctccgtgtactacgtgggcaccatcctgcccgtgaccctgatcctgctgggctacgtgatcaagcccggcaagtcccccc gctccaaggcctccaaggagcagTGActcgag SEQIDNO:122CavigPLA2-1 ggtaccATGaacttcgacttcctgtccaacatcccctggttcggcgccaaggcctccgacaacgccggctcctcatcggctccg ccaccatcgtgatccagcagcccccccccgtgtcccgcggcttcgacatccgccactggggctggccctggtccgtgctgtccgtg ctgccctggggcaagcccggctgcgacgagctgcgcgccccccccaccaccatcaaccgccgcctgaagcgcaacgccacct ccatgcactcctccgccgtgcgcggcaacgccgaggccgcccgcgtgcgcttccgcccctacgtgtccaaggtgccctggcaca ccggcttccgcggcctgctgtcccagctgttcccccgctacggccactactgcggccccaactggtcctccggcaagaacggcgg ctcccccgtgtgggaccagcgccccatcgactggctggactactgctgctactgccacgacatcggctacgacacccacgacca ggccaagctgctggaggccgacctggccttcctggagtgcctggagcgcccctcctaccccaccaagggcgacgcccacgtgg cccacatgtacaagaccatgtgcgtgaccggcctgcgcaacgtgctgatcccctaccgcacccagctgctgcgcctgaactcccg ccagcccctgatcgacttcggctggctgtccaacgccgcctggaagggctggaacgcccagaagtccTGActcgag SEQIDNO:123CignPLA2-1 ggtaccATGaacctggacttcctgtccaagatcccctggttcgaggccaaggcctccgagaaccccggcctgaacctgggctcc accaccatcgtgatcaagcagccccgccagggcttcgacatccgccactggggctggccctggtccgtgctgacctggggcaac cgcgtgaccgacgaggtgcacgccccccccaccaccatcaaccgccgcctgaagcgcaacgccaccggccccgccgtgcag ggcgacaccgaggccgcccgcctgcgcttccgcccctacgtgtccaaggtgccctggcacaccggcttccgcggcctgctgtccc agctgttcccccgctacggccactactgcggccccaactggtcctccggcaagaacggcggctcccccgtgtgggaccagcgcc ccatcgactggctggactactgctgctactgccacgacatcggctacgacacccacgaccaggccaagctgctggaggccgacc tggccttcctggagtgcctggagcgcccctcctaccccaccaccggcgacgcccacgtggcccacatgtacaagaccatgtgcgt gaccggcctgcgcaacgtgctgatcccctaccgcacccagctgctgcgcctgaacttccgccagcccctgatcgacttcggctggc tgtccaacgccgcctggaagggctggtccgcccagaagaccTGActcgag SEQIDNO:124CuPSR23PLA2-2 ggtaccATGgtgcacctgccccacaccctgaagctgggcctggtgatcgccatctccatctccggcctgtgcactcctccacccc cgcccgcgccctgaacgtgggcatccaggccgccggcgtgaccgtgtccgtgggcaagggctgctcccgcaagtgcgagtccg acactgcaaggtgccccccacctgcgctacggcaagtactgcggcctgatgtactccggctgccccggcgagaagccctgcgac ggcctggacgcctgctgcatgaagcacgacgcctgcgtgcaggccaagaacaacgactacctgtcccaggagtgctcccagaa cctgctgaactgcatggcctccaccgcatgtccggcggcaagcagacaagggctccacctgccaggtggacgaggtggtggac gtgctgaccgtggtgatggaggccgccctgctggccggccgctacctgcacaagcccTGActcgag SEQIDNO:125CprocPLA2-2 ggtaccATGgtgcacctgccccacaccctgaagctgggcctggtgatcgccatctccatctccggcctgtgcctgtcctccacccc cgcccgcgccctgaacgtgggcatccaggccgccggcgtgaccgtgtccgtgggcaagggctgctcccgcaagtgcgagtccg acactgcaaggtgccccccacctgcgctacggcaagtactgcggcctgatgtactccggctgccccggcgagaagccctgcgac ggcctggacgcctgctgcatgaagcacgacgcctgcgtgcaggccaagaacgacgactacctgtcccaggagtgctcccagaa cctgctgaactgcatggcctccaccgcatgtccggcggcaagcagacaagggctccacctgccaggtggacgaggtggtggac gtgctgaccgtggtgatggaggccgccctgctggccggccgctacctgcacaagcccTGActcgag
[0156] The constructs containing the codon optimized genes described above driven by the UTEX 1453 SAD2 promoter, were transformed into strain S7858 or S8714. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as described herein. The transgenic strains were selected for their ability to grow on melibiose. Stable transformants were grown under standard lipid production conditions at pH5 (for transgenic strains generated in the strain S7858) or at pH7 (for the transgenic strains generated in the strain S8174) for fatty acid analysis.
Expression of LPAATs
[0157] In WO2013/158938 we disclosed that Cocos nucifera LPAAT enzymes exhibit chain length specificity for the fatty acid acyl-CoA that it attach to the glycerol backbone. We disclosed the impact of expressing CnLPAAT in a transgenic strain also expressing a laurate specific thioesterase. In this example we transformed 5 LPAAT enzymes derived from C8-C10 rich Cuphea species and the CnLPAAT into S7858, and the remaining 8 LPAAT enzymes were transformed into S8174. The resulting fatty acid profiles from a set of representative transgenic lines arising from these transformations are shown in Tables 16 and 17. Expression of these genes as shown in Table 16 resulted in increases in C8:0 and/or- C10:0 fatty acid accumulation.
TABLE-US-00035 TABLE 16 Fatty acid profiles of representative transgenic strains of S7858 expressing optimized versions of the CpauLPAAT1, CpalLPAAT1, CignLPAAT1, CprocLPAAT1, ChookLPAAT1 and CnLPAAT1. Sample ID C8:0 C10:0 C12:0 C8-C10 S6165 0.00 0.00 0.05 0.00 S7858 11.70 23.36 0.48 35.06 CpauLPAAT1 @ SAD2-1vD locus CprocLPAAT1 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S7858; D4289-7 12.69 25.06 0.51 37.75 S7858; D4292-15 11.86 24.05 0.46 35.91 S7858; D4289-12 11.98 24.54 0.48 36.52 S7858; D4292-11 11.49 24.01 0.48 35.50 S7858; D4289-2 11.68 24.14 0.49 35.82 S7858; D4292-22 11.49 23.81 0.47 35.30 S7858; D4289-13 11.53 24.18 0.49 35.71 S7858; D4292-3 11.46 23.76 0.46 35.22 S7858; D4289-11 11.47 23.85 0.46 35.32 S7858; D4292-24 11.38 23.64 0.46 35.02 CpaiLPAAT1 @ SAD2-1vD locus ChookLPAAT1 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S7858; D4290-3 13.43 25.04 0.52 38.47 S7858; D4293-4 11.09 24.48 0.51 35.57 S7858; D4290-25 12.98 24.75 0.51 37.73 S7858; D4293-16 12.03 24.24 0.48 36.27 S7858; D4290-5 12.27 25.00 0.52 37.27 S7858; D4293-6 11.83 23.79 0.48 35.62 S7858; D4290-12 11.98 24.21 0.48 36.19 S7858; D4293-2 11.81 23.69 0.47 35.50 S7858; D4290-22 11.91 23.86 0.49 35.77 S7858; D4293-12 11.65 23.11 0.49 34.76 CignLPAAT1 @ SAD2-1vD locu CnLPAAT1 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S7858; D4291-13 12.95 24.78 0.52 37.73 S7858; D4404-11 12.30 24.31 0.47 36.61 S7858; D4291-20 12.13 24.63 0.49 36.76 S7858; D4404-6 12.03 24.02 0.46 36.05 S7858; D4291-15 12.12 24.35 0.47 36.47 S7858; D4404-13 11.48 23.98 0.46 35.46 S7858; D4291-22 11.94 24.50 0.47 36.44 S7858; D4404-2 11.54 23.71 0.46 35.25 S7858; D4291-7 12.11 23.14 0.50 35.25 S7858; D4404-1 11.76 23.36 0.48 35.12
TABLE-US-00036 TABLE 17 Fatty acid profiles of representative transgenic strains of S8174 expressing CavigLPAAT1, CavigLPAAT2, CpalLPAAT1, CuPSR23LPAAT1, CkoeLPAAT1, CkoeLPAAT2, CprocLPAAT1 and CprocLPAAT2 before lipase treatment. Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.32 9.24 0.37 33.56 CavigLPAAT1 @ SAD2-1vD locus CkoeLPAAT1 @ SAD2-1vD locus C8- C8- Sample ID C8:0 C10:0 C12:0 C10 Sample ID C8:0 C10:0 C12:0 C10 S8174: D4517-23 25.42 9.63 0.39 35.05 S8174; D4728-8 25.44 10.31 0.46 35.75 S8174: D4517-9 25.44 9.61 0.39 35.05 S8174; D4728-10 24.15 9.51 0.43 33.66 S8174: D4517-8 25.09 9.84 0.39 34.93 S8174; D4728-5 23.88 9.56 0.45 33.44 S8174: D4517-18 25.20 9.65 0.39 34.85 S8174; D4728-6 23.58 9.28 0.40 32.86 S8174: D4517-2 25.20 9.57 0.37 34.77 S8174; D4728-9 23.47 9.25 0.40 32.72 Cavig LPAAT2 @ SAD2-1vD locus CkoeLPAAT2-1 @ SAD2-1vD locus C8- C8- Sample ID C8:0 C10:0 C12:0 C10 Sample ID C8:0 C10:0 C12:0 C10 S8174: D4518-2 24.25 9.97 0.42 34.22 S8174; D4729-2 25.20 9.81 0.43 35.01 S8174: D4518-45 24.09 9.65 0.39 33.74 S8174; D4729-1 23.49 10.60 0.46 34.09 S8174: D4518-34 23.94 9.71 0.38 33.65 S8174; D4729-4 22.25 9.45 0.40 31.70 S8174: D4518-10 24.11 9.50 0.37 33.61 S8174; D4729-5 18.24 8.22 0.35 26.46 S8174: D4518-4 23.93 9.59 0.39 33.52 CpalLPAAT1 @ SAD2-1vD locus CprocLPAAT2 @ SAD2-1vD locus C8- C8- Sample ID C8:0 C10:0 C12:0 C10 Sample ID C8:0 C10:0 C12:0 C10 S8174: D4519-27 25.06 9.75 0.37 34.81 S8174; D4730-14 24.97 9.92 0.41 34.89 S8174: D4519-4 23.05 10.74 0.47 33.79 S8174; D4730-13 23.26 10.72 0.49 33.98 S8174: D4519-28 24.11 9.54 0.37 33.65 S8174; D4730-1 23.79 10.15 0.49 33.94 S8174: D4519-10 23.57 9.51 0.38 33.08 S8174; D4730-7 23.42 10.13 0.36 33.55 S8174: D4519-12 23.55 9.49 0.38 33.04 S8174; D4730-5 23.69 9.49 0.42 33.18 CuPSR23LPAAT2-1 @ SAD2-1vD locus CuPSR23LPAAT4 @ SAD2-1vD locus C8- C8- Sample ID C8:0 C10:0 C12:0 C10 Sample ID C8:0 C10:0 C12:0 C10 S8174; D4690-2 25.88 10.62 0.43 36.50 S8174; D4731-1 25.94 10.87 0.56 36.81 S8174; D4690-1 24.60 9.82 0.44 34.42 S8174; D4731-3 22.79 11.52 0.59 34.31 S8174; D4690-3 24.13 9.62 0.47 33.75 S8174; D4731-5 22.89 11.22 0.53 34.11 S8174; D4690-4 23.38 9.97 0.41 33.35 S8174; D4731-2 22.99 11.07 0.45 34.06 S8174; D4731-4 21.15 9.63 0.43 30.78
[0158] To assess the regiospecific activity of novel LPAAT enzymes, oil extracted from some of these transformants were treated with porcine pancreatic lipase, which selectively hydrolyzes the fatty acids at the sn-1 and sn-3 positions from the glycerol unit of the triacylglycerol, leaving monoacylglycerols (MAGs) with fatty acids located only at the sn-2 position. The resulting mixture of monoacylglycrols (2-MAGs), were isolated by solid phase extraction on an amino propyl cartridge followed by transesterifcation to generate fatty acid methyl esters (FAMEs). The fatty acid profiles of these FAMEs, which represent the profile of fatty acids at the sn-2 position of the various TAGs, were determined by GC-FID. When compared to the fatty acid profiles from transesterification of the oil without lipase treatment, the sn-2 fatty acid profiles show that the expressed LPAAT are selective for the sn-2 position.
[0159] The sn-2 analyses after lipase treatment disclosed in Table 18 show that CavigLPAAT1, CpaiLPAAT exhibit selectivity for either C8:0 fatty acids and CpauLPAAT, CignLPAAT are selective for C10:0 fatty acids, demonstrating that the heterologous LPAATs expressed in these transgenic strains have activities that acylate at the sn-2 position with preference for C8:0 or C10:0.
TABLE-US-00037 TABLE 18 Fatty acid profiles & sn-2 analysis of representative transgenic strains of S7858 & S8174 expressing codon optimized versions of the CnLPAAT1, CpauLPAAT1, CpaiLPAAT1, CignLPAAT1, ChookLPAAT1 and CavigLPAAT1, CavigLPAAT2, CpalLPAAT1 ![text missing or illegible when filed]()
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Expression of GPATs, DGATs, LPCATs and PLA2s:
[0160] The constructs expressing the other acyltransferases (GPAT, DGAT, LPCAT, and PLA2) were transformed into S8174. Stable transformants were grown under standard lipid production conditions at pH7 and analyzed for fatty acid profiles. Similar to the transgenic lines expressing LPAATs, expression of these genes (GPAT, DGAT, LPCAT, and PLA2) also resulted in increases in C8:0-C10:0 fatty acid accumulation (Tables 19a, 19b, and 20). The data presented shows that we have identified novel GPATs, DGATs, LPCATs and PLA2s that show high specificity for C8-C10 fatty acids. To determine the regiospecificity of the novel GPAT, DGAT, LPCAT, and PLA2 enzymes, sn-2 analysis is performed as disclosed in this example and elsewhere herein.
TABLE-US-00038 TABLE 19a Fatty acid profiles of representative transgenic strains of S8174 expressing GPATs and DGATs Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.61 9.10 0.42 33.71 CavigGPAT9 @ SAD2-1vD locus CignGPAT9-2 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4551-8 24.52 9.05 0.36 33.57 S8174; D4554-9 24.49 9.13 0.45 33.62 S8174; D4551-7 24.24 9.04 0.36 33.28 S8174; D4554-3 24.28 8.90 0.42 33.18 S8174; D4551-2 23.93 8.92 0.37 32.85 S8174; D4554-7 23.86 8.96 0.43 32.82 S8174; D4551-6 23.63 8.92 0.41 32.55 S8174; D4554-8 23.99 8.81 0.39 32.80 S8174; D4551-3 23.35 8.90 0.43 32.25 S8174; D4554-4 23.87 8.78 0.4 32.65 ChookGPAT9-1 @ SAD2-1vD locus CpalGPAT9-1 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4552-6 23.57 9.00 0.36 32.57 S8174; D4724-6 25.61 9.52 0.39 35.13 S8174; D4552-4 23.62 8.87 0.37 32.49 S8174; D4724-7 24.91 9.36 0.41 34.27 S8174; D4552-9 23.39 8.97 0.40 32.36 S8174; D4724-2 24.43 9.46 0.39 33.89 S8174; D4552-8 23.28 8.80 0.40 32.08 S8174; D4724-5 24.01 9.25 0.39 33.26 S8174; D4552-11 23.18 8.80 0.44 31.98 S8174; D4724-4 24.30 8.93 0.39 33.23 CignGPAT9-1 @ SAD2-1vD locus CpalGPAT9-2 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4553-12 25.19 9.42 0.40 34.61 S8174; D4725-5 24.24 10.30 0.48 34.54 S8174; D4685-1 24.33 10.24 0.46 34.57 S8174; D4725-6 24.81 9.29 0.41 34.10 S8174; D4553-15 25.11 9.33 0.41 34.44 S8174; D4725-7 24.35 9.51 0.42 33.86 S8174; D4553-1 24.56 9.50 0.44 34.06 S8174; D4725-8 24.37 9.39 0.40 33.76 S8174; D4553-6 24.74 9.16 0.40 33.90 S8174; D4725-9 24.28 9.29 0.41 33.57
TABLE-US-00039 TABLE 19b Fatty acid profiles of representative transgenic strains of S8174 expressing DGATs Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.61 9.10 0.42 33.71 Cavig DGAT1 @ SAD2-1vD locus S8174; D4549-7 24.89 9.28 0.36 34.17 S8174; D4549-6 24.53 9.04 0.47 33.57 S8174; D4549-4 23.93 8.99 0.41 32.92 S8174; D4549-1 23.93 8.97 0.38 32.90 S8174; D4549-3 23.76 8.9 0.36 32.66 Chook DGAT1 @ SAD2-1vD locus S8174; D4550-1 24.67 9.12 0.41 33.79 S8174; D4550-3 24.64 9.06 0.42 33.70 S8174; D4682-1 23.72 9.68 0.5 33.40 S8174; D4682-2 23.49 9.66 0.41 33.15 S8174; D4550-2 22.42 8.81 0.41 31.23
TABLE-US-00040 TABLE 20 Fatty acid profiles of representative transgenic strains of S8174 expressing LPCATs and PLA2s Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.61 9.10 0.42 33.71 Cavig LPCAT @ SAD2-1vD locus Cavig PLA2-1 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4555-1 26.6 9.38 0.47 35.98 S8174; D4732-1 26.31 11.24 0.60 37.55 S8174; D4555-3 26.4 9.47 0.39 35.87 S8174; D4732-2 25.30 11.88 0.50 37.18 S8174; D4688-1 25.95 9.67 0.44 35.62 S8174; D4732-3 25.29 11.01 0.48 36.30 S8174; D4688-3 25.47 9.89 0.44 35.36 S8174; D4732-4 25.30 11.00 0.47 36.30 S8174; D4555-2 25.52 9.55 0.36 35.07 S8174; D4732-5 25.07 11.20 0.44 36.27 Cpau LPCAT @ SAD2-1vD locus CignPLA2-1 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4556-3 25.55 9.21 0.43 34.76 S8174; D4734-6 26.39 11.34 0.47 37.73 S8174; D4556-4 25.24 9.46 0.41 34.70 S8174; D4734-1 26.17 10.90 0.46 37.07 S8174; D4689-7 24.63 9.86 0.43 34.49 S8174; D4734-5 25.58 11.12 0.57 36.70 S8174; D4556-1 25.18 9.13 0.42 34.31 S8174; D4734-4 25.48 11.17 0.57 36.65 S8174; D4689-6 24.05 9.89 0.48 33.94 S8174; D4734-2 24.75 11.32 0.46 36.07 Cpal LPCAT @ SAD2-1vD locus CuPSR23PLA2-2 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4726-4 26.34 9.76 0.41 36.10 S8174; D4735-5 25.81 11.16 0.44 36.97 S8174; D4726-2 25.92 9.9 0.44 35.82 S8174; D4735-1 25.95 10.92 0.47 36.87 S8174; D4726-3 26.15 9.62 0.41 35.77 S8174; D4735-8 25.54 10.91 0.42 36.45 S8174; D4726-5 26.09 9.55 0.41 35.64 S8174; D4735-7 25.45 10.95 0.44 36.40 S8174; D4726-1 25.64 9.57 0.39 35.21 S8174; D4735-6 25.51 10.88 0.41 36.39 Cschu LPCAT @ SAD2-1vD locus Cproc PLA2-2 @ SAD2-1vD locus Sample ID C8:0 C10:0 C12:0 C8-C10 Sample ID C8:0 C10:0 C12:0 C8-C10 S8174; D4727-1 26.24 9.95 0.45 36.19 S8174; D4736-2 25.60 10.87 0.42 36.47 S8174; D4727-7 26.26 9.84 0.42 36.10 S8174; D4736-4 25.55 10.76 0.40 36.31 S8174; D4727-9 26.13 9.87 0.42 36.00 S8174; D4736-3 25.40 10.87 0.36 36.27 S8174; D4727-11 25.99 9.97 0.44 35.96 S8174; D4736-5 25.45 10.46 0.39 35.91 S8174; D4727-16 26.28 9.68 0.44 35.96 S8174; D4736-1 24.34 11.06 0.48 35.40
Example 7: Expression of LPAAT and/or DGAT in Prototheca to Produce High SOS and Low Trisaturated TAGs
[0161] In this example we describe genetically engineered Prototheca moriformis strains in which we have modified fatty acid and triacylglycerol biosynthesis to maximize the accumulation of Stearoyl-Oleoyl-Stearoyl (SOS) TAGs, and minimize the production of trisaturated TAGs. Tailored oils from these strains resemble plant seed oils known as structuring fats, which have high proportions of Saturated-Oleate-Saturated TAGs and low levels of trisaturates. These structuring fats (often called butters) are generally solid at room temperature but melt sharply between 35-40 C.
[0162] High-SOS strains were obtained by three successive transformations beginning with strain S5100, a classically improved derivative, of a wild type isolate of Prototheca moriformis, S376. Strain S5100 was transformed with plasmid pSZ5654 to generate strain S8754, which produces an oil with increased stearic acid (C18:0) content, lower palmitic acid (C16:0) and reduced linoleic acid (C18:2cis9,12) content relative to S5100. In turn, strain S8754 was transformed with plasmid pSZ5868 to generate strain S8813, which produces oil with higher C18:0, lower C16:0 and improved sn-2 selectivity compared to S8754. Finally, strain S8813 was transformed with plasmids pSZ6383 or pSZ6384 to generate strains S9119, S9120 and S9121, producing oils rich in C18:0 with reduced levels of C18:2cis9,12 and improved sn-3 selectivity.
[0163] Construct Used for SAD2 Knockout in S5100
[0164] The first intermediate strains were prepared by transformation of strain S5100 with integrative plasmid pSZ5654 (SAD2-1vD::PmKASII-1tp_PmKASII-1_FLAG-CvNR:CrTUB2-PmFAD2hpA-CvNR:PmHXT1-2v2-ScarMEL1-PmPGK::SAD2-1vE). The construct targeted ablation of allele 1 of the endogenous stearoyl-ACP desaturase 2 gene (SAD2), concomitant with expression of the PmKASII gene encoding P. moriformis -keto-acyl-ACP synthase, and a RNAi hairpin sequence to down-regulate fatty acid desaturase (FAD2) gene expression. Deletion of one allele of SAD2 reduced SAD activity, resulting in elevated levels of C18:0. Overexpression of PmKASII stimulated elongation of C16:0 to C18:0, further increasing C18:0. FAD2 is responsible for the conversion of C18:1cis9 (oleic) to C18:2cis9,12 (linoleic) fatty acids, and RNAi of FAD2 resulted in decreased C18:2. Thus, the first intermediate strains had higher levels of C18:0 and decreased C16:0 and C18:2 fatty acid levels relative to the S5100 parent. The Saccharomyces carlsbergensis MEL1 gene, encoding a secreted melibiase served as a selectable marker as part of plasmid pSZ5654, enabling the strain to grow on melibiose.
[0165] The sequence of the pSZ5654 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlining and are 5-3 PmeI, SpeI, AscI, ClaI, SacI, AvrII, EcoRV, EcoRI, SpeI, BsiWI, XhoI, SacI, KpnI, SnaBI, BspQI and PmeI, respectively. PmeI sites delimit the 5 and 3 ends of the transforming DNA. Bold, lowercase sequences represent SAD2-1 5 genomic DNA that permit targeted integration at the SAD2-1 locus via homologous recombination. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent SAD2-1 5 genomic DNA sequences that permit targeted integration at the FATA-1 locus via homologous recombination. The initiator ATG of the sequence encoding the P. moriformis KASII-1 transit peptide (PmKASII-1tp) is indicated by uppercase, bold italics, and the PmKASII-1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The PmKASII-1 coding region is indicated by lowercase italics. A sequence encoding a 3FLAG tag fused to the C-terminus of PmKASII-1 is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The Chlorella vulgaris nitrate reductase (NR) gene 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The C. reinhardtii TUB2 promoter, driving expression of the PmFAD2hpA sequence is indicated by boxed text. Bold italics denote the PmFAD2hpA sequence followed by lowercase underlined text representing C. vulgaris nitrate reductase 3 UTR. A second spacer sequence is represented by lowercase text. The P. moriformis HXT1 promoter driving the expression of the S. carlbergensis MEL1 gene is indicated by boxed text. The initiator ATG and terminator TGA for MEL1 gene are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis PGK 3 UTR is indicated by lowercase underlined text. The SAD2-1 3 genomic region indicated by bold, lowercase text.
TABLE-US-00041 NucleotidesequenceoftransformingDNAcontainedinpSZ5654 SEQIDNO:126 gtttaaacgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacgg aaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgaca cgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagc agttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcg caggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcgg aacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttgg cgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcagg catcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtg gcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactc [00043]![embedded image]()
gccactacttcaacacccacctacccaccacctcccaccacaccatacaccacacctaatcccacatcacccacgggcgcgccgc cgccgccgccgacgccaaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccag accatcgagcagttctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacacc accaccatcgccggcgagatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtga tcaagtacgtgtacatcgccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccgg cctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgac ccgcggcggcgtgcgcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatc ggcttcatgggccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgcc gcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaag gccctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagg gcgccggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcg ccgccacctccgacgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggag cgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccg cgccatccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccgg cgccgtggaggccgtggccgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccg gcgtggaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttc ggcggccacaactcctgcgtgatcttccgcaagtacgacgagATGGACTACAAGGACCACGACGGCGACTACAA [00044]![embedded image]()
cactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcc tcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctc gtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgca cagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggga tgggaacacaaatggagagctccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcg gcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcg [00045]![embedded image]()
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cacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacag cctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccc tcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcg cacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgg gatgggaacacaaatggaaagctgtagagctcgatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccat gtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggca [00057]![embedded image]()
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ctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgc tggacacggccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctcc ggccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcaca acaactccttcctgttcggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggagg aggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcc cgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccctgtgcaactg gggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcggagttcacgc gccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctga acaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaac ctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaaca acctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcg cgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggc gaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaac ctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccat cctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgac acccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttct [00064]![embedded image]()
ttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgcaattaattgtgtgatgaagaaagggtggcacaaga tggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctggctcaatcttgtcgcatgtccggcgcaat gtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactgatcgcattgccatcccgtcaa ctcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaagcgtcaggaaatcg tctcggcagctggaagcgcatggaatgcggagcggagatcgaatcaggatccttagggagcgacgagtgtgcgtgcggggctggc gggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacga agaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaa gctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctc cgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgct cgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtcctgggcgaagaacgagggaatttg tgggtaaaacaagcatcgtctctcaggccccggcgcagtggccgttaaagtccaagaccgtgaccaggcagcgcagcgcgtccgt gtgcgggccctgcctggcggctcggcgtgccaggctcgagagcagctccctcaggtcgccttggacggcctctgcgaggccggtga gggcctgcaggagcgcctcgagcgtggcagtggcggtcgtatccgggtcgccggtcaccgcctgcgactcgccatccgaagagcg tttaaac
[0166] Construct pSZ5654 was transformed into S5100. Primary transformants were clonally purified and screened under standard lipid production conditions at pH 5. Integration of pSZ5654 at the SAD2-1 locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 21). S8754 was selected as the lead strain for additional rounds of genetic engineering. As shown in Table 21, C16:0 decreased from 17.6% to less than 6%, C18:0 increased from 4.3% to about 28%, C18:2 decreased from 5.8% to 1.3%.
TABLE-US-00042 TABLE 21 Fatty acid profiles of SAD2-1 ablation strains. Sample ID S5100 S8741 S8742 S8743 S8744 S8745 S8746 S8752 S8753 S8754 C14:0 0.7 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 C16:0 17.6 5.9 5.9 5.8 5.9 5.9 5.9 5.9 5.8 5.9 C16:1 cis-9 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 C18:0 4.3 28.2 28.1 27.7 27.8 27.4 28.2 28.3 28.3 28.1 C18:1 69.8 60.1 60.2 60.6 60.5 60.9 60.0 60.0 60.0 60.0 C18:2 5.8 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.2 1.3 C18:3 0.5 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 C20:0 0.3 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2 saturates 23.2 37.5 37.5 37.1 37.2 36.8 37.7 37.7 37.7 37.6 lipid (g/L) 13.5 12.8 12.5 12.5 12.5 12.3 12.3 12.3 12.4 12.3
Construct Used for FATA-1 Knockout in S8754
[0167] The second intermediate strains were prepared by transformation of strain S8754 with integrative plasmid pSZ5868 (FATA-1vB::CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1:PmG3PDH-1-TcLPAT2-PmATP:CrTUB2-ScSUC2-PmPGH::FATA-1vC). This construct targeted ablation of allele 1 of the endogenous fatty acyl-ACP thioesterase gene (FATA-1), and contained expression modules for GarmFATA1(G108A), encoding a variant of the Garcinia mangostana FATA1 thioesterase with improved activity, and TcLPAT2 encoding the Theobroma cacao lysophosphatidic acid acyltransferase (LPAAT). Deletion of one copy of FATA-1 reduced endogenous thioesterase activity, further reducing C16:0 accumulation. Expression of GarmFATA1(G108A) stimulated C18:0-ACP hydrolysis, further increasing C18:0. TcLPAT2 had superior specificity for transfer of C18:1 to the sn-2 position of triacylglycerides than the endogeneous LPAAT, leading to reduced accumulation of trisaturates. The second intermediate strains had increased C18:0 and lower C16:0 compared their parent, S8754. The S. cerevisiae SUC2 gene encoding a secreted sucrose invertase, served as a selectable marker as part of plasmid pSZ5868 and enabled the strain to grow on sucrose.
[0168] The sequence of the pSZ5868 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlining and are 5-3 BspQI, PmeI, SpeI, AscI, ClaI, SacI, AvrII, NdeI, NsiI, AfIII, KpnI, XbaI, MfeI, BamHI, BspQI and PmeI, respectively. BspQI and PmeI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FATA-1 5 genomic DNA that permit targeted integration at the FATA-1 locus via homologous recombination. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis G3PDH-1 promoter, driving expression of the TcLPAT2 sequence is indicated by boxed text. The initiator ATG and terminator TGA codons of the TcLPAT2 gene are indicated by uppercase, bold italics, while the remainder of the coding region is represented with italics. Lowercase underlined text represents the P. moriformis ATP 3 UTR. A second spacer sequence is represented by lowercase text. The C. reinhardtii TUB2 promoter driving the expression of the S. cerevisiae SUC2 gene is indicated by boxed text. The initiator ATG and terminator TGA for SUC2 are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis PGH 3 UTR is indicated by lowercase underlined text. The FATA-1 3 genomic region indicated by bold, lowercase text.
TABLE-US-00043 NucleotidesequenceoftransformingDNAcontainedinpSZ5868 SEQIDNO:127 gaagagcgcccaatgtttaaacctcttttgctgcgtctcctcaggcttgggggcctccttgggcttgggtgccgccatgatctgcgcg catcagagaaacgttgctggtaaaaaggagcgcccggctgcgcaatatatatataggcatgccaacacagcccaacctcactcg ggagcccgtcccaccacccccaagtcgcgtgccttgacggcatactgctgcagaagcttcatgagaatgatgccgaacaagaggg gcacgaggacccaatcccggacatccttgtcgataatgatctcgtgagtccccatcgtccgcccgacgctccggggagcccgccga tgctcaagacgagagggccctcgaccaggaggggctggcccgggcgggcactggcgtcgaaggtgcgcccgtcgttcgcctgca gtcctatgccacaaaacaagtcttctgacggggtgcgtttgctcccgtgcgggcaggcaacagaggtattcaccctggtcatgggg agatcggcgatcgagctgggataagagatacggtcccgcgcaaggatcgctcatcctggtctgagccggacagtcattctggcaa gcaatgacaacttgtcaggaccggaccgtgccatatatttctcacctagcgccgcaaaacctaacaatttgggagtcactgtgcca ctgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtcagcaccgccagccggccgaggacccgagtcata [00065]![embedded image]()
ggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctc ctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgacc gaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacc atcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccacc atgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtgga gatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggt gatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcga cgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagct ggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtg acctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccg ccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccaca acggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAAGGACCACGACGGCG [00066]![embedded image]()
gcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatc gagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgcca gagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaact tgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggc gagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtgagctccgcgtctcgaaca gagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttg gttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggt [00067]![embedded image]()
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tcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttcg tgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggc tggtggactggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccct ggtggtggccaaccaccgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccct ggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcct gggccaaggacgagaacaccctgaaggccggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtg gagggcacccgcttcacccaggccaagttcctggccgcccaggagtacgccgcctcccagggcctgcccatcccccgcaacgt gctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgcgctccttcgtgcccgccatctacgacatgaccgtggcc atccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctccgtggtgcacgtgcacatcaagcgctgcct gatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgttcgtggagaaggacaagctgctgg acaagcacatcgccgaggacaccactccgaccagcccatgcaggacctgggccgccccatcaagtccctgctggtggtggcc tcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcgccacttc ctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtgg [00073]![embedded image]()
agggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttcccaac gccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcgtc tggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggagagc gtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcgctg ttaggctgtattaatcaaggagcgtatcaataattaccgaccctatacctttatctccaacccaatcgcggcttaaggatctaagtaa gattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacg tgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagatacagcgcgagccagacacggagtg [00074]![embedded image]()
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ccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgc caagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgactggggccacgccacgtccgacg acctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtg gactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccg gagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccg ccaactccacccagaccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccag gactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcacctcgg ctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccat caaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaa ccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccct gggcatcgcgtgggcctccaactgggagtactccgccacgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaag ttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagca acgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcac cggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctgg ttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaac agcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaac gacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccacc aacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgaca [00079]![embedded image]()
cgaaacaagcccctggagcatgcgtgcatgatcgtctctggcgccccgccgcgcggtttgtcgccctcgcgggcgccgcggccgcg ggggcgcattgaaattgttgcaaaccccacctgacagattgagggcccaggcaggaaggcgttgagatggaggtacaggagtcaa gtaactgaaagtttttatgataactaacaacaaagggtcgtttctggccagcgaatgacaagaacaagattccacatttccgtgtag aggcttgccatcgaatgtgagcgggcgggccgcggacccgacaaaacccttacgacgtggtaagaaaaacgtggcgggcactgtc cctgtagcctgaagaccagcaggagacgatcggaagcatcacagcacaggatcctgaggacagggtggttggctggatggggaa acgctggtcgcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtgcacacaca caacccaaccggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatcctcctccctgcc gcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacaggccccttgtgcgcagtg ctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtgtggccaagcgggctgctgggcgc gccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttctgtaagccacgcgcttgctgctttgggaagagaagg gggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttggggaggcagggagagttggaaaatgt aagtggcacgacgggcaaggagaatggtgagcatgtgcatggtgatgtcgttggtcgaggacgatcctgcacgcgtgtatctgat gtagaatacggcaatcaccctagtctacatctataccttctccgtataacgccctttccaaatgccctcccgtttctctcctattcttg atccacatgatgaccctggcactatttcaagggctggagaagagcgtttaaac
[0169] Construct pSZ5868 was transformed into S8754. Primary transformants were clonally purified and screened under standard lipid production conditions at pH 5. Integration of pSZ5868 at the FATA-1 locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 22). S8813 was selected as the lead strain for the final round of genetic engineering. As shown in Table 22 as compared to strain S8754, C16:0 decreased from 5.9% to 3.4%, and C18:0 increased from 27.3% to about 45%. C18:2 increased slightly from 1.3% to about 1.6% due to the activity of the T. cacao LPAAT.
TABLE-US-00044 TABLE 22 Fatty acid profiles of FATA-1 ablation strains. Strain 55100 58754 58813 58814 C14:0 0.7 0.6 0.5 0.5 C16:0 18.8 5.9 3.4 3.4 C16:1 cis-9 0.5 0.0 0.0 0.0 C18:0 4.0 27.3 45.3 44.8 C18:1 68.3 60.9 45.9 46.3 C18:2 6.3 1.3 1.5 1.6 C18:3 0.6 0.3 0.3 0.3 C20:0 0.3 2.4 2.0 2.1 saturates 24.2 37.0 52.0 51.5 lipid (g/L) 12.7 11.9 11.9 11.9
Constructs Used for FAD2 Knockout in S8813
[0170] The high-SOS strains were generated by transformation of strain S8813 with integrative plasmid pSZ6383 (FAD2-1vA::PmLDH1-AtTHIC-PmHSP90:PmSAD2-2v2-TcDGAT1-CvNR:PmSAD2-1v3-CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1::FAD2-1vB), plasmid pSZ6384 (FAD2-1vA::PmLDH1-AtTHIC-PmHSP90:PmSAD2-2v2-TcDGAT2-CvNR:PmSAD2-1v3-CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1::FAD2-1vB), or plasmid pSZ6377 (FAD2-1vA::PmLDH1-AtTHIC-PmHSP90: PmSAD2-1v3-CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1::FAD2-1vB). These constructs targeted ablation of allele 1 of the endogenous fatty acid desaturase 2 gene (FAD2-1), and contained expression modules for a second copy of GarmFATA1(G108A), and either TcDGAT1 encoding the Theobroma cacao diacylglycerol O-acyltransferase 1 (pSZ6383) or TcDGAT2 encoding the Theobroma cacao diacylglycerol O-acyltransferase 2 (pSZ6384). Deletion of one allele of FAD2 further reduced C18:2 accumulation. Expression of GarmFATA1(G108A) stimulated C18:0-ACP hydrolysis, further increasing C18:0. TcDGAT1 and TcDGAT2 had superior specificity for transfer of C18:0 to the sn-3 position of triacylglycerides than the endogeneous DGAT, leading to an increase in C18:0 and lipid titer, and a reduction in trisaturated TAGs. The final strains had higher C18:0, lower C16:0 and lower C18:2 than their parent, S8813. The Arabidopsis thaliana THIC gene (AtTHIC) catalyzes the conversion of 5-aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethylpyrimidine (HMP), providing the pyrimidine ring structure for the biosynthesis of thiamine. AtTHIC served as a selectable marker as part of plasmids pSZ6383 and pSZ6384, allowing the strains to grow in the absence of exogenous thiamine.
[0171] The sequence of the pSZ6383 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlined text, and are 5-3 BspQI, KpnI, XbaI, SnaBI, BamHI, AvrII, SpeI, ClaI, AflII, EcoRI, SpeI, AscI, ClaI, SacI and BspQ I, respectively. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FAD2-1 5 genomic DNA that permits targeted integration at the FAD2-1 locus via homologous recombination. The P. moriformis LDH1 promoter driving the expression of the Arabidopsis thaliana THIC gene is indicated by boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis HSP90 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis SAD2-2 promoter, driving expression of the TcDGAT1 sequence is indicated by boxed text. The initiator ATG and terminator TGA codons of the TcDGAT1 gene are indicated by uppercase, bold italics, while the remainder of the coding region is represented with italics. Lowercase underlined text represents the C. vulgaris NR 3 UTR. A second spacer sequence is represented by lowercase text. The P. moriformis SAD2-1 promoter, indicated by boxed italicized text, is utilized to drive the expression of the G. mangostana FATA1 gene. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. The FAD2-1 3 genomic region is indicated by bold, lowercase text.
TABLE-US-00045 NucleotidesequenceoftransformingDNAcontainedinpSZ6383 SEQIDNO:128 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00080]![embedded image]()
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ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00092]![embedded image]()
cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00093]![embedded image]()
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[00098]![embedded image]()
[00099]![embedded image]()
[00100]![embedded image]()
[00101]![embedded image]()
cgagatcctgggctccaccgccaccgtgacctcctcctcccactccgactccgacctgaacctgctgtccatccgccgccgcacct ccaccaccgccgccgcccgcgcccccgaccgcgacgactccggcaacggcgaggccgtggacgaccgcgaccgcgtggagt ccgccaacctgatgtccaacgtggccgagaacgccaacgagatgcccaactcctccgacacccgcttcacctaccgcccccgcg tgcccgcccaccgccgcatcaaggagtcccccctgtcctccggcgccatcttcaagcagtcccacgccggcctgttcaacctgtgc atcgtggtgctggtggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggctggctgatccgctccggcttctggt tctcctcccgctccctgtccgactggcccctgttcatgtgctgcctgaccctgcccatcttccccctggccgccttcgtggtggagaa gctggtgcagcgcaactacatctccgagcccgtggtggtgttcctgcacgccatcatctccaccaccgccgtgctgtaccccgtg atcgtgaacctgcgctgcgactccgccttcctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtc ctacgcccacaccaacaacgacatgcgcgccctggccaagtccgccgagaagggcgacgtggacccctcctacgacgtgtcct tcaagtccctggcctacttcatggtggcccccaccctgtgctaccagcagtcctacccccgcacccccgccgtgcgcaagtcctgg gtggtgcgccagttcatcaagctgatcgtgttcaccggcctgatgggcttcatcatcgagcagtacatcaaccccatcgtgcag aactcccagcaccccctgaagggcaacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaacctgtacgtgtgg ctgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgctgcgcttcggcgaccgcgagttctacaagga ctggtggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgccacatctac ttcccctgcctgcgcaacggcatccccaagggcgtggccatcgtgatcgccttcctggtgtccgccgtgttccacgagctgtgcat cgccgtgccctgccacatgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctgatcaccaactacctgc aggacaagttccgctcctccatggtgggcaacatgatcttctggttcatcttctccatcctgggccagcccatgtgcgtgctgctgt [00102]![embedded image]()
gacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaac agcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttc cctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccct cgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagt gggatgggaacacaaatggacttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgta gtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggacca ggcatcgcgagatacagcgcgagccagacacggagtgccgagctatgcgcacgctccaactagatatcatgtggatgatgagcat [00103]![embedded image]()
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aatgcccactgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgc catccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctgg ccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggc atcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctact ccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctaca agtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactgga tcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctg cagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaaca actcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctgg acatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacga gctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccg aggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaactt cctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcAT GGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAA [00110]![embedded image]()
tcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagat ccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtcca cagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattat cttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagt caatgaatggtgagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgccttgtcttttgcacgcgcgactccgt cgcttcgcgggtggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtacccccaaccacccacctgcacct ctattattggtattattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttcagctggctcccaccattgtaaatt cttgctaaaatagtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggttttcgtgctgatctcgggcacaag gcgtcgtcgacgtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcctttactccgcactccaaacgact gtcgctcgtatttttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaaaggcctcgcggccgtgctcgtgg tgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcatggtgagtgcgcatcacaagatg catgtcttgttgtctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgctttggtcattacagaaattgcacaag ggcgtcctccgggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcaggagcgcggcgcatgacgacctacc cacatgcgaagagc
[0172] The sequence of the pSZ6384 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlined text, and are 5-3 BspQI, KpnI, XbaI, SnaBI, BamHI, AvrII, SpeI, ClaI, AfIII, EcoRI, SpeI, AscI, ClaI, SacI and BspQ I, respectively. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FAD2-1 5 genomic DNA that permits targeted integration at the FAD2-1 locus via homologous recombination. The P. moriformis LDH1 promoter driving the expression of the Arabidopsis thaliana THIC gene is indicated by boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis HSP90 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis SAD2-2 promoter, driving expression of the TcDGAT2 sequence is indicated by boxed text. The initiator ATG and terminator TGA codons of the TcDGAT2 gene are indicated by uppercase, bold italics, while the remainder of the coding region is represented with italics. Lowercase underlined text represents the C. vulgaris NR 3 UTR. A second spacer sequence is represented by lowercase text. The P. moriformis SAD2-1 promoter, indicated by boxed italicized text, is utilized to drive the expression of the G. mangostana FATA1 gene. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. The FAD2-1 3 genomic region is indicated by bold, lowercase text.
TABLE-US-00046 NucleotidesequenceoftransformingDNAcontainedinpSZ6384 SEQIDNO:129 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00111]![embedded image]()
[00112]![embedded image]()
[00113]![embedded image]()
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[00119]![embedded image]()
[00120]![embedded image]()
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ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00123]![embedded image]()
cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00124]![embedded image]()
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gaggagcgcaaggccaccggctaccgcgagactccggccgccacgagacccctccaacaccatgcacgccctgctggccat gggcatctggctgggcgccatccacttcaacgccctgctgctgctgactccttcctgacctgcccttctccaagttcctggtggtgt tcggcctgctgctgctgacatgatcctgcccatcgacccctactccaagttcggccgccgcctgtcccgctacatctccaagcacg cctgctcctacttccccatcaccctgcacgtggaggacatccacgccttccaccccgaccgcgcctacgtgttcggcttcgagccc cactccgtgctgcccatcggcgtggtggccctggccgacctgaccggcttcatgcccctgcccaagatcaaggtgctggcctcct ccgccgtgttctacacccccacctgcgccacatctggacctggctgggcctgacccccgccaccaagaagaacttctcctccctg ctggacgccggctactcctgcatcctggtgcccggcggcgtgcaggagaccaccacatggagcccggctccgagatcgccttc ctgcgcgcccgccgcggcttcgtgcgcatcgccatggagatgggctcccccctggtgcccgtgttctgcttcggccagtcccacgt gtacaagtggtggaagcccggcggcaagttctacctgcagttctcccgcgccatcaagttcacccccatcttcttctggggcatct tcggctcccccctgccctaccagcaccccatgcacgtggtggtgggcaagcccatcgacgtgaagaagaacccccagcccatc gtggaggaggtgatcgaggtgcacgaccgcttcgtggaggccctgcaggacctgttcgagcgccacaaggcccaggtgggc [00133]![embedded image]()
gctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgttt gatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcg cttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggt ttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacac aaatggacttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgt aagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagat [00134]![embedded image]()
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gcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgca tcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcg cctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagacc gccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggctt ctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctg gtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgacta cgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtgga cgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctga agaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaacca gcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagacc atcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccga ggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgct gcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAA [00142]![embedded image]()
ggagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccacccc gcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcaca tcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacacca aaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttc ctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggt gagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgccttgtcttttgcacgcgcgactccgtcgcttcgcggg tggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtacccccaaccacccacctgcacctctattattggta ttattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttcagctggctcccaccattgtaaattcttgctaaaat agtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggttttcgtgctgatctcgggcacaaggcgtcgtcgac gtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcctttactccgcactccaaacgactgtcgctcgtatt tttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaaaggcctcgcggccgtgctcgtggtgggggccgcg agcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcatggtgagtgcgcatcacaagatgcatgtcttgttg tctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgctttggtcattacagaaattgcacaagggcgtcctccg ggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcaggagcgcggcgcatgacgacctacccacatgcgaa gagc
[0173] The sequence of the pSZ6377 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlined text, and are 5-3 BspQI, KpnI, XbaI, SnaBI, BamHI, AvrII, SpeI, AscI, ClaI, SacI and BspQ respectively. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FAD2-1 5 genomic DNA that permits targeted integration at the FAD2-1 locus via homologous recombination. The P. moriformis LDH1 promoter driving the expression of the Arabidopsis thaliana THIC gene is indicated by boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis HSP90 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis SAD2-1 promoter, indicated by boxed italicized text, is utilized to drive the expression of the G. mangostana FATA1 gene. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. The FAD2-1 3 genomic region is indicated by bold, lowercase text.
TABLE-US-00047 NucleotidesequenceoftransformingDNAcontainedinpSZ6377 SEQIDNO:130 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00143]![embedded image]()
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ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00155]![embedded image]()
cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00156]![embedded image]()
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ccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggc ccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgag gccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatc gtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaac cacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgccc gcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaaga tcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatg aaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcc tggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctg gtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgcccca ggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactcc ctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaa cgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggc gcaagaagcccacccgcATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACA [00163]![embedded image]()
ctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgc ggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgct gccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttg caacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgc cctcgctgatcgagtgtacagtcaatgaatggtgagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgcctt gtcttttgcacgcgcgactccgtcgcttcgcgggtggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtac ccccaaccacccacctgcacctctattattggtattattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttca gctggctcccaccattgtaaattcttgctaaaatagtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggtt ttcgtgctgatctcgggcacaaggcgtcgtcgacgtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcc tttactccgcactccaaacgactgtcgctcgtatttttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaa aggcctcgcggccgtgctcgtggtgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcat ggtgagtgcgcatcacaagatgcatgtcttgttgtctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgcttt ggtcattacagaaattgcacaagggcgtcctccgggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcagg agcgcggcgcatgacgacctacccacatgcgaagagc
[0174] Constructs pSZ6383, pSZ6384 and pSZ6377 were transformed into S8813. Primary transformants were clonally purified and screened under standard lipid production conditions at pH 5. Integration of pSZ6383 or pSZ6384 at the FAD2-1 locus was verified by DNA blot analysis. The fatty acid profiles, sn-2 profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 23). FAD2-1 ablation reduced C18:2 to <1% in most strains. Expression of a second copy of GarmFATA1(G108A) and TcDGAT1 (S8990, S8992, S8998 & S8999), or TcDGAT2 (S8994, S9000 & S9047) elevated C18:0 to >56%. The D5393-28 strain, expressing a second copy of GarmFATA1(G108A) without either of the cocoa DGAT genes (pSZ6377) had a similar fatty acid profile, but lower lipid titer. As shown in Table 23, as compared to strain S8813, for strains expressing either TcDGAT1 or TcDGAT2, C16:0 increased from 3.2% to 3.7%-4.0%, C18:0 increased from 45.8% to about 56%, C18:2 decreased from 1.4% to about 1.0%.
TABLE-US-00048 TABLE 23 Fatty acid profiles of FAD2-1 ablation strains. Strain S8813 D5393-28 S8990 S8992 S8998 S8999 S8994 S9000 S9047 C12:0 0.1 0.2 0.2 0.2 0.1 0.2 0.1 0.1 0.2 C14:0 0.4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 C16:0 3.2 3.8 3.7 3.8 3.9 4.0 3.7 3.8 3.5 C16:1 cis-7 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C16:1 cis-9 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C17:0 0.1 0.2 0.2 0.1 0.2 0.1 0.2 0.2 0.2 C18:0 45.8 56.0 56.6 56.0 56.2 56.0 56.3 56.4 56.5 C18:1 45.9 35.8 35.4 35.9 35.7 35.5 35.9 35.7 35.9 C18:2 1.4 1.0 0.9 1.0 0.9 1.1 0.9 0.9 0.8 C18:3 0.3 0.3 0.3 0.2 0.3 0.2 0.2 0.3 0.3 C20:0 2.0 1.6 1.6 1.5 1.6 1.5 1.5 1.5 1.5 C22:0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 C24:0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 saturates 52.1 62.6 63.1 62.6 62.9 62.8 62.8 62.9 62.7
[0175] Liquid chromatography and mass spectrometry were used to analyze the TAG composition of final strains. The strains accumulated 68-71% SOS, with trisaturates ranging from 2.5-2.8%. The D5393-28 strain, expressing a second copy of GarmFATA1(G108A) without either of the cocoa DGAT genes had similar SOS content but slightly higher trisaturates. The TAG composition of a typical Shea stearin and a sample of Kokum butter are shown for comparison
TABLE-US-00049 TABLE 24 LC/MS TAG profiles of FAD2-1 ablation strains. Shea Kokum Strain D5393-28 S8990 S8992 S8998 S8999 S8994 S9000 S9047 stearin butter OOL 0.4 LLS 0.2 POL 0.3 OOO 1.3 1.7 SOL 1.0 0.4 LaOS + MOP 0.2 0.3 0.3 0.2 0.3 0.3 0.4 0.2 OOP 0.5 0.2 0.3 0.2 0.2 0.4 0.3 0.2 0.8 0.7 PLS (+SLnS) 0.6 0.7 0.7 0.7 0.7 0.6 0.6 0.4 0.6 0.3 POP (+MOS) 1.1 1.0 1.0 1.1 1.1 1.0 1.2 0.8 0.7 0.4 OOS 10.5 10.3 11.3 11.0 11.0 10.9 10.1 10.6 6.4 11.8 SLS (+PLA) 1.9 1.7 2.0 1.6 2.1 1.8 1.9 1.5 5.5 1.4 POS 8.4 8.5 8.4 8.7 8.9 8.4 10.0 7.7 6.3 4.8 MaOS 0.3 SOG 0.4 0.5 0.5 0.6 0.3 0.5 0.4 0.5 OOA 0.5 0.3 0.4 0.4 0.4 0.4 0.4 0.4 0.2 0.2 SOS (+POA) 68.4 69.7 68.7 69.1 68.3 69.4 68.0 71.4 69.7 76.6 SSP (+MSA) 0.5 0.5 0.5 0.4 0.5 0.5 0.5 0.4 0.2 SOA + POB 3.9 3.8 3.5 3.6 3.4 3.5 3.5 3.4 4.0 1.0 SSS (+PSA) 2.6 2.3 2.2 2.1 2.3 2.2 2.3 2.1 2.0 0.5 SOB + LgOP + AOA 0.4 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.4 SSA (+PBS) 0.2 SOLg (+POHx) 0.3 SUM (area %) 99.8 99.9 99.8 99.9 99.8 99.9 100.0 99.9 100.0 100.0 Sat-Sat-Sat 3.1 2.8 2.7 2.5 2.7 2.7 2.8 2.5 2.4 0.5 Sat-U-Sat 84.9 85.9 84.7 85.3 85.1 85.0 86.0 85.8 87.5 84.7 Sat-O-Sat 82.4 83.5 82.0 82.9 82.3 82.6 83.4 83.9 81.4 83.1 Sat-L-Sat 2.5 2.4 2.6 2.3 2.8 2.4 2.6 1.9 6.1 1.6 U-U-U/Sat 11.8 11.3 12.4 12.2 12.0 12.2 11.3 11.7 10.6 14.8 La = laurate (C12:0), M = myristate (C14:0), P = palmitate (C16:0), Ma = margarate (C17:0), S = stearate (C18:0), O = oleate (C18:1), L = linoleate (C18:2), Ln = -linolenate (C18:3 ), A = arachidate (C20:0), G = (C20:1), B = behenate (C22:0), Lg = lignocerate (C24:0), Hx = hexacosanoate (C26:0). Sat = saturated, U = unsaturated
Example 8 Variant Brassica napus Thioeserase
[0176] In this example, we demonstrate the modification of the enzyme specificity of a FATA thioesterase originally isolated from Brassica napus (BnOTE, accession CAA52070), by site directed mutagenesis targeting two amino acids positions D124 and D209).
[0177] To determine the impact of each amino acid substitution on the enzyme specificity of the BnOTE, the wild-type and the mutant BnOTE genes were cloned into a vector enabling expression and expressed in P. moriformis strain S8588. Strain S8588 is a strain in which the endogenous FATA1 allele has been disrupted and expresses a Prototheca moriformis KASII gene and sucrose invertase. Recombinant strains with FATA1 disruption and co-expression of P. moriformis KASII and invertase were previously disclosed in co-owned applications WO2012/106560 and WO2013/15898, herein incorporated by reference.
[0178] Strains that express wild type or mutant BnOTE enzymes, constructs pSZ6315, pSZ6316, pSZ6317, or pSZ6318 were expressed in S8588. In these constructs, the Saccharomyces carlsbergensis MEL1 gene (Accession no: AAA34770) was utilized as the selectable marker to introduce the wild-type and mutant BnOTE genes into the FAD2-2 locus of P. moriformis strain S8588 by homologous recombination using previously described transformation methods (biolistics). The constructs that have been expressed in S8588 are listed in Table 25.
TABLE-US-00050 TABLE 25 DNA lot# and plasmid ID of DNA constructs that expressing wild-type and mutant BnOTE genes DNA Solazynne Lot# Plasmid Construct D5309 pSZ6315 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE-PmSAD2-1 utr::FAD2-2 D5310 pSZ6316 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE(D124A)-PmSAD2-1 utr::FAD2-2 D5311 pSZ6317 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE(D209A)-PmSAD2-1 utr::FAD2-2 D5312 pSZ6318 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE(D124A, D209A)- PmSAD2-1 utr::FAD2-2
[0179] pSZ6315
[0180] The construct psZ6315 can be written as FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE-PmSAD2-1 utr::FAD2-2. The sequence of the pSZ6315 transforming DNA is provided below. Relevant restriction sites in pSZ6315 are indicated in lowercase, bold and underlining and are 5-3 SgrAI, Kpn I, SnaBI, AvrII, SpeI, AscI, ClaI, Sac I, SbfI, respectively. SgrAI and SbfI sites delimit the 5 and 3 ends of the transforming DNA. Bold, lowercase sequences represent FAD2-2 genomic DNA that permit targeted integration at FAD2-2 locus via homologous recombination. Proceeding in the 5 to 3 direction, the P. moriformis HXT1 promoter driving the expression of the Saccharomyces carlsbergensis MEL1 gene is indicated by boxed text. The initiator ATG and terminator TGA for MEL1 gene are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis PGK 3 UTR is indicated by lowercase underlined text followed by the P. moriformis SAD2-2 V3 promoter, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the wild-type BnOTE are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics in lower case. The three-nucleotide codon corresponding to the target amino acids, D124 and D209, are in lower case, italicized, bolded and wave underlined. The P. moriformis SAD2-1 3UTR is again indicated by lowercase underlined text followed by the FAD2-2 genomic region indicated by bold, lowercase text.
TABLE-US-00051 NucleotidesequenceoftransformingDNAcontainedinpSZ6315 SEQIDNO:131 caccggcgcgctgcttcgcgtgccgggtgcagcaatcagatccaagtctgacgacttgcgcgcacgcgccggatccttcaattccaaagtgtcg tccgcgtgcgcttcttcgccttcgtcctcttgaacatccagcgacgcaagcgcagggcgctgggcggctggcgtcccgaaccggcctcggcgcac gcggctgaaattgccgatgtcggcaatgtagtgccgctccgcccacctctcaattaagtttttcagcgcgtggttgggaatgatctgcgctcatg gggcgaaagaaggggttcagaggtgctttattgttactcgactgggcgtaccagcattcgtgcatgactgattatacatacaaaagtacagctc gcttcaatgccctgcgattcctactcccgagcgagcactcctctcaccgtcgggttgcttcccacgaccacgccggtaagagggtctgtggcctc gcgcccctcgcgagcgcatattccagccacgtctgtatgattttgcgctcatacgtctggcccgtcgaccccaaaatgacgggatcctgcataa tatcgcccgaaatgggatccaggcattcgtcaggaggcgtcagccccgcgggagatgccggtcccgccgcattggaaaggtgtagagggggt [00164]![embedded image]()
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gcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggacacggccgacc gcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctg gtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgc gggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacct gaagtacgacaactgctacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaa gacgggccgccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtc cggcgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgc tccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcgg cgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtga acaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtct ggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtg gcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctccaagaagctga cctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccg gcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtc [00170]![embedded image]()
accggcgctgatgtggcgcggacgccgtcgtactctttcagactttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgc aattaattgtgtgatgaagaaagggtggcacaagatggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctg gctcaatcttgtcgcatgtccggcgcaatgtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactg atcgcattgccatcccgtcaactcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaag cgtcaggaaatcgtctcggcagctggaagcgcatggaatgcggagcggagatcgaatcaggatcccgcgtctcgaacagagcgcgcagagga acgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgt [00171]![embedded image]()
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tcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcgg tggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaaca cttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcacta ttatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaat gaatggtgagctccgcgcctgcgcgaggacgcagaacaacgctgccgccgtgtcttttgcacgcgcgactccggcgcttcgctggtggcacccc cataaagaaaccctcaattctgtttgtggaagacacggtgtacccccacccacccacctgcacctctattattggtattattgacgcgggagtgg gcgttgtaccctacaacgtagcttctctagttttcagctggctcccaccattgtaaattcatgctagaatagtgcgtggttatgtgagaggtatag tgtgtctgagcagacggggcgggatgcatgtcgtggtggtgatctttggctcaaggcgtcgtcgacgtgacgtgcccgatcatgagagcaatac cgcgctcaaagccgacgcatagcctttactccgcaatccaaacgactgtcgctcgtattifttggatatctattttaaagagcgagcacagcgcc gggcatgggcctgaaaggcctcgcggccgtgctcgtggtgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggag gaacgcatggtgcgtgcgcaatataagatacatgtattgttgtcctgcagg NucleotidesequenceofBnOTE(D124A)inpSZ6316 SEQIDNO:132 [00192]![embedded image]()
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[0181] The sequence of the pSZ6317 transforming DNA is same as pSZ6315 except the D209A point mutation, the BnOTE D209A DNA sequence is provided below. The three-nucleotide codon corresponding to the target two amino acids, D124 and D209, are in lower case, italicized, bolded and wave underlined. pSZ6317 is written as FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE (D209A)-PmSAD2-1 utr::FAD2-2
TABLE-US-00052 SEQIDNO:133NucleotidesequenceofBnOTE(D209A)inpSZ6317: ![custom-character]()
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atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaagg acgacgacgacaag![custom-character]()
[0182] The sequence of the pSZ6318 transforming DNA is same as pSZ6315 except two point mutations, D124A and D209A, the BnOTE (D124A, D209A) DNA sequence is provided below. The three-nucleotide codon corresponding to the target two amino acids, D124 and D209, are in lower case, italicized, bolded and wave underlined. pSZ6318 is written as FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE (D124A, D209A)-PmSAD2-1 utr::FAD2-2
TABLE-US-00053 SEQIDNO:134NucleotidesequenceofBnOTE(D124A,D209A)inpSZ6318 ![custom-character]()
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atggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaagg acgacgacgacaag![custom-character]()
[0183] The DNA constructs containing the wild-type and mutant BnOTE genes were transformed into the parental strain S8588. Primary transformants were clonally purified and grown under standard lipid production conditions at pH5.0. The resulting profiles from representative clones arising from transformations with pSZ6315, pSZ6316, pSZ6317, and pSZ6318 into S8588 are shown in Table 26. The parental strain S8588 produces 5.4% C18:0, when transformed with the DNA cassette expressing wild-type BnOTE, the transgenic lines produce 11% C18:0. The BnOTE mutant (D124A) increased the amount of C18:0 by at least 2 fold compared to the wild-type protein. In contrast, the BnOTE D209A mutation appears to have no impact on the enzyme activity/specificity of the BnOTE thioesterase. Finally, expression of the BnOTE (D124A, D209A) resulted in very similar fatty acid profile to what we observed in the transformants from S8588 expressing BnOTE (D124A), again indicating that D209A has no significant impact on the enzyme activity.
TABLE-US-00054 TABLE 26 Fatty acid profiles in S8588 and derivative transgenic lines transformed with wild-type and mutant BnOTE genes Fatty Acid Area % Transforming DNA Sample ID C16:0 C18:0 C18:1 C18:2 pH5; S8588 (parental strain) 3.00 5.43 81.75 6.47 D5309, pSZ6315; pH5; S8588, D5309-6; 3.86 11.68 76.51 5.06 wild-type BnOTE pH5; S8588, D5309-2; 3.50 11.00 77.80 4.95 pH5; S8588, D5309-9 ; 3.51 10.72 78.03 5.00 pH5; S8588, D5309-10; 3.55 10.69 78.06 4.96 pH5; S8588, D5309-11; 3.61 10.69 78.05 4.95 D5310, pSZ6316, pH5; S8588, D5310-6; 4.27 31.55 55.31 5.30 BnOTE (D124A) pH5; S8588, D5310-1; 4.53 30.85 54.71 6.03 pH5; S8588, D5310-5; 5.21 20.75 65.43 5.02 pH5; S8588, D5310-10; 4.99 19.18 67.75 5.00 pH5; S8588, D5310-2; 4.90 18.92 68.17 4.98 D5311, pSZ6317, pH5; S8588, D5311-3; 3.50 11.90 76.95 4.98 BnOTE (D209A) pH5; S8588, D5311-4; 3.63 11.35 77.44 4.94 pH5; S8588, D5311-14; 3.47 11.23 77.68 4.98 pH5; S8588, D5311-10; 3.60 11.20 77.53 5.00 pH5; S8588, D5311-12; 3.53 11.12 77.59 5.09 D5312, pSZ6318, pH5; S8588, D5312-20; 4.79 37.97 47.74 6.01 BnOTE (D124A, pH5; S8588, D5312-40; 5.97 22.94 62.20 5.11 D209A) pH5; S8588, D5312-39; 6.07 22.75 62.24 5.17 pH5; S8588, D5312-16; 5.25 18.81 67.36 5.09 pH5; S8588, D5312-26; 4.93 18.70 68.37 4.96
Example 9 Variant Garcinia mangostana Thioeserase
[0184] In this example, we demonstrate the ability to modify the activity and specificity of a FATA thioesterase originally isolated from Garcinia mangostana (GmFATA, accession 004792), using site directed mutagenesis targeting six amino acid positions within the enzyme and various combinations thereof. Facciotti et al (NatBiotech 1999) had previously altered three of the amino acids (G108, S111, V193). The remaining three amino acids targeted are L91, G96, and T156.
[0185] To test the impact of each mutation on the activity of the GmFATA, the wild-type and mutant genes were cloned into a vector enabling expression within the P. moriformis strain S3150. Table 27 summarizes the results from a three day lipid profile screen comparing the wild-type GmFATA with the 14 mutants. Three GmFATA mutants (DNA lot numbers D3998, D4000, D4003) increased the amount of C18:0 by at least 1.5 fold compared to the wild-type protein (DNA lot number D3997). D3998 and D4003 were mutations that had been described by Facciotti et al (NatBiotech 1999) as substitutions that increased the activity of the GmFATA. Strain S3150 expressing the mutations contained in DNA lot number D4000 was based on research at Solazyme which demonstrated this position influenced the activity of the FATB thioesterases. All of the constructs were codon optimized to reflect UTEX 1435 codon usage. Non-mutated GmFATA increases the fatty acid content of C18:0 and decreases the fatty acid content of C18:1 and C18:2. As can be seen in Table 27 the G90A mutant GmFATA increases the fatty acid content of C18:0 and decreases the fatty acid content of C18:1 and C18:2 when compared to the wild-type GmFATA.
TABLE-US-00055 TABLE 27 Algal Strain DNA # GmFATA C14:0 C16:0 C18:0 C18:1 C18:2 P. S3150 1.63 29.82 3.08 55.95 7.22 moriformis D3997 Wild-Type 1.79 29.28 7.32 52.88 6.21 S3150 pSZ5083 GmFATA D3998 S111A, 1.84 28.88 11.19 49.08 6.21 pSZ5084 V193A D3999 S111V, 1.73 29.92 3.23 56.48 6.46 pSZ5085 V193A D4000 G96A 1.76 30.19 12.66 45.99 6.01 pSZ5086 D4001 G96T 1.82 30.60 3.58 55.50 6.28 pSZ5087 D4002 G96V 1.78 29.35 3.45 56.77 6.43 pSZ5088 D4003 G108A 1.77 29.06 12.31 47.86 6.08 pSZ5089 D4007 G108V 1.81 28.78 5.71 55.05 6.26 pSZ5093 D4004 L91F 1.76 29.60 6.97 53.04 6.13 pSZ5090 D4005 L91K 1.87 28.89 4.38 56.24 6.35 pSZ5091 D4006 L91S 1.85 28.06 4.81 56.45 6.47 pSZ5092 D4008 T156F 1.81 28.71 3.65 57.35 6.31 pSZ5094 D4009 T156A 1.72 29.66 5.44 54.54 6.26 pSZ5095 D4010 T156K 1.73 29.95 3.17 56.86 6.21 pSZ5096 D4011 T156V 1.80 29.17 4.97 55.44 6.27 pSZ5097
[0186] Nucleotide sequence of the GmFATA wild-type parental gene expression vector is shown below (D3997, pSZ5083). The plasmid pSZ5083 can be written as THI4a::CrTUB2-NeoR-PmPGH:PmSAD2-2Ver3-CpSAD1tp_GarmFATA1 FLAG-CvNR::THI4a. The 5 and 3 homology arms enabling targeted integration into the Thi4 locus are noted with lowercase; the CrTUB2 promoter is noted in uppercase italic which drives expression of the neomycin selection marker noted with lowercase italic followed by the PmPGH 3UTR terminator highlighted in uppercase. The PmSAD2-1 promoter (noted in bold text) drives the expression of the GmFATA gene (noted with lowercase bold text) and is terminated with the CvNR 3UTR noted in underlined, lower case bold. Restriction cloning sites and spacer DNA fragments are noted as underlined, uppercase plain lettering. The nucleotide sequence for all of the GmFATA constructs disclosed in this example is identical to that of pSZ5083 with the exception of the encoded GmFATA. The promoter, 3UTR, selection marker and targeting arms are the same as described for pSZ5083. The individual GmFATA mutant sequences are shown below. The amino acid sequence of the unmutagenized GmFATA is showing in FIG. 1. The amino acid sequences of the altered GmFATA proteins are shown below.
TABLE-US-00056 SEQ ID NO: 135 pSZ5083 ccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttg gcacggctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcg tccagagacggcaagtcgtccatcctctgcgtgtgtggcgcgacgctgcagcagtccctctg cagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttctta aagcaaatgactgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagat agcgaccatgttgctgcgtctgaaaatctggattccgaattcgaccctggcgctccatccat gcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccactta gattcccgatcacccacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctc aagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgccgctggctgcaggg tctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggt cacttgccttccagattgccaataaactcaattatggcctctgtcatgccatccatgggtct gatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccctgccccgccca ccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaGGTACCCTTTCTTGCGCTA TGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACAC CGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCA GGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAA GCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCA CTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTCTAGAATATC Aatgatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcg gctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcc cagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcagga cgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacg tggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctg tcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgca caccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgccc gcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctg gcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggt gacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttca tcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgac atcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgc ccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttctgaCAATTGACGC CCGCGCGGCGCACCTGACCTGTTCTCTCGAGGGCGCCTGTTCTGCCTTGCGAAACAAGCCCC TGGAGCATGCGTGCATGATCGTCTCTGGCGCCCCGCCGCGCGGTTTGTCGCCCTCGCGGGCG CCGCGGCCGCGGGGGCGCATTGAAATTGTTGCAAACCCCACCTGACAGATTGAGGGCCCAGG CAGGAAGGCGTTGAGATGGAGGTACAGGAGTCAAGTAACTGAAAGTTTTTATGATAACTAAC AACAAAGGGTCGTTTCTGGCCAGCGAATGACAAGAACAAGATTCCACATTTCCGTGTAGAGG CTTGCCATCGAATGTGAGCGGGCGGGCCGCGGACCCGACAAAACCCTTACGACGTGGTAAGA AAAACGTGGCGGGCACTGTCCCTGTAGCCTGAAGACCAGCAGGAGACGATCGGAAGCATCAC AGCACAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTC GCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCAT TAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGT GGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGTGAAAACTCGCTCGACCGCCCGC GTCCCGCAGGCAGCGATGACGTGTGCGTGACCTGGGTGTTTCGTCGAAAGGCCAGCAACCCC AAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGCTTGGACCAGATCCCCCACGATGC GGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATTGGTGTCC GATACCTTGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGC TACCAGGGTTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACCGGCGCAGAGGGTGAGT TGATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGTGTGTCTGTTTTCGGCTGCAC AATTTCAATAGTCGGATGGGCGACGGTAGAATTGGGTGTTGCGCTCGCGTGCATGCCTCGCC CCGTCGGGTGTCATGACCGGGACTGGAATCCCCCCTCGCGACCCTCCTGCTAACGCTCCCGA CTCTCCCGCCCGCGCGCAGGATAGACTCTAGTTCAACCAATCGACAACTAGTatggccaccg catccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccggg ccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgt ggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcc tggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttc atcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacct gctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctcca ccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatc tacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaa gatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcg ccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggac gtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaa caactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcc tggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggc tgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccct ggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccct ccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgcc aacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagat caaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacg gcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaATCGATgcagca gcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccaca cttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgat cttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccaccccca gcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctg ctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctc cgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaag tagtgggatgggaacacaaatggaAAGCTTGAGCTCcagcgccatgccacgccctttgatgg cttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaata atacacttgatttcttgctcacggcaatctcggcttgtccgcaggttcaaccccatttcgga gtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgagg tgagctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagc acccaagcaaaatgtcaagtgcgttccgatttgcgtccgcaggtcgatgttgtgatcgtcgg cgccggatccgccggtctgtcctgcgcttacgagctgaccaagcaccctgacgtccgggtac gcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcg cgaaactgtgctcgattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgag cagggcgttgctcccggcggcggcgcctggctggggggacagctgttctcggccatgtgtgt acgtagaaggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttca gactattgttgaatgtttttagatttcttaggatgcatgatttgtctgcatgcgact SEQIDNO:136AminoacidsequenceofGmFATAwild-typeparentalgene; D3997,pSZ5083.ThealgaltransitpeptideisunderlinedandtheFLAGepitopetagis uppercasebold MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:137AminoacidsequenceofGmFATAS111A,V193Amutantgene; D3998,pSZ5084.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheS111A,V193Aresiduesarelower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFaTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:138AminoacidsequenceofGmFATAS111V,V193Amutantgene; D3999,pSZ5085.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheS111V,V193Aresiduesarelower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFvTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:139AminoacidsequenceofGmFATAG96Amutantgene;D4000, pSZ5086.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheG96Aresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVaCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:140AminoacidsequenceofGmFATAG96Tmutantgene;D4001, pSZ5087.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheG96Tresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVtCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:141AminoacidsequenceofGmFATAG96Vmutantgene;D4002, pSZ5088.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheG96Vresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVvCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:142AminoacidsequenceofGmFATAG108Amutantgene; D4003,pSZ5089.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheG108Aresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTaGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:143AminoacidsequenceofGmFATAL91Fmutantgene;D4004, pSZ5090.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheL91Fresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANfLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:144AminoacidsequenceofGmFATAL91Kmutantgene;D4005, pSZ5091.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheL91Kresidueislower-casebold MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANkLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:145FIG.10.AminoacidsequenceofGmFATAL915mutant gene;D4006,pSZ5092.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheL91Sresidueislower-casebold MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANsLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:146AminoacidsequenceofGmFATAG108Vmutantgene; D4007,pSZ5093.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheG108Vresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTvGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:147AminoacidsequenceofGmFATAT156Fmutantgene; D4008,pSZ5094.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheT156Fresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGfRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:148AminoacidsequenceofGmFATAT156Amutantgene; D4009,pSZ5095.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheT156Aresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGaRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:149AminoacidsequenceofGmFATAT156Kmutantgene;D4010, pSZ5096.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheT156Kresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGkRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:150AminoacidsequenceofGmFATAT156Vmutantgene; D4011,pSZ5097.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheT156Vresidueislower-casebold. MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGvRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK SEQIDNO:151NucleotidesequenceoftheGmFATAS111A,V193Amutantgene (D3998,pSZ5084).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesame aspSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:152NucleotidesequenceoftheGmFATAS111V,V193Amutantgene (D3999,pSZ5085).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethe sameaspSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttcgtcaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:153NucleotidesequenceoftheGmFATAG96Amutantgene(D4000, pSZ5086).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:154NucleotidesequenceoftheGmFATAG96Tmutantgene(D4001, pSZ5087).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgacgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:155NucleotidesequenceoftheGmFATAG96Vmutantgene(D4002, pSZ5088).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtggtgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:156NucleotidesequenceoftheGmFATAG108Amutantgene (D4003,pSZ5089).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethe sameaspSZ50836. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgcc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:157NucleotidesequenceoftheGmFATAL91Fmutantgene(D4004, pSZ5090).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacttcctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:158NucleotidesequenceoftheGmFATAL91Kmutantgene(D4005, pSZ5091).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacaagctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:159NucleotidesequenceoftheGmFATAL91Smutantgene(D4006, pSZ5092).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaactcgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:160NucleotidesequenceoftheGmFATAG108Vmutantgene (D4007,pSZ5093).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethe sameaspSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgtc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:161NucleotidesequenceoftheGmFATAT156Fmutantgene(D4008, pSZ5094).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcttccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:162NucleotidesequenceoftheGmFATAT156Amutantgene(D4009, pSZ5095).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcgcgcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:163NucleotidesequenceoftheGmFATAT156Kmutantgene(D4010, pSZ5096).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcaagcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga SEQIDNO:164NucleotidesequenceoftheGmFATAT156Vmutantgene(D4011, pSZ5097).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcgtgcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga
TABLE-US-00057 SEQUENCES SEQIDNO:1 gcgaggggtc tgcctgggcc agccgctccc tctgaacacg ggacgcgtgg tccaattcgg 60 gcttcgggac cctttggcgg tttgaacgcc tgggagaggg cgcccgcgag cctggggacc 120 ccggcaacgg cttccccaga gcctgccttg caatctcgcg cgtcctctcc ctcagcacgt 180 ggcggttcca cgtgtggtcg ggcgtcccgg actagctcac gtcgtgacct agcttaatga 240 acccagccgg gcctgcagca ccaccttaga ggttttgatt atttgattag accaatctat 300 tcacc 305 SEQIDNO:2 ggcgaataga ttggtataat gaaataatca aaacctctta ggcggtgcta caggcccggc 60 tgggttcatt aagctaggtc acgacgcgag ctagtccggg aagcccgacc acacgtggaa 120 ccgccacgtg ctgagggaga ggacgcgcga gattgcaagg caggctctgg ggaagccgtt 180 gccggggtcc ccaggctcgc gggcgcccca tccctggcgt tcaaaccgcc aaagggtccc 240 gaagcccgaa ttggaccacg cgtcccgtgt ttagagggag cggctggccc aggcagaccc 300 ctcgc 305 SEQIDNO:3 ggtgaataga ttggtctaat caaataatca aaacctctaa ggtggtgctg caggcccggc 60 tgggttcatt aagctaggtc acgacgtgag ctagtccggg acgcccgacc acacgtggaa 120 ccgccacgtg ctgagggaga ggacgcgcga gattgcaagg caggctctgg ggaagccgtt 180 gccggggtcc ccaggctcgc gggcgccctc tcccaggcg tcaaaccgcc aaagggtccc 240 gaagcccgaa ttggaccacg cgtcccgtgt tcagagggag cggctggccc aggcagaccc 300 ctcgc 305 SEQIDNO:4 gtgatgggtt ctttagacga tccagcccag gatcatgtgt tgcccacatg gagcctatcc 60 acgctggcct agaaggcaag cacatttcaa ggtgaaccca cgtccatgga gcgatggcgc 120 caatatctcg cctctagacc aagcggttct caccccaact gcgtcatttg tatgtatggc 180 tgcaaagttg tcggtacgat agaggccgcc aacctggcgg cgagggcgag gagctggttg 240 ccgatctgtg cccaagcatg tgtcggagct cggctgtctc ggcagcgagc tcctgtgcaa 300 ggggcttgca tcgagaatgt caggcgatag acactgcacg ttggggacac ggaggtgccc 360 ctgtggcgtg tcctggatgc cctcgggtcc gtcgcgagaa gctctggcga ccagcacccg 420 gccacaaccg cagcaggcgt tcacccacaa gaatcttcca gatcgtgatg cgcatgtatc 480 gtgacacgat tggcgaggtc cgcaggacgc acacggactc gtccactcat cagaactggt 540 cagggcaccc atctgcgtcc cttttcagga accacccacc gctgccaggc accttcgcca 600 gcggcggact ccacacagag aatgccttgc tgtgagagac catggccggc aagtgctgtc 660 ggatctgccc gcatacggtc agtccccagc acaaggaagc caagagtaca ggctgttggt 720 gtcgatggag gagtggccgt tcccacaagt agtgagcggc agctgctcaa cggcttcccc 780 ctgttcatct tggcaaagcc agtgacttcc tacaagtatg tgatgcagat cggcactgca 840 atctgtcggc atgcgtacag aacatcggct cgccagggca gcgttgctcg ctctggatga 900 gctgcttggg aggaatcatc ggcacacgcc cgtgccgtgc ccgcgccccg cgcccgtcgg 960 gaaaggcccc cggttaggac actgccgcgt cagccagtcg tgggatcgat cggacgtggc 1020 gaatcctcgc ccggacaccc tcatcacacc ccacatttcc ctgcaagcaa tcttgccgac 1080 aaaatagtca agatccattg ggtttaggga acacgtgcga gactgggcag ctgtatctgt 1140 ccttgccccg cgtcaaattc ctgggcgtga cgcagtcaca ggagaatcta ttagaccctg 1200 gacttgcagc tcagtcatgg gcgtgagtgg ctaaagcacc taggtcaggc gagtaccgcc 1260 ccttccccag gattcactct tctgcgattg acgttgagcc tgcatcgggc tgcttcgtca 1320 cc 1322 SEQIDNO:5 tcggagctaa agcagagact ggacaagact tgcgttcgca tactggtgac acagaatagc 60 tcccatctat tcatacgcct ttgggaaaag gaacgagcct tgtggcctct gcattgctgc 120 ctgctttgag gccgaggacg gtgcgggacg ctcagatcca tcagcgatcg ccccaccctc 180 agagcacctc cgatccaagg caatactatc aggcaaagtt tccaaattca aacattccaa 240 aatcacgcca gggactggat cacacacgca gatcagcgcc gttttgctct ttgcctacgg 300 gcgactgtgc cacttgtcga cccctggtga cgggagggac cacgcctgcg gttggcatcc 360 acttcgacgg acccagggac ggtttctcat gccaaacctg agatttgagc acccagatga 420 gcacattatg cgttttagga tgcctgagca gcgggcgtgc aggaatctgg tctcgccaga 480 ttcaccgaag atgcgcccat cggagcgagg cgagggcttt gtgaccacgc aaggcagtgt 540 gaggcaaaca catagggaca cctgcgtctt tcaatgcaca gacatctatg gtgcccatgt 600 atataaaatg ggctacttct gagtcaaacc aacgcaaact gcgctatggc aaggccggcc 660 aaggttggaa tcccggtctg tctggatttg agtttgtggg ggctatcacg tgacaatccc 720 tgggattggg cggcagcagc gcacggcctg ggtggcaatg gcgcactaat actgctgaaa 780 gcacggctct gcatcccttt ctcttgacct gcgattggtc cttttcgcaa gcgtgatcat 840 c 841 SEQIDNO:6 tcggagctaa agcagaaact gaacaagact tgcgttcgca tacttgtgac actgaatagg 60 ttcaatctat tcatacgcct ttgggaaact gaacgagcct tgtggcctct gcattgctgc 120 ctgctttgag gccgaggacg gcgcggaacg cacagatcca tcagcgatcg ccccaccctc 180 agagtacatc cgatccaagg caatactatc aggcaaagtt tccaaattca aacattccaa 240 aattacgtca gggactggat cacacacgca gatcagcgcc gttttgctct ttgcctacgg 300 gcgactgtgc cacttgtcga cgcctggtga cgggagggac cacgcctgcg gttggcatcc 360 acttcgacgg acccagggac ggtctcacat gccaaacctg agatttgagc accaagatga 420 gcacattatg cgtttttgga tgcctgagca gcgggcgtgc aggaatctgg tctcgccaga 480 ttcaccgaag atgcggccat cggagcgagg cgagggctgt gtggccacgc caggcagtgt 540 gaggcaaaca cacagggaca tctgcttctt tcgatgcaca gacatctatg ttgcccgtgc 600 atataaaatg ggctacttct gaatcaaacc aacgcaaact tcgctatggc aaggccggcc 660 aaggttggaa tcccggtctg tctggatttg agtttgtggg ggctatcacg tgacaatccc 720 tgggattggg cggcagcagc gcacggcctg gatggcaatg gcgcactaat actgctgaaa 780 gcacggctct gcatcccttt ctcttgacct gcgattggtc cttttcgcaa gcgtgatcat 840 c 841 SEQIDNO:7 caccgatcac tccgtcgccg cccaagagaa atcaacctcg atggagggcg aggtggatca 60 gaggtattgg ttatcgttcg ttcttagtct caatcaatcg tacaccttgc agttgcccga 120 gtttctccac acatacagca cctcccgctc ccagcccatt cgagcgaccc aatccgggcg 180 atcccagcga tcgtcgtcgc ttcagtgctg accggtggaa agcaggagat ctcgggcgag 240 caggaccaca tccagcccag gatcttcgac tggctcagag ctgaccctca cgcggcacag 300 caaaagtagc acgcacgcgt tatgcaaact ggttacaacc tgtccaacag tgttgcgacg 360 ttgactggct acattgtctg tctgtcgcga gtgcgcctgg gcccttacgg tgggacactg 420 gaactccgcc ccgagtcgaa cacctagggc gacgcccgca gcttggcatg acagctctcc 480 ttgtgttcta aataccttgc gcgtgtggga ga 512 SEQIDNO:8 atccaccgat cactccgtcg ccgcccaaga gaattcaacc tcgatggagg gcaaggtgga 60 tcagaggtat tggttatcgt tcgctattag tctcaatcaa tcgtgcacct tgcagttgct 120 cgagtttctc cacacataca gcacctcccg ctcccagccc attcgagcga cccaatccgg 180 gcgatcccag cgatcgtcgt cgcttcagtg ctgaccggtg gaaagcagga gatctcgggc 240 gagcaggacc acatccagca caggatcttc gactggctca gagctgaccc tcacgcggca 300 cagcaaaagt agcccgcacg cgttatgcaa acaggttaca acctgtccaa cactgttgcg 360 acgttgactg gctacattgt ctgtctgtcg cgagtacgcc tggaccctta cggtgggaca 420 ctggaactcc gccccgagtc gaacacctag ggcgacgccc gcagcttggc atgacagctc 480 tccttgtatt ctaaatacct cgcgcgtgtg ggagaa 516 SEQIDNO:9 atgatgcgcg tgtacgacta tcaaggaaga aagaggactt aatttcttac cttctaacca 60 ccatattctt tttgctggat gcttgctcgt ctcgatgaca attgtgaacc tcttgtgtga 120 ccctgaccct gctgcaaggc tctccgaccg cacgcaaggc gcagccggcg cgtccggagg 180 cgatcggatc caatccagtc gtcctcccgc agcccgggca cgtttgccca tgcaggccct 240 tccacaccgc tcaagagact cccgaacacc gcccactcgg cactcgcttc ggctgccgag 300 tgcgcgtttg agtttgccct gccacagaag acacc 335 SEQIDNO:10 atgatgcgcg tgtacgacta tcaaggaaga aagaggactt aatttcttac cttctaacca 60 ccatattctt tttgctggat gcttgctcgt ctcgatgaca attgtgaacc tcttgtgtga 120 ccctgaccct gctgcaaggc tctccgaccg cacgcaaggc gcagccggcg cgtccggagg 180 cgatcggatc caatccagtc gtcctcccgc agcccgggca cgtttgccca tgcaggccct 240 tccacaccgc tcaagagact cccgaacacc gcccactcgg cactcgcttc ggctgccgag 300 tgcgcgtttg agtttgccct gccacaggag acatc 335 SEQIDNO:11 cccgggcgag ctgtacgcct acggagcgag gcctggtgtg accgttgcga tctcgccagc 60 agacgtcgcg gagcctcgtc ccaaaggccc tttctgatcg agcttgtcgt ccactggacg 120 ctttaagttg cgcgcgcgat gggataaccg agctgatctg cactcagatt ttggtttgtt 180 ttcgcgcatg gtgcagcgag gggaggtact acgctggggt acgagatcct ccggattccc 240 agaccgtgtt gccggcattt acccggtcat cgccagcgat tcgggacgac aaggccttat 300 cctgtgctga gacgctcgag cacgtttata aaattgtggg taccgcggta tgcacagcgt 360 tcaacacgcg ccacgccgaa attggttggt gggggagcac gtatgggact gacgtatggc 420 cagcagcgaa cactcaccga acaagtgcca atgtatacct tgcatcaatg atgctccggc 480 agcttcgatt gactgtctcg aaaaagtgtg agcaagcaga tcatgtggcc gctctgtcgc 540 gcagcacctg acgcattcga cacccacggc aatgcccagg ccagggaata gagagtaaga 600 caactcccat tgttcagcaa aacattgcac tgcagtgcct tcacaactat acaatgaatg 660 ggagggaata tgggctctgc atgggacagc ttagctggga cattcggcta ctgaacaaga 720 aaaccccacg agaaccaatt ggcgaaacct gccgggagga ggtgatcgtt tctgtaaatg 780 gcttacgcat tcccccccgg cggctcacga ggggtgtggt gaaccctgcc agctgatcaa 840 gtgcttgctg acgtcggcca gggaggtgta tgtgattggg ccgtggggcg tgagttatcc 900 taccgccgga cccgcgaagt cacatgacga atggccgtgc gggatgacga gagcacgact 960 cgctctttct tcgccggccc ggcttcatgg aggacaataa taaagggtgg ccaccggcaa 1020 cagccctcca tacctgaacc gattccagac ccaaacctct tgaattttga gggatccagt 1080 tcaccggtat agtcacg 1097 SEQIDNO:12 atccccgggc gagctgtacg cctacggagc gaggcctggt gtgaccgttg cgatctcgcc 60 agcagacgtc gcggagcctc gtcccaaagg ccctttctga tcgagcttgt cgtccactgg 120 acgctttaag ttgcgcgcgc gatgggataa ccgagctgat ctgcactcag attttggttt 180 gttttcgcgc atggtgcagc gaggggaggt actacgctgg ggtacgagat cctccggatt 240 cccagaccgt gttgccggca tttacccggt catcgccagc gattcgggac gacaaggcct 300 tatcctgtgc tgagacgctc gagcacgttt ataaaattgt ggtcaccgtg gtacgcacag 360 cgtccaacac gcgccacgcc gaaattcgtt ggtgggggag cacgtatcgg actgacgtat 420 ggccagcagc gaacactcac caaacaggtg ccaatgtata gcttgcatca atgatgctct 480 ggcagcttcg attgactgtc tcgaaaaagt gtgtgcaaac agattatgtg gccgctctgt 540 ggccgcgcag cacctgacgc actcgacacc cacggcaatg cccaggccaa ggaacagaga 600 gtaagacaac tcccattgtt cagtaaaaca ttgcactgca gtgccttcac aaacatacaa 660 cgaatgggag ggaatatggg cttcgaatgg gacagcttag ctgggacatt cggttactga 720 acaagaaaac cccacgagaa ccaactggcg aaacctgccg ggaggaggtg atcgtttttg 780 taaatggctt acgcattccc cccccggcgg ctcacggggg gtgtggtgaa ccctgccagc 840 tgatcaagtg cttgctgacg tcggccaggg aggtgtatgt gatttggccg tggggcgtga 900 gttatcctac cgccggaccc gcgaagtcac atgacgaatg gccgtgcggg atgacgagag 960 cagggctcgc tctttcttcg ccggcccggc ttcatggagg acaataataa agggtggcca 1020 ccggcaacag ccctccatac ctgaaccga ttccagaccca aacctcttga attttgaggg 1080 atccagttca ccggtatagt cacga 1105 SEQIDNO:13 gcgagtggtt ttgctgccgg gaagggagtg gggagcgtcg agcgagggac gcggcgctcg 60 aggcgcacgt cgtctgtcaa cgcgcgcggc cctcgcggcc cgcggcccca cccagctcta 120 atcatcgaaa actaagaggc tccacacgcc tgtcgtagaa tgcatgggat tcgccagtag 180 accacgatct gcgccgaaga agctggtcta cccgacgttt tttgttgctc ctttattctg 240 aatgatatga agatagtgtg cgcagtgcca cgcataggca tcaggagcaa gggaggacgg 300 gtcaacttga aagaaccaaa ccatccatcc gagaaatgcg catcatcttt gtagtaccat 360 caaacgcctt ggccaatgtc ttctgcatgg acaacacaac ctgctcctgg ccacacggtc 420 gacttggagc gccccatgcg cccaggtcgc cacgacccgc ggcccagcgc gcggcgattc 480 gcctcacgag atcccggcgg acccggcacg cccgcgggcc gacggtgcgc ttggcgatgc 540 tgctcattaa cccacggccg tcacccgatc cacatgctct ttttcaacac atccacattg 600 gaatagagct ctaccagggt gagtactgca ttctttgggg ctgggaggac cccactcgac 660 acctggtcct tcatcggccg aaagcccgaa cctgagcgct tccccgcccc gttcctcatc 720 cccgactttc cgatggccca ttgcagtttc aaac 754 SEQIDNO:14 atctgggtgg aggactggga gtaagatgta aggatattaa ttaaacattc tagtttgttg 60 atggcacaac agtcaatgca tttcagtcgt cttgctcctt ataacctatg cgtgtgccat 120 cgccggccat gcacctgtgg cgtggtaccg accatcgggg agaggcccga gattcggagg 180 tacctcccgc cctgggcgag cccttcacgt gacggcacaa gtcccttgca tcggcccgcg 240 agcacggaat acagagcccc gtgcccccca cgggccctca catcatccac tccattgttc 300 ttgccacacc gatcagca 318 SEQIDNO:15 tgggtggagg actgggaaga agatgtaagg atatcaattt aacattctag tttgttgatg 60 gcacaacagt cactgaatac cgggcgtctg gctgctaaaa tagccggagc gtgtgccatc 120 gccggccatg catctgtggc gtggtaccga ccatcaggga gaggcccgag attcggaggt 180 acctcccgcc ctgggcgagc ccttcacgtg acggcacaag tcccttgcat cggcccgcga 240 gcacggaata cagagccccg tgctccccac gggccctcac atcatccact ccattgttct 300 tgccacaccg atcagc 316 SEQIDNO:16 ataacgaggc acaatgatcg atatttctat cgaacaactg tatttagccc tgtacgtacc 60 ccgctcttgg gccagcccgt ccgtgcttgc cttcggaaaa ttgcatggcg cctcatgcaa 120 actcgcgctc tcacagcaga tctcgcccag ctcccgggag agcaatcgcg ggtggggccc 180 ggggcgaatc caggacgcgc cccgcggggc cgctccactc gccagggcca atgggcggct 240 tatagtcctg gcatgggctc tgcatgcaca gtatcgcagt ttgggcgagg tgttgccccc 300 gcgatttcga atacgcgacg cccggtactc gtgcgagaac agggttcttg Protothecamoriformis(UTEX1435)Amt02promoter SEQIDNO:17 TCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATG GCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCC CTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGGGGCTGGCGGGAAAACAGGC TTCGTGTGCTCAGGTTATGGGAGGTGCAGGACAGCTCATTAAACGCCAACAATCGCACAATTCATGGCAAGCTAA TCAGTTATTTCCCATTAACGAGCTATAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGC CCTTCCCTCAACCCTAGGTATGCGCACATGCGGTCGCCGCGCAACGCGCGCGAGGGCCGAGGGTTTGGGACGGGC CGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAA ATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGTGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCG ACTGCCCTAGTATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACA TTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGATCCCCAACCTCCTTATTGTTCTGTCGACAGAGTG GGCCCAGAGGCCGGTCGCAGCC Protothecamoriformis(UTEX1435)Amt03promoter SEQIDNO:18 Ggccgacaggacgcgcgtcaaaggtgctggtcgtgtatgccctggccggcaggtcgttgctgctgctggttagtg attccgcaaccctgattttggcgtcttattttggcgtggcaaacgctggcgcccgcgagccgggccggcggcgat gcggtgccccacggctgccggaatccaagggaggcaagagcgcccgggtcagttgaagggctttacgcgcaaggt acagccgctcctgcaaggctgcgtggtggaattggacgtgcaggtcctgctgaagttcctccaccgcctcaccag cggacaaagcaccggtgtatcaggtccgtgtcatccactctaaagagctcgactacgacctactgatggccctag attcttcatcaaaaacgcctgagacacttgcccaggattgaaactccctgaagggaccaccaggggccctgagtt gttccttccccccgtggcgagctgccagccaggctgtacctgtgatcgaggctggcgggaaaataggcttcgtgt gctcaggtcatgggaggtgcaggacagctcatgaaacgccaacaatcgcacaattcatgtcaagctaatcagcta tttcctcttcacgagctgtaattgtcccaaaattctggtctaccgggggtgatccttcgtgtacgggcccttccc tcaaccctaggtatgcgcgcatgcggtcgccgcgcaactcgcgcgagggccgagggtttgggacgggccgtcccg aaatgcagttgcacccggatgcgtggcaccttttttgcgataatttatgcaatggactgctctgcaaaattctgg ctctgtcgccaaccctaggatcagcggcgtaggatttcgtaatcattcgtcctgatggggagctaccgactaccc taatatcagcccgactgcctgacgccagcgtccacttttgtgcacacattccattcgtgcccaagacatttcatt gtggtgcgaagcgtccccagttacgctcacctgtttcccgacctccttactgttctgtcgacagagcgggcccac aggccggtcgcagcc pSZ3840/D2554transformingconstruct(CpauLPAAT1) SEQIDNO:19 gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatga aatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgt cttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaa ttacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggc ttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagcca ggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggtatgggaaccacgggg [00206]![embedded image]()
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gcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggacaggccga ccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctccggccgcgactccga cggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgtt cggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacgcccagact tcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcccgagatctcctacca ccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccctgtgcaactggggccaggacctga ccactactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcggagttcacgcgccccgactcccgct gcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctgaacaaggccgccccc atgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaacctgacggacgacga ggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaacaacctgaaggcctcct cctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtctggcgctacta cgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtggc gctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctccaagaa gctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgcaaca agaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttcgg ccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttctaccgcctgcgcccc [00217]![embedded image]()
acacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagtt gctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacg ctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtact [00218]![embedded image]()
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[00230]![embedded image]()
[00231]![embedded image]()
atcaacctgttccaggccctgtgcttcgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccg agctgctgctgtccgagctgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttcc gcctgatgggcaaggagcacgccctggtgatcatcaaccacatgaccgagctggactggatgctgggctgggtgatgggcca gcacctgggctgcctgggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttct ccgagtacctgtacatcgagcgctcctgggccaaggaccgcaccaccctgaagtcccacatcgagcgcctgaccgactacccc ctgcccactggatggtgatcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcct ccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgccc gccgtgtacgacgtgaccgtggccttccccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcgtgc tgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagtt cgtggagaaggacgccctgctggacaagcacaacgccgaggacaccttctccggccaggaggtgcaccgcaccggctcccg ccccatcaagtccctgctggtggtgatctcctgggtggtggtgatcaccttcggcgccctgaagttcctgcagtggtcctcctgga agggcaaggccttctccgtgatcggcctgggcatcgtgaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctc [00232]![embedded image]()
[00233]![embedded image]()
gacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtg ctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatcc ctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaac cagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttgagctcagcggcgacggtcctgctacc gtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcct ggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgc cagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactct cgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaag ggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttctt agctcttc pSZ3841/D2555(CpaiLPAAT1) SEQIDNO:20 [00234]![embedded image]()
gccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgacgccgagctgctgcccctgga gacctgtggctgttccactggtgcgccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaagga gcacgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctg ggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccggctacctgttcctg gagcgctcctgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgcccttctggctgatc atcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgcccc gcaacgtgctgatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgacc gtggccttccccaagacctcccccccccccaccatgctgaagctgacgagggccagtccgtggagctgcacgtgcacatcaag cgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgcc ctgctggacaagcacaactccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggt ggtgatctcctgggtggtggtgatcatcttcggcgccctgaagttcctgctgtggtcctccctgctgtcctcctggaagggcaagg ccactccgtgatcggcctgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgaggg [00235]![embedded image]()
[00236]![embedded image]()
pSZ3842/D2556(CigneaLPAAT1) SEQIDNO:21 [00237]![embedded image]()
gccctgtgcttcgtgctgatctggcccctgtccaagaacgtgtaccgccgcatcaaccgcgtgacgccgagctgctgctgatgg acctgctgtgcctgttccactggtgggccggcgccaagatcaagctgacaccgaccccgagaccttccgcctgatgggcatgg agcacgccctggtgatcatgaaccacaagaccgacctggactggatggtgggctggatcctgggccagcacctgggctgcct gggctccatcctgtccatcgccaagaagtccaccaagttcatccccgtgctgggctggtccgtgtggactccgagtacctgttcc tggagcgctcctgggccaaggacaagtccaccctgaagtcccacatggagaagctgaaggactaccccctgcccttctggctg gtgatcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgc cccgcaacgtgctgatcccccacaccaagggcttcgtgtcctgcgtgtccaacatgcgctccacgtgcccgccgtgtacgacgt gaccgtggccttccccaagtcctcccccccccccaccatgctgaagctgttcgagggccagtccatcgtgctgcacgtgcacatc aagcgccacgccctgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggac gccctgctggacaagcacaacgccgaggacaccttctccggccaggaggtgcaccacatcggccgccccatcaagtccctgct ggtggtgatcgcctgggtggtggtgatcatcttcggcgccctgaagttcctgcagtggtcctccctgctgtccacctggaagggc aaggccttctccgtgatcggcctgggcatcgccaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctccaacc [00238]![embedded image]()
pSZ3844/D2557(ChookLPAAT1) SEQIDNO:22 [00239]![embedded image]()
gccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgacgccgagctgctgcccctgga gacctgtggctgttccactggtgcgccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaagga gcacgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctg ggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccgagtacctgttcctg gagcgctcctgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgcccttctggctgatc atcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgcccc gcaacgtgctgatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgacc gtggccttccccaagacctcccccccccccaccatgctgaagctgacgagggccagtccgtggagctgcacgtgcacatcaag cgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgcc ctgctggacaagcacaactccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggt ggtgatctcctgggtggtggtgatcatcttcggcgccctgaagttcctgctgtggtcctccctgctgtcctcctggaagggcaagg ccactccgtgatcggcctgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgaggg [00240]![embedded image]()
[00241]![embedded image]()
CpauLPAAT1 SEQIDNO:23 MAIPAAAVIFLFGLLFFTSGLIINLFQALCFVLVWPLSKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMLGWVMGQHLGCLGSILSVAKKSTKFLPVLGWSMWFSEYLYIERSWAKDRTT LKSHIERLTDYPLPFWMVIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVYDVT VAFPKTSPPPTLLNLFEGQSIVLHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHRTG SRPIKSLLVVISWVVVITFGALKFLQWSSWKGKAFSVIGLGIVTLLMHMLILSSQAERSSNPAKVAQAKLKTELS ISKKATDKENSEQIDNO: CprocL2AAT1 SEQIDNO:24 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTQTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVYDLT VAFPKTSPPPTLLNLFEGQSVVLHVHIKRHAMKDLPESDDEVAQWCRDKEVEKDALLDKHNAEDTFSGQELQHTG RRPIKSLLVVISWVVVIAFGALKFLQWSSWKGKAFSVIGLGIVTLLMHMLILSSQAERSKPAKVAQAKLKTELSI SKTVTDKEN CprocL2AAT1b SEQIDNO:25 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTQTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVYDLT VAFPKTSPPPTLLNLFEGQSVVLHVHIKRHAMKDLPESDDEVAQWCRDKEVEKSEQIDNO: CprocL2AAT2a SEQIDNO:26 IVNLVQAVCFVLVRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDI DWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPFWLALFV EGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSS VLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNADDTFSGQELQDTGRPIKSLLVVISWAVLEVFGAV KFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKAKIEGESSKTEMEKEK CprocL2AAT2b SEQIDNO:27 IVNLVQAVCFVLVRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDI DWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPFWLALFV EGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSS VLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNADDTFSGQELQDTGRPIKSLLV CpaiLPAAT1 SEQIDNO:28 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVFAELLPLEFLWLFHWCAGAKLKLFTDP ETFRLMGKEHALVIINHKIELDWMVGWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSGYLFLERSWAKDKIT LKSHIESLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPAIYDVT VAFPKTSPPPTMLKLFEGQSVELHVHIKRHAMKDLPESDDAVAQWCRDKEVEKDALLDKHNSEDTFSGQEVHHVG RPIKALLVVISWVVVIIFGALKFLLWSSLLSSWKGKAFSVIGLGIVAGIVTLLMHILILSSQAEGSNPVKAAPAK LKTELSSSKKVTNKEN ChookLPAAT1 SEQIDNO:29 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVFAELLPLEFLWLFHWCAGAKLKLFTDP ETFRLMGKEHALVIINHKIELDWMVGWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSEYLFLERSWAKDKIT LKSHIESLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPAIYDVT VAFPKTSPPPTMLKLFEGQSVELHVHIKRHAMKDLPESDDAVAQWCRDKEVEKDALLDKHNSEDTFSGQEVHHVG RPIKALLVVISWVVVIIFGALKFLLWSSLLSSWKGKAFSVIGLGIVAGIVTLLMHILILSSQAEGSNPVKAAPAK LKTELSSSKKVTNKEN ChookL2AAT2a SEQIDNO:30 LSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETFNLMGKEHA LVVCNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDY PLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSVPPT MLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLLVVIS WAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKNEGESSKTEMEKE H ChookL2AAT2b SEQIDNO:31 QIKVFTDHETFNLMGKEHALVVCNHKSDIDWLVGWVLAQWSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLER SWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSF VPAIYDVTVAIPKTSVPPTMLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFS GQELQDIGRPIKSLLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKV APAKLKKEGESSKPETDKQN ChookL2AAT3a SEQIDNO:32 LSLLFFVSGLIVNLVQAVCFVLIRPLLKNTYRRINRVVAELLWLELVWLIDWWAGIKIKVFTDHETFHLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSQMRSFVPAIYDVTVAIPKTSPPPT LLRMFKGQSSVLHVHLKRHLMNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVVIS WATLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKPKNEGESSKTEMEKE H ChookL2AAT3b SEQIDNO:33 LSLLFFVSGLIVNLVQAVCFVLIRPLLKNTYRRINRVVAELLWLELVWLIDWWAGIKIKVFTDHETFHLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSQMRSFVPAIYDVTVAIPKTSPPPT LLRMFKGQSSVLHVHLKRHLMNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLLVVIS WAVLEIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKPETDKE N CigneaLPAAT1 SEQIDNO:34 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVFAELLLMDLLCLFHWWAGAKIKLFTDP ETFRLMGMEHALVIMNHKTDLDWMVGWILGQHLGCLGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKDKST LKSHMEKLKDYPLPFWLVIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSCVSNMRSFVPAVYDVT VAFPKSSPPPTMLKLFEGQSIVLHVHIKRHALKDLPESDDAVAQWCRDKEVEKDALLDKHNAEDTFSGQEVHHIG RPIKSLLVVIAWVVVIIFGALKFLQWSSLLSTWKGKAFSVIGLGIATLLMHMLILSSQAERSNPAKVAK CigneaLPAAT2 SEQIDNO:35 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVFAELLLMDLLCLFHWWAGAKIKLFTDP ETFRLMGMEHALVIMNHKTDLDWMVGWILGQHLGCLGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKDEST LKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPKNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSAPPTLLRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELHDIG RPVKSLLVVISWAMLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKQKNN EGESSKTEMEKEH DcLPAAT1 SEQIDNO:36 SGLVVNLIQAFFEVLVRPFSKNAYRKINRVVAELLWLELIWLIDWWAGVKIQLYTDPETFKLMGKEHALVICNHK SDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDENTLKSGFQRLRDFPHAFWLA LEVEGTRFTQAKLLAAQEYASSMGLPAPRNVLIPRTKGFVTAVTHMRPFVPAVYDVTLAIPKTSPPPTMLRLFKG QSSVVHIHLKRHLMSDLPKSDDSVAQWCKDAFVVKDNLLDKHKENDSFGDGVLQDTGRPLNSLVVVISWACLLIF GALKFFQWSSILSSWKGLAFSAVGLGIVTVLMQILIQFSQSERSNRPMPSKHAK DcLPAAT2 SEQIDNO:37 MAIPTAAYVVPLGAIFFFSGLLVNLIQAFFFITVWPLSKKTYIRINKVIVELLWLEFVWLADWWAGLKIEVYADA ETFQLMGKEHALVICNHKSDIDWLVGWILAQRAGCLGSSFAVTKKSARYLPVVGWSIWFSGAIFLERSWEKDENT LKAGFQRLREFPCAFWLGLFVEGTRFTQAKLLAAQEYASTMGLPFPRNVLIPRTKGFIAAVNHMREFVPAIYDLT FAFPKDSPPPTMLRLLKGQPSVVHVHIKRHLMKDLPEKNEAVAQWCKDVFLVKDKLLDKHKDDGSFGDGELHEIG RPLKSLVVVTTWACLLILGTLKFLLWSSLLSSWKGLIFSATGLAVLTVLMQFLIQSTQSERSNPASLSK CcrLPAATla SEQIDNO:38 LGLLFFISGLAVNLIQAVCFVFLRPLSKNTYRKINRVLAELLWLQLVWLVDWWAGVKIKVFADRESFNLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSSLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKEGLRRLKDF PRPFWLALFVEGTRFTQAKLLAAQEYATSQGLPVPRNVLIPRTKVHVHVKRHLMKELPETDEAVAQWCKDLFVEK DKLLDKHVAEDTFSDQPLQDIGRPVKPLLVVSSWACLVAYGALKFLQWSSLLSSWKGIAVSAVALAIVTILMQIM ILFSQSERSIPAKVA CcrLPAAT1b SEQIDNO:39 LGLLFFISGLAVNLIQAVCFVFLRPLSKNTYRKINRVLAELLWLQLVWLVDWWAGVKIKVFADRESFNLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSSLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKEGLRRLKDF PRPFWLALFVEGTRFTQAKLLAAQEYATSQGLPVPRNVLIPRTKGFVSAVSHMRSFVPAVYDMTVAIPKSSPSPT MLRLFKGQSSVVHVHVKRHLMKELPETDEAVAQWCKDLFVEKDKLLDKHVAEDTFSDQPLQDIGRPVKPLLVVSS WACLVAYGALKFLQWSSLLSSWKGIAVSAVALAIVTILMQIMILFSQSERSIPTKVA CcrL2AAT2a SEQIDNO:40 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVFAELLLLELLWLFHWRAGAKLKLFADP ETFRLFGKEHALVICNHRTDLDWMVGWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKDKST LKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKLHVHIKRYAMKDLPESDDAV AQWCRDIYVEKDAFLDKHNAEDTFSGQEVHHIGRPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIG LGIVTLLVNILILSSQAERSNPAKVAPAKLKTELSPSKKVTNKEN CcrL2AAT2b SEQIDNO:41 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVFAELLLLELLWLFHWRAGAKLKLFADP ETFRLFGKEHALVICNHRTDLDWMVGWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKDKST LKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSMSHMRSFVPAVYDLT VAFPKTSPPPTLLKLFEGQSVVLHVHIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFSGQEVHHIG RPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIGLGIVTLLVNILILSSQAERSNPAKVAPAKLKTE LSPSKKVTNKEN BrLPAATla SEQIDNO:42 AAAVIVPLGILFFISGLVVNLLQAICYVLIRPLSKNTYRKINRVVAETLWLELVWIVDWWAGVKIQVFADNETFN RMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSG LQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIP KTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQNIGRPIK SLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIITLCMQILIRSSQSERSTPAKVVPAKPKDNHNDS GSSSQTE BrLPAAT1b SEQIDNO:43 AAAVIVPLGILFFISGLVVNLLQAVCYVLVRPMSKNTYRKINRVVAETLWLELVWIVDWWAGVKIQVFADDETFN RMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSG LQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIP KTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQNIGRPIK SLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIITLCMQILIRSSQSERSTPAKVVPAKPKDNHNDS GSSSQTE BrLPAAT1c SEQIDNO:44 MAIAAAVIVPLGLLFFISGLLMNLLQAICYVLVRPLSKNTYRKINRVVAETLWLELVWIVDWWAGVKIKVFADNE TFSRMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTL KSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTV AIPKTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQNIGR PIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIITLCMQILIRSSQSERSTPAKVVPAKPKDNH NDSGSSSQTE BjLPAAT1a SEQIDNO:45 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMK KSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELP VPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPESDDAIAQ WCRDQFVAKDALLDKHIAADTFPGQKEQNIGRPIKSLAVSLIKTFPWLHPHQLTNIFVLFQVVVSWACLLTLGAM KFLHWSNLFSSWKGIALSAFGLGIITLCMQILIRSSQSERSTPAKVAPAKPK BjLPAAT1b SEQIDNO:46 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMK KSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELP VPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPEPEDEIAQ WCRDQFVAKDALLDKHIAADTFPGQKEQNIGRPIKSLAVVVSWACLLTLGAMKFLHWSNLFSSWKGIALSAFGLG IITLCMQILIRSSQSERSTPAKVAPAKPK BjLPAAT1c SEQIDNO:47 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMK KSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELP VPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPESDDAIAQ WCRDQFVAKDALLDKHIAADTFPGQQEQNIGRPIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLG IITLCMQILIRSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE BjLPAAT1d SEQIDNO:48 INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMK KSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELP VPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPESDDAIAQ WCRDQFVAKDALLDKHIAADTFPGQQEQNIGRPIKSLAVSLS CcLPAAT1a SEQIDNO:49 MAIGVAAIVVPLGLLFILSGLMVNLIQAICFILVRPLSKNMYRRVNRVVVELLWLELIWLIDWWGGVKVDVYADS ETFQSLGKEHALVVSNHRSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLRRLKDFPRPFWLALFVEGTRFTQAKLLAAREYAASTGLPIPRNVLIPRTKGFVSAVSNMRSFVPAIYDVT VAIPKTQPSPTMLRIFNRQPSVVHVHIKRHSMNQLPQTDEGVGQWCKDIFVAKDALLDRHLAE CcLPAAT1b SEQIDNO:50 MAIGVAAIVVPLGLLFILSGLMVNLIQAICFILVRPLSKNMYRRVNRVVVELLWLELIWLIDWWGGVKVDVYADS ETFQSLGKEHALVVSNHRSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLRRLKDFPRPFWLALFVEGTRFTQAKLLAAREYAASTGLPIPRNVLIPRTKGFVSAVSNMRSFVPAIYDVT VAIPKTQPSPTMLRIFNRQPSVVHVHIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEKEFKRIR RPIKSLLVISSWSFLLMFGVFKFLKWSALLSTWKGVAVSTTVLLLVTVVMYMFILFSQSERSSPRKVAPSGPENG UcLPAAT1a SEQIDNO:51 MAIGVAAIVVPLGLLFILSGLIINLIQAICFILVRPLSKNMYRKVNRVVVELLWLELIWLIDWWGGVKVDVYADS ETFQSLGKEHALVVSNHRSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIPRNVLIPRTKGEVSAVSNMRSFVPAIYDVT VAIPKTQPSPTMLRIFNRQPSVVHVHIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEKEFKLIR RPIKSLLVISSWSFLLMFGVFKFLKWSALLSTWKGVAVSTAVLLLVTVVMYMFILFSQSERSSPRKVAPIGPENG UcLPAAT1b SEQIDNO:52 MAIGVAAIVVPLGLLFILSGLIINLIQAICFILVRPLSKNMYRKVNRVVVELLWLELIWLIDWWGGVKVDVYADS ETFQSLGKEHALVVSNHRSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIPRNVLIPRTKGEVSAVSNMRSFVPAIYDVT VAIPKTQPSPTMLRIFNRQPSVVHVHIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAE LdLPAAT1 SEQIDNO:53 SLLFFMSGLVVNFIQAVFYVLVRPISKNTYRRINTLVAELLWLELVWVIDWWAGVKVQLYTDTESFRLMGKEHAL LICNHRSDIDWLIGWVLAQRCGCLSSSIAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDENTLKSGLQRLNDFP KPFWLALFVEGTRFTKAKLLAAQEYAASAGLPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDLTVAIPKTTEQPTM LRLFRGKSSVVHVHLKRHLMKDLPKTDDGVAQWCKDQFISKDALLDKHVAEDTFSGLEVQDIGRPMKSLVVVVSW MCLLCLGLVKFLQWSALLSSWKGMMITTFVLGIVTVLMHILIRSSQSEHSTPAK CaequLPAAT1a SEQIDNO:54 QRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRFTQAK LLAAQQYAASSGLPVPRNVLIPRTKGEVSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRH LMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLVVISWAVLVIFGAVKFLQWSSLL SSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTETEKEN CaequLPAAT1b SEQIDNO:55 DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSE YLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGEVSSV SHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHN AEDTFSGQELQDIGRPVKSLLV CaequLPAAT1c SEQIDNO:56 DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSE YLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGEVSSV SHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHN AEDTFSGQELQDIGRPVKSLLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSER STPAKVAPAKPKKEGESSKTETEKEN CaequLPAAT1d SEQIDNO:57 QRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRFTQAK LLAAQQYAASSGLPVPRNVLIPRTKGEVSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRH LMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLV CglutLPAAT1a SEQIDNO:58 LSLLFFVSGLFVNLVQAVCFVLIRPFSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETLSLMGKEHA LVISNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDY PLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKMSTPPT MLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLVVIS WAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTETEKE N CglutLPAAT1b SEQIDNO:59 QAVCFVLIRPFSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVG WVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRF TQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVH LKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLVVISWAVLVIFGAVKFLQW SSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTETEKEN CprLPAAT1 SEQIDNO:60 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVFAELLLLELLWLFHWRAGAKLKLFADP ETFRLFGKEHALVICNHRTDLDWMVGWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKDKST LKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSMSHMRSFVPAVYDLT VAFPKTSPPPTLLKLFEGQSVVLHVHIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFSGQEVHHIG RPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIGLGIVTLLVNILILSSQAERSNPAKVVPAKLKTE LSPSKKVTNKEN ChsLPAAT1 SEQIDNO:61 MAIPSAAVVFLFGLLFFASGLIINLVQAVCFVLIWPLSKNTCRRINIVFQDMLLSELLWLFHWRAGAKLKFFTDP ETYRHMGKEHALVITNHRTDLDWMIGWVLGEHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWFGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPAVYETT MTFPKTSPPPTLLKLFEGQPLVLHIHMKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDTFGGLEVHIGR SIKSLMVVICWVVVIIFGALKFLQWSSLLSSWKGIAFIGIGLGIVNLLVHVLILSSQAERSAPTKVAPAKLKTKL LSSKKITNKEN ChsLPAAT2 SEQIDNO:62 MAIPSAAVVFLFGLLFFASGLIINLVQAVCFVLIWPLSKNTCRRINIVFQDMLLSELLWLFHWRAGAKLKFFTDP ETYRHMGKEHALVITNHRTDLDWMIGWVLGEHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWFGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTMLRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIG RPIKSLVVVISWAALVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKPKRE GESSKTEMDKEN CcalcLPAAT1a SEQIDNO:63 MAIPAAAVVFLFGLLFFPSGLIINLFQAVCFVLIWPFSRNTCRRINIVFQEMLLSELLWLFHWRAGAKLKLFADP ETYRHMGKEHALLITNHRTDLDWMIGWALGQHLGCLGSILSVVKKSTKFLPSHIERLEDFPQPFWMAIFVEGTRF TRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVYETTMTFPKTSPPPTLLKLFEGQPIVLHVH MKRHAMKDIPESDEAVAQWCRDKFVEKDSLLDKHNAGDTFSCQEIHIGRPIKSLMVVISWVVVIIFGALKFLQWS SLLSSWKGIAFSGIGLGIVTLLVHILILSSQAERSTPAKVAPAKLKTELSSSTKVTNKEN CcalcLPAAT1b SEQIDNO:64 MAIPAAAVVFLFGLLFFPSGLIINLFQAVCFVLIWPFSRNTCRRINIVFQEMLLSELLWLFHWRAGAKLKLFADP ETYRHMGKEHALLITNHRTDLDWMIGWALGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVYETT MTFPKTSPPPTLLKLFEGQPIVLHVHMKRHAMKDIPESDEAVAQWCRDKFVEKDSLLDKHNAGDTFSCQEIHIGR PIKSLMVVISWVVVIIFGALKFLQWSSLLSSWKGIAFSGIGLGIVTLLVHILILSSQAERSTPAKVAPAKLKTEL SSSTKVTNKEN Cca1cLPAAT2 SEQIDNO:65 LSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETFRLMGTEHA LVISNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPT MLRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLVVVIS WAALVVFGAVKFLQWSSLLSSWKGLAFSGIALGIITLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTETDKE N ChtLPAAT1a SEQIDNO:66 MAIPAAAVIFLFSILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVFQEMLLSELLGLFHWRAGAKLKLYTDP ETYPLLGKEHALLMINHRTDLDWMIGWVLGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKGFVSTVSHMRSFVPAVYDTT LTFPKTSPPPTLLNLFAGQPIVLHIHIKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDAFSDQEFPISR SIKSLMVVISWVMVIIFGALKFLQWSSLLSSWKGKAFSVIAVGIVTLLMHMSILSSQAERSNPAKVALPKLKTEL PSSKKVLNKEN ChtLPAAT1b SEQIDNO:67 MAIPAAAVIFLFSILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVFQEMLLSELLGLFHWRAGAKLKLYTDP ETYPLLGKEHALLMINHRTDLDWMIGWVLGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKGFVSTVSHMRSFVPAVYDTT LTFPKTSPPPTLLNLFAGQPIVLHIHIKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDAFSDQEFPISR SIKSLMVVISWVMVIIFGALKFLQWSSLLSSWKGIAFSGIGLGIVTLLMHILILSSQAERSTPAKVAQAKVKTEL PSSTKVTNKGN CwLPAAT1 SEQIDNO:68 MAIPAAAVIFLFGILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVFQEMLLSELLWLFHWRAGAELKLFTDP ETYRLLGKEHALVMTNHRTDLDWMIGWVTGQHLGCLGSILSIAKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVCHMRSFVPAVYDTT LTFPKNSPPPTLLNLFAGQPIVLHIHIKRHAMKDMPKSDDAVAQWCRDKFVKKDALLDKHNTEDTFSDQEFPIGR PIKSLMVVISWVVVIIFGTLKFLQWSSLLSSWKGIAFSGIGLGIVTLLVHILILSSQAERSTPPKVAPAKLKTEL SSTTKVINKGN CwLPAAT2b SEQIDNO:69 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRLNRVVAELLWLELVWLIDWWAGVKIKVFTDHETFHLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPT MLRMFKGQSSVDALLDKHNADDTFSGQELHDIGRPIKSLLVVISWAVLVVFGAVKFLQWSSLLSSWKGIAFSGIG LGIVTLLVHILILSSQAERSTSAKVAQAKVKTELSSSKKVKNKGN CwLPAAT2a SEQIDNO:70 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRLNRVVAELLWLELVWLIDWWAGVKIKVFTDHETFHLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPT MLRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDVLLDKHNAEDTFSGQELQDIGRPVKSLLVVIS WTLLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKMETDKE N CgLPAAT1a SEQIDNO:71 LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDYPLPFWLALFVEGT RFTRAKLLAAQQYAASLGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSSVLH VHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVVISWAVLEVFGAVKFL QWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKPKNEGESSKAEMEKEK CgLPAAT1b SEQIDNO:72 LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDYPLPFWLALFVEGT RFTRAKLLAAQQYAASLGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSSVLH VHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVRCFLVLSLIYLNGIML KLRGPCLQVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKPKN EGESSKAEMEKEK CgLPAAT1c SEQIDNO:73 LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDYPLPFWLALFVEGT RFTRAKLLAAQQYAASLGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSSVLH VHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVVTSWAVLVISGAVKFL QWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEGESSKTEKDKEN CpalLPAAT1 SEQIDNO:74 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHETLSLMGKEHA LVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDENTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYATSSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPT MLRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVVIS WAVLVIFGAVKFLQWSSLLSSWKGLAFSGVGLGIITLLMHILILFSQSERSTPAKVAPAKPKKDGESSKTEIEKE N CaLPAAT1 SEQIDNO:75 MAIAAAAVIVPVSLLFFVSGLIVNLVQAVCFVLIRPLFKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDH ETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDEST LKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTLLRMFKGQSSVLHVHLKRHQMNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLIVISWAVLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGVITLLMHILILFSQSERSTPAKVAPAKPKIE GESSKTEMEKEH CaLPAAT3 SEQIDNO:76 MTIASAAVVFLFGILLFTSGLIINLFQAFCSVLVWPLSKNAYRRINRVFAEFLPLEFLWLFHWWAGAKLKLFTDP ETFRLMGKEHALVIINHKIELDWMVGWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSEYLFLERNWAKDKKT LKSHIERLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASAGLPVPRNVLIPHTKGFVSSVSHMRSFVPAIYDVT VAFPKTSPPPTMLKLFEGHFVELHVHIKRHAMKDLPESEDAVAQWCRDKEVEKDALLDKHNAEDTFSGQEVHHVG RPIKSLLVVISWVVVIIFGALKFLQWSSLLSSWKGIAFSVIGLGTVALLMQILILSSQAERSIPAKETPANLKTE LSSSKKVTNKEN SalL2AAT1 SEQIDNO:77 MAIGAAAIVVPLGLLFMLSGLMVNLIQAICFILVRPLSKNMYRRVNRVVVELLWLELIWLIDWWGGVKVDVYADS ETFQSLGKEHALVVSNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIPRNVLIPRTKGFVSAVSNMRSFVPAIYDVT VAIPKTQPSPTMLRIFNRQPSVVHVRIKRHSMNQLPPTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEKEFKRIR RPIKSLLVISSWSFLLLFGVFKFLKWSALLSTWKGVAVSTAVLLLVTVVMYMFILFSQSERSSPRKVAPSGPENG C1eptL2AAT1 SEQIDNO:78 MAIPAAVVIFLFGLLFFSSGLIINLFQALCFVLIWPLSKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVNHMRSFVPAVYDLT VAFPKTSPPPTLLNLFEGQSVVLHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSSQEVHHTG SRPIKSLLVVISWVVVITFGALKFLQWSSWKGKAFSVIGLGIVTLLMHMLILSSQAERSKPAKVTQAKLKTELSI SKKVTDKEN ClopLPAAT1 SEQIDNO:79 MAIAAAAVIFLFGLLFFASGLIINLFQALCFVLIRPLSKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETLRLMGKEHALIIINHMTELDWMVGWVMGQHFGCLGSIISVAKKSTKFLPVLGWSMWFSEYLYLERSWAKDKST LKSHIERLKDYPLPFWLVIFVEGTRFTRTKLLAAQEYAASSGLPVPRNVLIPRTKGFVSCVNHMRSFVPAVYDVT VAFPKTSPQPTLLNLFEGRSIVLHVHIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHHTG RRPIKSLLVVMSWVVVTTFGALKFLQWSSWKGKAFSVIGLGIVTLLMHVLILSSQAERSNPAKVVQAELNTELSI SKKVTNKGN CcrasLPAAT1a SEQIDNO:80 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKAK CcrasLPAAT1b SEQIDNO:81 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVRCFLVLSLIYLNGIILKLCGLCLQVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLM HILILFSQSERSTPAKVAPAKAKSEQIDNO: CcrasLPAAT1c SEQIDNO:82 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKAKME GESSKTEMEMEK CcrasLPAAT1d SEQIDNO:83 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVRCFLVLSLIYLNGIILKLCGLCLQVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLM HILILFSQSERSTPAKVAPAKAKMEGESSKTEMEMEK CkoeLPAAT1 SEQIDNO:84 MAIAAAPVIFLFGLLFFASGLIINLFQAICFVLIWPLSKNAYRRINRVFAELLLSELLCLFDWWAGAKLKLFTDP ETFRLMGKEHALVITNHKIDLDWMIGWILGQHFGCLGSVISIAKKSTKFLPIFGWSLWFSEYLFLERNWAKDKRT LKSHIERMKDYPLPLWLILFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPAIYDVT VAIPKTSPPPTLIRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQETG RPIKSLLVVISWAVLEVYGAVKFLQWSSLLSSWKGLAFSGIGLGLITLLMHILILFSQSERSTPAKVAPAKPKKE GESSKTEMEKEK CkoeLPAAT2 SEQIDNO:85 MHVLLEMVTFRFSSFFVFDNVQALCFVLIWPLSKSAYRKINRVFAELLLSELLCLFDWWAGAKLKLFTDPETFRL MGKEHALVITNHKIDLDWMIGWILGQHFGCLGSVISIAKKSTKFLPIFGWSLWFSEYLFLERNWAKDKRTLKSHI ERMKDYPLPLWLILFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPAVYDVTVAFPK TSPPPTMLSLFEGQSVVLHVHIKRHAMKDLPDSDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHHVGRPIKS LLVVISWMVVIIFGALKFLQWSSLLSSWKGKAFSAIGLGIATLLMHVLVVFSQADRSNPAKVPPAKLNTELSSSK KVTNKEN pSZ2624PmKASII SEQIDNO:86 gtttaaacGCCGGTCACCACCCGCATGCTCGTACTACAGCGCACGCACCGCTTCGTGA TCCACCGGGTGAACGTAGTCCTCGACGGAAACATCTGGTTCGGGCCTCCTGCTTG CACTCCCGCCCATGCCGACAACCTTTCTGCTGTTACCACGACCCACAATGCAACG CGACACGACCGTGTGGGACTGATCGGTTCACTGCACCTGCATGCAATTGTCACAA GCGCTTACTCCAATTGTATTCGTTTGTTTTCTGGGAGCAGTTGCTCGACCGCCCGC GTCCCGCAGGCAGCGATGACGTGTGCGTGGCCTGGGTGTTTCGTCGAAAGGCCA GCAACCCTAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGTTTGGACC AGATCCGCCCCGATGCGGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCT TTCGTAAATGCCAGATTGGTGTCCGATACCTGGATTTGCCATCAGCGAAACAAGA CTTCAGCAGCGAGCGTATTTGGCGGGCGTGCTACCAGGGTTGCATACATTGCCCA TTTCTGTCTGGACCGCTTTACTGGCGCAGAGGGTGAGTTGATGGGGTTGGCAGGC ATCGAAACGCGCGTGCATGGTGTGCGTGTCTGTTTTCGGCTGCACGAATTCAATA GTCGGATGGGCGACGGTAGAATTGGGTGTGGCGCTCGCGTGCATGCCTCGCCCC GTCGGGTGTCATGACCGGGACTGGAATCCCCCCTCGCGACCATCTTGCTAACGCT CCCGACTCTCCCGACCGCGCGCAGGATAGACTCTTGTTCAACCAATCGACAactagt ATGcagaccgcccaccagcgcccccccaccgagggccactgcttcggcgcccgcctgcccaccgcctcccgccgcgccgtgc gccgcgcctggtcccgcatcgcccgcgggcgcgccgccgccgccgccgacgccaaccccgcccgccccgagcgccgcgtggt gatcaccggccagggcgtggtgacctccctgggccagaccatcgagcagactactcctccctgctggagggcgtgtccggcatct cccagatccagaagacgacaccaccggctacaccaccaccatcgccggcgagatcaagtccctgcagctggacccctacgtgc ccaagcgctgggccaagcgcgtggacgacgtgatcaagtacgtgtacatcgccggcaagcaggccctggagtccgccggcctg cccatcgaggccgccggcctggccggcgccggcctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggcat gacctccacgccgccggcgtggaggccctgacccgcggcggcgtgcgcaagatgaaccccactgcatccccactccatctcca acatgggcggcgccatgctggccatggacatcggatcatgggccccaactactccatctccaccgcctgcgccaccggcaacta ctgcatcctgggcgccgccgaccacatccgccgcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcc cctccggcatcggcggcttcatcgcctgcaaggccctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggac gccgaccgcgacggatcgtgatgggcgagggcgccggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcg ccaccatcctggccgagctggtgggcggcgccgccacctccgacgcccaccacatgaccgagcccgacccccagggccgcgg cgtgcgcctgtgcctggagcgcgccctggagcgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacct ccacccccgccggcgacgtggccgagtaccgcgccatccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagt ccatgatcggccacctgctgggcggcgccggcgccgtggaggccgtggccgccatccaggccctgcgcaccggctggctgcac cccaacctgaacctggagaaccccgcccccggcgtggaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacc tggacgtggtgctgtccaactccttcggcttcggcggccacaactcctgcgtgatcttccgcaagtacgacgagatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggatcgacgacgacaagTGAatcgatAGATCTCTT AAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGAT GGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTAT CAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCT GCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGC TTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCT GCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCT GGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGG ATGGGAACACAAATGGAAAGCTTAATTAAgagctccgcgtctcgaacagagcgcgcagaggaacgct gaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagc gtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctaggtg atatccatcttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgta agtgggctggcgtaccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagatac agcgcgagccagacacggagtgccgagctatgcgcacgctccaactaggtaccagtttaggtccagcgtccgtggggggggacg ggctgggagcttgggccgggaagggcaagacgatgcagtccctctggggagtcacagccgactgtgtgtgttgcactgtgcggccc gcagcactcacacgcaaaatgcctggccgacaggcaggccctgtccagtgcaacatccacggtccctctcatcaggctcaccttgct cattgacataacggaatgcgtaccgctctttcagatctgtccatccagagaggggagcaggctccccaccgacgctgtcaaacttgctt cctgcccaaccgaaaacattattgtttgagggggggggggggggggcagattgcatggcgggatatctcgtgaggaacatcactgg gacactgtggaacacagtgagtgcagtatgcagagcatgtatgctaggggtcagcgcaggaagggggcctttcccagtctcccatgc cactgcaccgtatccacgactcaccaggaccagcttcttgatcggcttccgctcccgtggacaccagtgtgtagcctctggactccagg tatgcgtgcaccgcaaaggccagccgatcgtgccgattcctgggtggaggatatgagtcagccaacttggggctcagagtgcacact ggggcacgatacgaaacaacatctacaccgtgtcctccatgctgacacaccacagcttcgctccacctgaatgtgggcgcatgggcc cgaatcacagccaatgtcgctgctgccataatgtgatccagaccctctccgcccagatgccgagcggatcgtgggcgctgaatagatt cctgtttcgatcactgtttgggtcctttccillicgtctcggatgcgcgtctcgaaacaggctgcgtcgggctttcggatcccttttgctccct ccgtcaccatcctgcgcgcgggcaagttgcttgaccctgggctgataccagggttggagggtattaccgcgtcaggccattcccagcc cggattcaattcaaagtctgggccaccaccctccgccgctctgtctgatcactccacattcgtgcatacactacgttcaagtcctgatcca ggcgtgtctcgggacaaggtgtgcttgagtttgaatctcaaggacccactccagcacagctgctggttgaccccgccctcgcaatcta gaATGgccgcgtccgtccactgcaccctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaac tcctccctgctgcccggatcgacgtggtggtccaggccgcggccacccgatcaagaaggagacgacgaccacccgcgccacg ctgacgacgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacacca gcccatcccctccacgaggagtgatccccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcct gaaggtgcccaccgccgcgtgcacctgtccggcggcgagcccgccacgacaactacgacacgtccggcccccagaacgtcaa cgcccacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatg tactacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagacgtccgctc cgaggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagacc tggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccacc atgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgc ggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgacc gcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctga ccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcg cctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggct ccatctacgacgccaacgacacggcccagacgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaagga cgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgc aacgaggcgcccactacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcg gccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgt gaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtggga cgacgcgctgtccaaggcgcgatcgagaccgctggatggaccagacgcgctgtccctggaccccatgacggcgatgtccacc acgacgagacgctgcccgcggacggcgcgaaggtcgcccacactgctccatgtgcggccccaagactgctccatgaagatca cggaggacatccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgt ccgaggagacaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtc ctacgtcaaggccgcgcagaagTGAcaattgACGGAGCGTCGTGCGGGAGGGAGTGTGCCGAG CGGGGAGTCCCGGTCTGTGCGAGGCCCGGCAGCTGACGCTGGCGAGCCGTACGC CCCGAGGGTCCCCCTCCCCTGCACCCTCTTCCCCTTCCCTCTGACGGCCGCGCCTG TTCTTGCATGTTCAGCGACggatccTAGGGAGCGACGAGTGTGCGTGCGGGGCTGGC GGGAGTGGGACGCCCTCCTCGCTCCTCTCTGTTCTGAACGGAACAATCGGCCACC CCGCGCTACGCGCCACGCATCGAGCAACGAAGAAAACCCCCCGATGATAGGTTG CGGTGGCTGCCGGGATATAGATCCGGCCGCACATCAAAGGGCCCCTCCGCCAGA GAAGAAGCTCCTTTCCCAGCAGACTCCTTCTGCTGCCAAAACACTTCTCTGTCCA CAGCAACACCAAAGGATGAACAGATCAACTTGCGTCTCCGCGTAGCTTCCTCGG CTAGCGTGCTTGCAACAGGTCCCTGCACTATTATCTTCCTGCTTTCCTCTGAATTA TGCGGCAGGCGAGCGCTCGCTCTGGCGAGCGCTCCTTCGCGCCGCCCTCGCTGAT CGAGTGTACAGTCAATGAATGGTCCTGGGCGAAGAACGAGGGAATTTGTGGGTA AAACAAGCATCGTCTCTCAGGCCCCGGCGCAGTGGCCGTTAAAGTCCAAGACCG TGACCAGGCAGCGCAGCGCGTCCGTGTGCGGGCCCTGCCTGGCGGCTCGGCGTG CCAGGCTCGAGAGCAGCTCCCTCAGGTCGCCTTGGACGGCCTCTGCGAGGCCGG TGAGGGCCTGCAGGAGCGCCTCGAGCGTGGCAGTGGCGGTCGTATCCGGGTCGC CGGTCACCGCCTGCGACTCGCCATCCgaagagcgtttaaac pSZ3204GarmFATA SEQIDNO:87 gctettcGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTT GGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGG TCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATG AGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCC AGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCC GCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTA CAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCT GGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCG CACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGC GCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCG CGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACC CCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGG CCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCA CGCTCAggtaccctttcttgcgctatgacacttccagcaaaaggtagggcgggctgcgagacggcttcccggcgctgcatgcaa caccgatgatgcttcgaccccccgaagctccttcggggctgcatgggcgctccgatgccgctccagggcgagcgctgtttaaatagc caggcccccgattgcaaagacattatagcgagctaccaaagccatattcaaacacctagatcactaccacttctacacaggccactcga gcttgtgatcgcactccgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaactctagaatatcaATGctgctgcag gccttcctgttcctgctggcggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcactt cacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacaccagt acaacccgaacgacaccgtctgggggacgccatgactggggccacgccacgtccgacgacctgaccaactgggaggaccag cccatcgccatcgccccgaagcgcaacgactccggcgccactccggctccatggtggtggactacaacaacacctccggcactt caacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcct acagcctggacggcggctacaccacaccgagtaccagaagaaccccgtgctggccgccaactccacccagaccgcgacccg aaggtcactggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccg acgacctgaagtcctggaagctggagtccgcgacgccaacgagggcacctcggctaccagtacgagtgccccggcctgatcga ggtccccaccgagcaggaccccagcaagtcctactgggtgatgacatctccatcaaccccggcgccccggccggcggctcatc aaccagtacttcgtcggcagatcaacggcacccacttcgaggccacgacaaccagtcccgcgtggtggacttcggcaaggact actacgccctgcagaccacttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactc cgccacgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagactccctcaacaccgagtaccaggccaacccgg agacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggacgccaccaacac cacgagacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagacgagctggtgtacgccgtcaa caccacccagacgatctccaagtccgtgacgcggacctctccctctggacaagggcctggaggaccccgaggagtacctccgc atgggatcgaggtgtccgcgtcctccacacctggaccgcgggaacagcaaggtgaagacgtgaaggagaacccctacttcac caaccgcatgagcgtgaacaaccagccatcaagagcgagaacgacctgtcctactacaaggtgtacggcagctggaccaga acatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgt gaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCAGCA GCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGC CGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCT CAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTA TTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCA ACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTC ACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAA CCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACAC AAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcata caccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtgg caggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctagggatatcctgaagaatgggaggcaggtgttgttgattat gagtgtgtaaaagaaaggggtagagagccgtcctcagatccgactactatgcaggtagccgctcgcccatgcccgcctggctgaata ttgatgcatgcccatcaaggcaggcaggcatactgtgcacgcaccaagcccacaatcttccacaacacacagcatgtaccaacgcac gcgtaaaagttggggtgctgccagtgcgtcatgccaggcatgatgtgctcctgcacatccgccatgatctcctccatcgtctcgggtgtt tccggcgcctggtccgggagccgttccgccagatacccagacgccacctccgacctcacggggtactittcgagcgtctgccggtag tcgacgatcgcgtccaccatggagtagccgaggcgccggaactggcgtgacggagggaggagagggaggagagagagggggg ggggggggggggatgattacacgccagtctcacaacgcatgcaagacccgtttgattatgagtacaatcatgcactactagatggatg agcgccaggcataaggcacaccgacgttgatggcatgagcaactcccgcatcatatttcctattgtcctcacgccaagccggtcaccat ccgcatgctcatattacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacggaaacatctggctcgggcctcgt gctggcactccctcccatgccgacaacctttctgctgtcaccacgacccacgatgcaacgcgacacgacccggtgggactgatcggtt cactgcacctgcatgcaattgtcacaagcgcatactccaatcgtatccgtttgatttctgtgaaaactcgctcgaccgcccgcgtcccgc aggcagcgatgacgtgtgcgtgacctgggtgtttcgtcgaaaggccagcaaccccaaatcgcaggcgatccggagattgggatctg atccgagcttggaccagatcccccagatgcggcacgggaactgcatcgactcggcgcggaacccagctttcgtaatgccagattg gtgtccgataccttgatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgctaccagggttgcatacattgc ccatttctgtctggaccgctttaccggcgcagagggtgagttgatggggttggcaggcatcgaaacgcgcgtgcatggtgtglgtgtct gttttcggctgcacaatttcaatagtcggatgggcgacggtagaattgggtgttgcgctcgcgtgcatgcctcgccccgtcgggtgtcat gaccgggactggaatcccccctcgcgaccctcctgctaacgctcccgactctcccgcccgcgcgcaggatagactctagttcaacca atcgacaactagtATGgccaccgcatccactactcggcgacaatgcccgctgcggcgacctgcgtcgctcggcgggctccggg ccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaagg tgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgt cctacaaggagaagacatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgc aggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcactccaccacccccaccatgcgcaagctgcgcc tgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggcca gggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctcc aagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgccc ccgcgagctgcgcctggccaccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactc caagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctgg agtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacga cgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgacaaccacaacggcaccaacggctccgcca acgtgtccgccaacgaccacggctgccgcaacacctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggcc gcaccgagtggcgcaagaagcccacccgcATGGACTACAAGGACCACGACGGCGACTACAAGGA CCACGACATCGACTACAAGGACGACGACGACAAGTGAategatagatctcttaagGCAGCAG CAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCC GCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTC AGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTAT TTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAA CCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCA CTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAAC CTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACA AATGGAaagcttaattaagagctcTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTC TCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAA AGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGG GAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTT CGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCA GTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCA GGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACC TCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTC CATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACC GGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAG AATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGA GCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCC GCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAG TCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTgaagagc pSZ4198(BnLPAT2) SEQIDNO:88 gctcttccgctAACGGAGGTCTGTCACCAAATGGACCCCGTCTATTGCGGGAAACCACG GCGATGGCACGTTTCAAAACTTGATGAAATACAATATTCAGTATGTCGCGGGCGG CGACGGCGGGGAGCTGATGTCGCGCTGGGTATTGCTTAATCGCCAGCTTCGCCCC CGTCTTGGCGCGAGGCGTGAACAAGCCGACCGATGTGCACGAGCAAATCCTGAC ACTAGAAGGGCTGACTCGCCCGGCACGGCTGAATTACACAGGCTTGCAAAAATA CCAGAATTTGCACGCACCGTATTCGCGGTATTTTGTTGGACAGTGAATAGCGATG CGGCAATGGCTTGTGGCGTTAGAAGGTGCGACGAAGGTGGTGCCACCACTGTGC CAGCCAGTCCTGGCGGCTCCCAGGGCCCCGATCAAGAGCCAGGACATCCAAACT ACCCACAGCATCAACGCCCCGGCCTATACTCGAACCCCACTTGCACTCTGCAATG GTATGGGAACCACGGGGCAGTCTTGTGTGGGTCGCGCCTATCGCGGTCGGCGAA GACCGGGAAggtaccgcggtgagaatcgaaaatgcatcgtttctaggttcggagacggtcaattccctgctccggcgaatctg tcggtcaagctggccagtggacaatgttgctatggcagcccgcgcacatgggcctcccgacgcggccatcaggagcccaaacagc gtgtcagggtatgtgaaactcaagaggtccctgctgggcactccggccccactccgggggcgggacgccaggcattcgcggtcggt cccgcgcgacgagcgaaatgatgattcggttacgagaccaggacgtcgtcgaggtcgagaggcagcctcggacacgtctcgctag ggcaacgccccgagtccccgcgagggccgtaaacattgtttctgggtgtcggagtgggcattttgggcccgatccaatcgcctcatgc cgctctcgtctggtcctcacgttcgcgtacggcctggatcccggaaagggcggatgcacgtggtgttgccccgccattggcgcccac gtttcaaagtccccggccagaaatgcacaggaccggcccggctcgcacaggccatgctgaacgcccagatttcgacagcaacacca tctagaataatcgcaaccatccgcgttttgaacgaaacgaaacggcgctgtttagcatgtttccgacatcgtgggggccgaagcatgct ccggggggaggaaagcgtggcacagcggtagcccattctgtgccacacgccgacgaggaccaatccccggcatcagccttcatcg acggctgcgccgcacatataaagccggacgcctaaccggtttcgtggttatgactagtATGttcgcgttctacttcctgacggcctgc atctccctgaagggcgtgacggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaaca cgacgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacatgggctaca agtacatcatcctggacgactgctggtcctccggccgcgactccgacggcacctggtcgccgacgagcagaagaccccaacgg catgggccacgtcgccgaccacctgcacaacaactccacctgacggcatgtactcctccgcgggcgagtacacgtgcgccggct accccggctccctgggccgcgaggaggaggacgcccagacttcgcgaacaaccgcgtggactacctgaagtacgacaactgc tacaacaagggccagacggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccg ccccatcactactccctgtgcaactggggccaggacctgaccactactggggctccggcatcgcgaactcctggcgcatgtccgg cgacgtcacggcggagacacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcacc actgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaa cctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacactccatgtgggccatggtgaagtcccccctgat catcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactcc aacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtc cggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggagg agatcacttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaa ctccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacg gcctgtccaagaacgacacccgcctgacggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccg cccacggcatcgcgactaccgcctgcgcccctcctccTGAtacgtactcgagGCAGCAGCAGCTCGGATAGT ATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTG CCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATC TTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCAC CCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCT ACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCAC AGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGC ACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAagctgtag aattcctggctcgggcctcgtgctggcactccctcccatgccgacaacctttctgctgtcaccacgacccacgatgcaacgcgacacg acccggtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcatactccaatcgtatccgtttgatttctgtgaaaactcg ctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtgacctgggtgtttcgtcgaaaggccagcaaccccaaatcgcaggc gatccggagattgggatctgatccgagcttggaccagatcccccacgatgcggcacgggaactgcatcgactcggcgcggaaccca gctttcgtaaatgccagattggtgtccgataccttgatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgct accagggttgcatacattgcccatttctgtctggaccgctttaccggcgcagagggtgagttgatggggttggcaggcatcgaaacgc gcgtgcatggtgtgtgtgtctgttttcggctgcacaatttcaatagtcggatgggcgacggtagaattgggtgttgcgctcgcgtgcatgc ctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccctcctgctaacgctcccgactctcccgcccgcgcgcag gatagactctagttcaaccaatcgacaactagtATGgccatggccgccgccgtgatcgtgcccctgggcatcctgttcttcatctcc ggcctggtggtgaacctgctgcaggccatctgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcg tggtggccgagaccctgtggctggagctggtgtggatcgtggactggtgggccggcgtgaagatccaggtgacgccgacaacg agaccttcaaccgcatgggcaaggagcacgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcc tggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaagacctgcccgtgatcggctggtccatgt ggactccgagtacctgacctggagcgcaactgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgactt cccccgccccactggctggccctgacgtggagggcacccgatcaccgaggccaagctgaaggccgcccaggagtacgccgc ctcctccgagctgcccgtgccccgcaacgtgctgatcccccgcaccaagggatcgtgtccgccgtgtccaacatgcgctccacgt gcccgccatctacgacatgaccgtggccatccccaagacctcccccccccccaccatgctgcgcctgacaagggccagccctcc gtggtgcacgtgcacatcaagtgccactccatgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcgacca gacgtggccaaggacgccctgctggacaagcacatcgccgccgacaccaccccggccagcaggagcagaacatcggccgc cccatcaagtccctggccgtggtgctgtcctggtcctgcctgctgatcctgggcgccatgaagacctgcactggtccaacctgactc ctcctggaagggcatcgccactccgccctgggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcctcccagtccga gcgctccacccccgccaaggtggtgcccgccaagcccaaggacaaccacaacgactccggctcctcctcccagaccgaggtg gagaagcagaagTGAatcgatagatctcttaagGCAGCAGCAGCTCGGATAGTATCGACACACT CTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGT GAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACG CGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCC CCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTG CTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTT GGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCT GATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagettaattaagagatcAGCGG CGACGGTCCTGCTACCGTACGACGTTGGGCACGCCCATGAAAGTTTGTATACCGA GCTTGTTGAGCGAACTGCAAGCGCGGCTCAAGGATACTTGAACTCCTGGATTGAT ATCGGTCCAATAATGGATGGAAAATCCGAACCTCGTGCAAGAACTGAGCAAACC TCGTTACATGGATGCACAGTCGCCAGTCCAATGAACATTGAAGTGAGCGAACTGT TCGCTTCGGTGGCAGTACTACTCAAAGAATGAGCTGCTGTTAAAAATGCACTCTC GTTCTCTCAAGTGAGTGGCAGATGAGTGCTCACGCCTTGCACTTCGCTGCCCGTG TCATGCCCTGCGCCCCAAAATTTGAAAAAAGGGATGAGATTATTGGGCAATGGA CGACGTCGTCGCTCCGGGAGTCAGGACCGGCGGAAAATAAGAGGCAACACACTC CGCTTCTTAgctcttc pSZ4198BnLPAT2(1.5) SEQIDNO:89 ATGgccatggccgccgccgccgtgatcgtgcccctgggcatcctgacttcatctccggcctggtggtgaacctgctgcaggccgt gtgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagaccctgtggctggagctg gtgtggatcgtggactggtgggccggcgtgaagatccaggtgacgccgacgacgagaccacaaccgcatgggcaaggagca cgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctcc gccctggccgtgatgaagaagtcctccaagacctgcccgtgatcggctggtccatgtggactccgagtacctgacctggagcgca actgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgacacccccgccccactggctggccctgacgtg gagggcacccgatcaccgaggccaagctgaaggccgcccaggagtacgccgcctcctcccagctgcccgtgccccgcaacgt gctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgctccttcgtgcccgccatctacgacatgaccgtggccat ccccaagacctcccccccccccaccatgctgcgcctgacaagggccagccctccgtggtgcacgtgcacatcaagtgccactcc atgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcgaccagacgtggccaaggacgccctgctggacaa gcacatcgccgccgacaccaccccggccagaaggagcacaacatcggccgccccatcaagtccctggccgtggtggtgtcctg ggcctgcctgctgaccctgggcgccatgaagacctgcactggtccaacctgactcctccctgaagggcatcgccctgtccgccctg ggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcctcccagtccgagcgctccacccccgccaaggtggcccccg ccaagcccaaggacaagcaccagtccggctcctcctcccagaccgaggtggaggagaagcagaagTGA pSZ4206TcLPAT2GhomLPAT2A SEQIDNO:90 ATGgccatcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttc gtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggctggtgg actggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccctggtggtggccaacc accgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctcc aagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagaacaccctgaaggc cggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtggagggcacccgcttcacccaggccaagttcctggccgc ccaggagtacgccgcctcccagggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgc gctccttcgtgcccgccatctacgacatgaccgtggccatccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctc cgtggtgcacgtgcacatcaagcgctgcctgatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgttcg tggagaaggacaagctgctggacaagcacatcgccgaggacaccttctccgaccagcccatgcaggacctgggccgccccatcaagtcc ctgctggtggtggcctcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcg ccttcttcctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggc ccccggcaagcccaagaacgacggcgagacctccgaggcccgccgcgacaagcagcagTGA NucleotidesequenceoftheGhomLPAT2Acodingsequence,usedinthe transformingDNAfrompSZ4412. SEQIDNO:91 ATGgccatccccgccgccatcgtgatcgtgcccgtgggcctgctgttcttcatctccggcctgatcgtgaacctgctgcaggccctgtgcttcg tgctgatccgccccctgtccaagtccgcctaccgcaccatcaaccgccagctggtggagctgctgtggctggagctggtgtgcatcgtggac tggtgggcccgcgtgaagatccagctgttcaccgacaaggagaccctgaactccatgggcaaggagcacgccctggtgatgtgcaacca ccgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccaccgtggccgtgatgaagaagtcctcca aggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagtcc ggcctgcagcgcctgcgcgacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgcc caggagtacgccgcctccaccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccatcacccgc tccttcgtgcccgtgatctacgacatcaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctccg tggtgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgacgtggcccagtggtgccgcgaccagttcgtgg tgaaggactccctgctggacaagcacatcgccgaggacaccttctccgaccaggagctgcaggacatcggccgccccatcaagtccctgg tggtgttcacctcctgggtgtgcatcatcaccttcggcgccctgaagttcctgcagtggtcctccctgctgcactcctggaagggcatcgccat ctccgcctccggcctggccatcgtgaccgtgctgatgcacatcctgatccgcttctcccagtccgagcactccacctccgccaagatcgccgcc gagaagcacaagaacggcggcgtgtcccaggagatgggccgcgagaagcagcacTGA NucleotidesequenceoftheGhomLPAT2Bcodingsequence,usedinthe transformingDNAfrompSZ4413. SEQIDNO:92 ATGgagatccccgccgtggccgtgatcgtgcccatcggcatcctgttcttcatctccggcctgatcgtgaacctgatgcaggccatctgcttc ttcctgatccgccccctgtccaagaacacccaccgcatcgtgaaccgccagctggccgagctgctgtggctggagctgatctggatcgtgga ctggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcacctgatgggcaaggagcacgccctggtgatctgcaacc actcctccgacatcgactggctggtgggctggctgctgtgccagcgctccggctgcctgggctccgccctggccgtgatgaagtcctcctcca aggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagtccaccctgaagtcc ggcctgcagcgcctgaaggacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccaggccaagctgctggccgc ccaggagtacgccatgtccgccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgc gctccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccgtgcagcccaccatgctgcgcctgttcaagggccagtcctc cgtggtgcaggtgcacctgaagcgccactccatgaaggacctgcccgagtccgaggacgacgtggcccagtggtgccgcgaccgcttcgt ggtgaaggactccctgctggacaagcacaaggtggaggacaccttcaccgaccaggagctgcaggacctgggccgccccatcaagtccc tggtggtggtgacctgctgggcctgcatcatcatcttcggcatcctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatggc catctccgcctccggcctggccgtggtgaccttcctgatgcagatcctgatccgcttctcccagtccgagcgctccacccccgccaagatcgcc cccgccaagcccaacaaggccggcaactcctccgagaccgtgcgcgacaagcaccagTGA NucleotidesequenceoftheGhomLPAt2Ccodingsequence,usedinthe transformingDNAfrompSZ4414. SEQIDNO:93 ATGgccatccccgccgccatcatcatcgtgcccctgggcctgatcttcttcacctccggcctgatcatcaacctgatccaggccgtgtgctacg tgctgatccgccccctgtccaagtccaccttccgccgcatcaaccgcgagctggccgagctgctgtggctggagctggtgtgggtggtggac tggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcactccatgggcaaggagcacgccctggtgatctgcaaccac cgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacttcttcctggagcgcaactgggccatggacgagtccaccctgaagtccg gcctgcagcgcctgaaggacttcccccagcccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgccc aggagtacgccgcctccgccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgaacatcatgcgc tccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctccg tggtgcacgtgcacctgaagcgccacctgatggaggacctgcccgagaccgacgacgacgtggcccagtggtgccgcgaccgcttcgtgg tgaaggactccctgctggacaagtacgtggccgaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccctgg tggtggtgacctcctgggtgtgcatcatcgccttcggctccctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgtgat ctccgccgcctccctggccgtggtgaccgtgctgatgcagatcctgatccgcttctcccagtccgagcgctccacctccgccaagatcgccgcc gccaagcgcaagaacgtgggcgagcacTGA NucleotidesequenceoftheGindPAT2Acodingsequence,usedinthetransforming DNAfrompSZ4415. SEQIDNO:94 ATGgccatccccgtggtggtggtgatcgtgcccgtgggcctgctgttcttcatctccggcctgatcgtgaacctgctgcaggccctgtgcttc gtgctgatccgccccctgtccaagtccgcctaccgcaccatcaaccgccagctggtggagctgctgtggctggagctggtgtgcatcgtgga ctggtgggcccgcgtgaagatccagctgttcatcgacaaggagaccctgaactccatgggcaaggagcacgccctggtgatgtgcaacc accgctcctacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccaccgtggccgtgatgaagaagtcctcc aaggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagt ccggcctgcagcgcctgcgcgacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccg cccaggagtacgccgcctccaccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccatcaccc gctccttcgtgcccgtgatctacgacatcaccgtggccatccccaagtcctcctcccagcccaccatgctgaagctgttcaagggccagtcctc cgtggtgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgacgtggcccagtggtgccgcgcccagttcgt ggtgaaggactccctgctggacaagcacatcgccgaggacaccttctccgaccaggagctgcaggacatcggccgccccatcaagtccct ggtggtgttcacctcctgggtgtgcatcatcaccttcggcgccctgaagttcctgcagtggtcctccctgctgcactcctggaagggcatcgcc atctccgcctccggcctggccatcgtgaccgtgctgatgcacatcctgatccgcttctcccagtccgagcactccacctccgccaagatcgccg ccgagaagcacaagaacggcggcgtgtcccaggagatgggccgcgagaagcagcacTGA NucleotidesequenceoftheGindPAT2Bcodingsequence,usedinthetransofrming DNAfrompSZ4416. SEQIDNO:95 ATGggcatccccgccgtggccgtgatcgtgcccatcggcatcctgttcttcatctccggcttcatcgtgaacctgatgcaggccatctgcttcg tgctgatccgccccctgtccaagaacacctaccgcatcgtgaaccgccagctggccgagttcctgtggctggagctgatctgggtggtggac tggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcacctgatgggcaaggagcacgccctggtgatctgcaacca ccgctccgacatcgactggctggtgggctggctgctgtgccagcgctccggctgcctgggctccgccctggccgtgatgaagtcctcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagtccaccctgaagctgg gcctgcagcgcctgaaggacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccaggccaagctgctggccgccc aggagtacgccatgtccgccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgc tccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccgtgcagcccaccatgctgggcctgttcaagggccagtcctgc gtggtgcaggtgcacctgaagcgccacctgatgaaggacctgcccgagtccgaggacgacgtggcccagtggtgccgcgagcgcttcgt ggtgaaggactccctgctggacaagcacaaggtggaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccct ggtggtggtgatctcctgggcctgcatcctgatcttctggatcctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgcc atctccgcctgcgccatggccgtgatcgccttcctgatgcagatcctgctgcgcttctcccagtccgagcgctccacccccgccaagatcgccc ccgccaagcccaacaacgcccgcaactcctccgagaccgtgcgcgacaagcaccagTGA NucleotidesequenceoftheGindPAT2Ccodingsequence,usedinthetransforming DNAfrompSZ4417. SEQIDNO:96 ATGgccatccccgccgccatcatcatcgtgcccctgggcctgatcttcttcacctccggcttcatcatcaacctgatccaggccgtgtgctacg tgctgatccgccccctgtccaagtccaccttccgccgcatcaaccgccagctggccgagctgctgtggctggagctggtgtgggtggtggac tggtgggccggcgtgaagatccagctgttcaccaacaaggagaccctgcactccatcggcaaggagcacgccctggtgatctgcaaccag cgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccatggacgagtccaccctgaagtccg gcctgcagtggctgaaggacttcccccagcccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgcc caggagtacgccgcctccgccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgaacatcatgcg ctccttcgtgcccgccgtgtacgacgtgaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctcc gtggtgcacgtgcacctgaagcgccacctgatggaggacctgcccgagaccgacgacgacgtggcccagtggtgccgcgaccgcttcgtg gtgaaggactccctgctggacaagcacctggccgaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccctg gtggtggtgacctcctgggtgtgcatcatcgccttcggcgccctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgtg atctccgccgcctccctggccgtggtgaccgtgctgatgcagatcctgatccgcttctcccagtccgagcgctccacctccgccaaggtggtg gccgagaagcgcaagaacgtgggcgagcacTGA pSZEX61TransormingDNAexpressingCnLPAAT. SEQIDNO:97 gtttaaacgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacgg aaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgaca cgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagc agttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcg caggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcgg aacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttgg cgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcagg catcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtg gcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactc tcccgaccgcgcgcaggatagactcttgttcaaccaatcgacaggtaccATGgacgcctccggcgcctcctccttcctgcgcggccgct gcctggagtcctgcttcaaggcctccttcggctacgtaatgtcccagcccaaggacgccgccggccagccctcccgccgccccgccgacgcc gacgacttcgtggacgacgaccgctggatcaccgtgatcctgtccgtggtgcgcatcgccgcctgcttcctgtccatgatggtgaccaccatc gtgtggaacatgatcatgctgatcctgctgccctggccctacgcccgcatccgccagggcaacctgtacggccacgtgaccggccgcatgct gatgtggattctgggcaaccccatcaccatcgagggctccgagttctccaacacccgcgccatctacatctgcaaccacgcctccctggtgg acatcttcctgatcatgtggctgatccccaagggcaccgtgaccatcgccaagaaggagatcatctggtatcccctgttcggccagctgtac gtgctggccaaccaccagcgcatcgaccgctccaacccctccgccgccatcgagtccatcaaggaggtggcccgcgccgtggtgaagaag aacctgtccctgatcatcttccccgagggcacccgctccaagaccggccgcctgctgcccttcaagaagggcttcatccacatcgccctccag acccgcctgcccatcgtgccgatggtgctgaccggcacccacctggcctggcgcaagaactccctgcgcgtgcgccccgcccccatcaccgt gaagtacttctcccccatcaagaccgacgactgggaggaggagaagatcaaccactacgtggagatgatccacgccctgtacgtggacc acctgcccgagtcccagaagcccctggtgtccaagggccgcgacgcctccggccgctccaactccTGAttaattaactcgagatgtggaga tgtagggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttccca acgccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcg tctggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggaga gcgtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcg [00242]![embedded image]()
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cgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccga gcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgact gctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagaccccaacggcatgggccacgtcgccgac cacctgcacaacaactccttcctgacggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggcc gcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagt tcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccct gtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcgg agttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatga acatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcg tcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaa cgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatcccc gccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctgg acaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttc gactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggc gtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtcca agaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggc atcgcgactaccgcctgcgcccctcctccTGAtacaacttattacgtattctgaccggcgctgatgtggcgcggacgccgtcgtac tctttcagactttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgcaattaattgtgtgatgaagaaaggg tggcacaagatggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctggctcaatcttgtcgcatgt ccggcgcaatgtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactgatcgcattgcc atcccgtcaactcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaagcgt caggaaatcgtctcggcagctggaagcgcatggaatgcggagcggagatcgaatcagatatcAAGCTCCATCgagctccagc cacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgaggg ccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctacgccaacatga tgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaacctcttcgccga cttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaag gtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgcc gagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagg gagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccg cgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcaca tcaaagggcccctccgccagagaagaagctcctttcccagcagactcctgaagagcgtttaaac CpauLPAAT1 SEQIDNO:98 ggtaccATGgccatccccgccgccgccgtgatcttcctgttcggcctgctgttcttcacctccggacctgatcatcaacctgttccaggccctgtg cttcgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgagctgctgtgcctgttc gactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagcacgccctggtgatcatcaac cacatgaccgagctggactggatgctgggctgggtgatgggccagcacctgggctgcctgggctccatcctgtccgtggccaagaagtcc accaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacatcgagcgctcctgggccaaggaccgcaccaccctgaa gtcccacatcgagcgcctgaccgactaccccctgcccttctggatggtgatcttcgtggagggcacccgcttcacccgcaccaagctgctggc cgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacat gcgctccttcgtgcccgccgtgtacgacgtgaccgtggccttccccaagacctcccccccccccaccctgctgaacctgttcgagggccagtcc atcgtgctgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttc gtggagaaggacgccctgctggacaagcacaacgccgaggacaccttctccggccaggaggtgcaccgcaccggctcccgccccatcaa gtccctgctggtggtgatctcctgggtggtggtgatcaccttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggccttctc cgtgatcggcctgggcatcgtgaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctcctccaaccccgccaaggtggccc aggccaagctgaagaccgagctgtccatctccaagaaggccaccgacaaggagaacTGActcgag CprocLPAAT1 SEQIDNO:99 ggtaccATGgccatccccgccgccgccgtgatcttcctgttcggcctgatcttcttcgcctccggcctgatcatcaacctgttccag gccctgtgcttcgtgctgatctggcccatctccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgag ctgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagc acgccaggtgatcatcaaccacatgaccgagaggactggatggtgggagggtgatgggccagcactteggagcctgggctc catcctgtccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttcaccgagtacctgtacatcgagcg ctcctggaacaaggacaagtccaccctgaagtcccacatcgagcgcctgaaggactaccccctgcccttctggctggtgatcttcg ccgagggcacccgatcacccagaccaagagaggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaac gtgctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgcccgccgtgtacgacctgaccgtggcct tccccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccgtggtgctgcacgtgcacatcaagcgccacgc catgaaggacctgcccgagtccgacgacgaggtggcccagtggtgccgcgacaagncgtggagaaggacgccagaggac aagcacaacgccgaggacaccttaccggccaggagagcagcacaccggccgccgccccatcaagtccctgaggtggtgat ctcctgggtggtggtgatcgccttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggccttctccgtgatcggcctg ggcatcgtgaccagagatgcacatgagatcctgtecteccaggccgagcgaccaagcccgccaaggtggcccaggccaag ctgaagaccgagctgtccatctccaagaccgtgaccgacaaggagaacTGActcgag CpaiLPAAT1 SEQIDNO:100 ggtaccATGgccatcccctccgccgccgtggtgttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccagg ccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgatcaaccgcgtgttcgccgagctgctgcccctggagtt cctgtggctgaccactggtgcgccggcgccaagctgaagctgacaccgaccccgagaccaccgcctgatgggcaaggagcac gccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctcca tcctgtccgtggccaagaagtccaccaagacctgcccgtgacggctggtccctgtggactccggctacctgacctggagcgctcc tgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgccataggctgatcatcacgtgga gggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgct gatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccttcccc aagacctcccccccccccaccatgctgaagctgacgagggccagtccgtggagctgcacgtgcacatcaagcgccacgccatg aaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaagc acaactccgaggacaccactccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtgatctcctgggt ggtggtgatcatcacggcgccctgaagacctgctgtggtcctccctgctgtcctcctggaagggcaaggccactccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgagggctccaaccccgtgaaggc cgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaggagaacTGActcgag ChookLPAAT1 SEQIDNO:101 ggtaccATGgccatcccctccgccgccgtggtgttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccagg ccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgcccctggagtt cctgtggctgaccactggtgcgccggcgccaagctgaagctgacaccgaccccgagaccaccgcctgatgggcaaggagcac gccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctcca tcctgtccgtggccaagaagtccaccaagacctgcccgtgacggctggtccctgtggactccgagtacctgacctggagcgctcc tgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgccataggctgatcatcacgtgga gggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgct gatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccttcccc aagacctcccccccccccaccatgctgaagctgacgagggccagtccgtggagctgcacgtgcacatcaagcgccacgccatg aaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaagc acaactccgaggacaccactccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtgatctcctgggt ggtggtgatcatcacggcgccctgaagacctgctgtggtcctccctgctgtcctcctggaagggcaaggccactccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgagggctccaaccccgtgaaggc cgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag CignLPAAT1 SEQIDNO:102 ggtaccATGgccatcgccgccgccgccgtgatcttcctgttcggcctgctgttcttcgcctccggcatcatcatcaacctgttccag gccctgtgcacgtgctgatctggcccctgtccaagaacgtgtaccgccgcatcaaccgcgtgacgccgagctgctgctgatggac ctgctgtgcctgaccactggtgggccggcgccaagatcaagctgacaccgaccccgagaccaccgcctgatgggcatggagca cgccctggtgatcatgaaccacaagaccgacctggactggatggtgggctggatcctgggccagcacctgggctgcctgggctc catcctgtccatcgccaagaagtccaccaagacatccccgtgctgggctggtccgtgtggactccgagtacctgacctggagcgc tcctgggccaaggacaagtccaccctgaagtcccacatggagaagctgaaggactaccccctgccataggctggtgatcacgt ggagggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgt gctgatcccccacaccaagggcttcgtgtcctgcgtgtccaacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggcctt ccccaagtcctcccccccccccaccatgctgaagctgacgagggccagtccatcgtgctgcacgtgcacatcaagcgccacgcc ctgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggacaa gcacaacgccgaggacaccactccggccaggaggtgcaccacatcggccgccccatcaagtccctgctggtggtgatcgcctg ggtggtggtgatcatcacggcgccctgaagacctgcagtggtcctccctgctgtccacctggaagggcaaggccactccgtgatc ggcctgggcatcgccaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctccaaccccgccaaggtggccaag TGActcgag CavigLPAAT1 SEQIDNO:103 ggtaccATGaccatcgcctccgccgccgtggtgttcctgttcggcatcctgctgttcacctccggcctgatcatcaacctgttccag gccttctgctccgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagttcctgcccctggag ttcctgtggctgttccactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagc acgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctc catcctgtccgtggccaagaagtccaccaagacctgcccgtgacggctggtccctgtggactccgagtacctgacctggagcgc aactgggccaaggacaagaagaccctgaagtcccacatcgagcgcctgaaggactaccccctgccataggctgatcatcttcg tggagggcacccgatcacccgcaccaagctgctggccgcccagcagtacgccgcctccgccggcctgcccgtgccccgcaac gtgctgatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggcct tccccaagacctcccccccccccaccatgctgaagctgacgagggccacttcgtggagctgcacgtgcacatcaagcgccacgc catgaaggacctgcccgagtccgaggacgccgtggcccagtggtgccgcgacaagacgtggagaaggacgccctgctggac aagcacaacgccgaggacaccactccggccaggaggtgcaccacgtgggccgccccatcaagtccctgctggtggtgatctcc tgggtggtggtgatcatcacggcgccctgaagacctgcagtggtcctccctgctgtcctcctggaagggcatcgccactccgtgat cggcctgggcaccgtggccctgctgatgcagatcctgatcctgtcctcccaggccgagcgctccatccccgccaaggagaccccc gccaacctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag CavigLPAAT2 SEQIDNO:104 ggtaccATGgccatcgccgccgccgccgtgatcgtgcccgtgtccctgctgttcttcgtgtccggcctgatcgtgaacctggtgca ggccgtgtgatcgtgctgatccgccccctgacaagaacacctaccgccgcatcaaccgcgtggtggccgagctgctgtggctgg agctggtgtggctgatcgactggtgggccggcgtgaagatcaaggtgacaccgaccacgagaccaccacctgatgggcaagg agcacgccctggtgatctgcaaccacaagtccgacatcgactggctggtgggctgggtgctggcccagcgctccggctgcctggg ctccaccctggccgtgatgaagaagtcctccaagacctgcccgtgatcggctggtccatgtggactccgagtacctgacctggag cgcaactgggccaaggacgagtccaccctgaagtccggcctgaaccgcctgaaggactaccccctgccataggctggccctgt tcgtggagggcacccgatcacccgcgccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgca acgtgctgatcccccgcaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtgg ccatccccaagacctcccccccccccaccctgctgcgcatgacaagggccagtcctccgtgctgcacgtgcacctgaagcgcca ccagatgaacgacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacatcacgtggagaaggacgccctgctgg acaagcacaacgccgaggacaccactccggccaggagctgcaggacaccggccgccccatcaagtccctgctgatcgtgatct cctgggccgtgctggtggtgacggcgccgtgaagacctgcagtggtcctccctgctgtcctcctggaagggcctggccactccgg catcggcctgggcgtgatcaccctgctgatgcacatcctgatcctgactcccagtccgagcgctccacccccgccaaggtggccc ccgccaagcccaagatcgagggcgagtcctccaagaccgagatggagaaggagcacTGActcgag CpalLPAAT1 SEQIDNO:105 ggtaccATGgccatcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcgtgtccggcctgatcgtgaacctggtgca ggccgtgtgcttcgtgctgatccgccccctgtccaagaacacctaccgccgcatcaaccgcgtggtggccgagctgctgtggctgg agctggtgtggctgatcgactggtgggccggcgtgaagatcaaggtgttcaccgaccacgagaccctgtccctgatgggcaagg agcacgccctggtgatctgcaaccacaagtccgacatcgactggctggtgggctgggtgctggcccagcgctccggctgcctggg ctccaccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgcccgagtcc gacgacgccgtggcccagtggtgccgcgacatcttcgtggagaaggacgccctgctggacaagcacaacgccgaggacacctt ctccggccaggagctgcaggacaccggccgccccatcaagtccctgctggtggtgatctcctgggccgtgctggtgatcttcggcg ccgtgaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggccttctccggcgtgggcctgggcatcatcaccctgct gatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggcccccgccaagccaagaaggacggcga gtcctccaagaccgagatcgagaaggagaacgttcctggagcgctcctgggccaaggacgagaacaccctgaagtccggcct gaaccgcctgaaggactaccccctgcccUctggctggccctgttcgtggagggcacccgcttcacccgcgccaagctgctggcc gcccagcagtacgccacctcctccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctccgtgtc ccacatgcgctcatcgtgcccgccatctacgacgtgaccgtggccatccccaagacctcccccccccccaccatgctgcgcatgtt caagggccagtcctccgtgctgcacgtgcacctgaagcgccacctgatgaaggacctTGActcgag CuPSR23LPAAT2 SEQIDNO:106 ggtaccATGgccatcgccgccgccgccgtgatcttcctgttcggcctgatcttcttcgcctccggcctgatcatcaacctgttccag gccctgtgcttcgtgctgatccgccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgag ctgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagc acgccctggtgatcatcaaccacatgaccgagctggactggatggtgggctgggtgatgggccagcacttcggctgcctgggctc catcatctccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacctggagcg ctcctgggccaaggacaagtccaccctgaagtcccacatcgagcgcctgatcgactaccccctgcccttctggctggtgatcttcgt ggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgtgtcctccggcctgcccgtgccccgcaacgt gctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctcatcgtgcccgccgtgtacgacgtgaccgtggccttc cccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcatgctgcacgtgcacatcaagcgccacgcca tgaaggacctgcccgagtccgacgacgccgtggccgagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaa gcacaacgccgaggacaccttctccggccaggaggtgtgccactccggctcccgccagctgaagtccctgctggtggtgatctcc tgggtggtggtgaccaccttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggccttctccgccatcggcctggg catcgtgaccctgctgatgcacgtgctgatcctgtcctcccaggccgagcgctccaaccccgccgaggtggcccaggccaagctg aagaccggcctgtccatctccaagaaggtgaccgacaaggagaacTGActcgag CkoeLPAAT1 SEQIDNO:107 ggtaccATGgccatccccgccgccgtggccgtgatccccatcggcctgctgttcatcatctccggcctgatcgtgaacctgatcca ggccgtggtgtacgtgctgatccgccccctgtccaagaacctgcaccgcaagatcaacaagcccatcgccgagctgctgtggctg gagctgatctggctggtggactggtgggccggcatcaaggtggaggtgtacgccgactcccagaccctggagctgatgggcaag gagcacgccctgctgatctgcaaccaccgctccgacatcgactggctggtgggctgggtgctggcccagcgcgcccgctgcctgg gctccgccctggccatcatgaagaagtccgccaagttcctgcccgtgatcggctggtccatgtggttctccgactacatcttcctgga ccgcacctgggccaaggacgagaagaccctgaagtccggcttcgagcgcctggccgacttccccatgcccttctggctggccctg ttcgtggagggcacccgcttcaccaaggccaagctgctggccgcccaggagtacgccgcctcccgcggcctgcccgtgccccag aacgtgctgatcccccgcaccaagggcttcgtgaccgccgtgacccacatgcgctcctacgtgcccgccatctacgactgcaccg tggacatctccaaggcccaccccgccccctccatcctgcgcctgatccgcggccagtcctccgtggtgaaggtgcagatcacccg ccactccatgcaggagctgcccgagaccgccgacggcatctcccagtggtgcatggacctgttcgtgaccaaggacggcttcctg gagaagtaccactccaaggacatcttcggctccctgcccgtgcagaacatcggccgccccgtgaagtccctgatcgtggtgctgtg ctggtactgcctgatggccttcggcctgttcaagttcttcatgtggtcctccctgctgtcctcctgggagggcatcctgtccctgggcctg atcctgctggccgtggccatcgtgatgcagatcctgatccagtccaccgagtccgagcgctccacccccgtgaagtccatccaga aggacccctccaaggagaccctgctgcagaacTGActcgag CkoeLPAAT2 SEQIDNO:108 ggtaccATGcacgtgctgctggagatggtgaccttccgcttctcctccttcttcgtgttcgacaacgtgcaggccctgtgcttcgtgct gatctggcccctgtccaagtccgcctaccgcaagatcaaccgcgtgttcgccgagctgctgctgtccgagctgctgtgcctgttcga ctggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagcacgccctggtgatcac caaccacaagatcgacctggactggatgatcggctggatcctgggccagcacttcggctgcctgggctccgtgatctccatcgcca agaagtccaccaagttcctgcccatchcggctggtccctgtggttctccgagtacctgttcctggagcgcaactgggccaaggaca agcgcaccctgaagtcccacatcgagcgcatgaaggactaccccctgcccctgtggctgatcctgttcgtggagggcacccgctt cacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgctgatcccccacac caagggcttcgtgtcctccgtgtcccacatgcgctcatcgtgcccgccgtgtacgacgtgaccgtggcchccccaagacctcccc cccccccaccatgctgtccctgttcgagggccagtccgtggtgctgcacgtgcacatcaagcgccacgccatgaaggacctgccc gactccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaagcacaacgccgagg acaccttctccggccaggaggtgcaccacgtgggccgccccatcaagtccctgctggtggtgatctcctggatggtggtgatcatct tcggcgccctgaagttcctgcagtggtcctccctgctgtcctcctggaagggcaaggccttctccgccatcggcctgggcatcgcca ccctgctgatgcacgtgctggtggtgttctcccaggccgaccgctccaaccccgccaaggtgccccccgccaagctgaacaccga gctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag CprocLPAAT2 SEQIDNO:109 ggtaccATGgccatccccgccgccgtggccgtgatccccatcggcctgctgttcatcatctccggcctgatcgtgaacctgatcca ggccgtggtgtacgtgctgatccgccccctgtccaagaacctgtaccgcaagatcaacaagcccatcgccgagctgctgtggctg gagctgatctggctggtggactggtgggccggcatcaaggtggaggtgtacgccgactccgagaccctggagtccatgggcaag gagcacgccctgctgatctgcaaccaccgctccgacatcgactggctggtgggctgggtgctggcccagcgcgcccgctgcctgg gctccgccctggccatcatgaagaagtccgccaagttcctgcccgtgatcggctggtccatgtggttctccgactacatcttcctgga ccgcacctgggagaaggacgagaagaccctgaagtccggcttcgagcgcctggccgachccccatgcccttctggctggccct gttcgtggagggcacccgcttcaccaaggccaagctgctggccgcccaggagttcgccgcctcccgcggcctgcccgtgcccca gaacgtgctgatcccccgcaccaagggcttcgtgaccgccgtgacccacatgcgctcctacgtgcccgccatctacgactgcacc gtggacatctccaaggcccaccccgccccctccatcctgcgcctgatccgcggccagtcctccgtggtgaaggtgcagatcaccc gccactccatgcaggagctgcccgagacccccgacggcatctcccagtggtgcatggacctgttcgtgaccaaggacgcttcct ggagaagtaccactccaaggacatcttcggctccctgcccgtgcacgacatcggccgccccgtgaagtccctgatcgtggtgctgt gctggtactccctgatggcchcggcactacaagttcttcatgtggtcctccctgctgtcctcctgggagggcatcctgtccctgggcct ggtgctgatcgtgatcgccatcgtgatgcagatcctgatccagtcctccgagtccgagcgctccacccccgtgaagtccgtgcaga aggacccctccaaggagaccctgctgcagaacTGActcgag CavigGPAT9 SEQIDNO:110 ggtaccATGgccaccggcggctccctgaagccctcctcctccgacctggacctggaccaccccaacatcgaggactacctgcc ctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccgc cggcgccatcgtggacgactcchcacccgctgatcaagtccatcccccgcgagccctggaactggaacctgtacctgttccccct gtggtgcatcggcgtgctgatccgctacttcatcctgttccccggccgcgtgatcgtgctgaccatgggctggatcaccgtgatctcct catcatcgccgtgcgcgtgctgctgaagggccacgacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tcatcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttchcatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctgc tgcagtccaccctgctggagtccgtgggctgcatctggacgaccgcgccgaggccaaggaccgcggcatcgtggccaagaagc tgtgggaccacgtgcacggcgagggcaacaaccccctgctgatchccccgagggcacctgcgtgaacaacaactactccgtga tgttcaagaagggcgcchcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatchcgtggacgcatctgg aactccaagaagcagtcchcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagcc ccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgcccgcgccggcctgaaga aggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagacchcgccgagtcc gtgctgcagcgcctggaggagTGActcgag ChookGPAT9-1 SEQIDNO:111 ggtaccATGgccaccgccggctccctgaagccctcccgctccgagctggacttcgaccgccccaacatcgaggactacctgcc ctccggctcctccatcatcgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccgcc ggcgccatcgtggacgactcatcacccgctgatcaagtccaacccccccgagccctggaactggaacatctacctgttccccct gtggtgatcggcgtgctgatccgctacctgatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcatatcctgtcctc atcatccccgtgcacctgctgctgaagggccacgacgccctgcgcatcaagctggagcgcctgctggtggagctgatctgctcat atcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaaccac acctccatgatcgacttatcatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctgctg cagtccaccctgctggagtccgtgggctgcatctggttcgaccgcgccgaggccaaggaccgcggcatcgtggccaagaagctg tgggaccacgtgcacggcgagggcaacaaccccctgctgatatccccgagggcacctgcgtgaacaacaactactccgtgatg ttcaagaagggcgcatcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatatcgtggacgcatctggaa ctccaagaagcagtcatcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagcccc agaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctgaagaag gtgccctgggacggctacctgaagtactcccgcccctcccccaagcacaccgagcgcaagcagcagaacttcgccgagtccgt gctgcagcgcctggagaagaagTGActcgag CignGPAT9-1 SEQIDNO:112 ggtaccATGgccaccggcggccgcctgaagccctcctcctccgagctggacctggaccgcgccaacaccgaggactacctgc cctccggctcctccatcaacgagcccgtgggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccg ccggcgccatcgtggacgactcatcacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgttccccc tgtggtgatcggcgtgctgatccgctacttcatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcaccgtgatctcct catcaccgccgtgcgatcctgctgaagggccacaacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tcatcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttcctgatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctg ctgcagtccaccctgctggagtccgtgggctgcatctggttcaaccgcgccgaggccaaggaccgcgagatcgtggccaagaag ctgtgggaccacgtgcacggcgagggcaacaaccccctgctgatatccccgagggcacctgcgtgaacaaccactactccgtg atgttcaagaagggcgcatcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatatcgtggacgcatctg gaactcccgcaagcagtcatcaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagc cccagaccctgaagcccggcgagaccgccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctgaag aaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagtccaagcagcagtcatcgccgagtcc gtgctgcgccgcctggaggagaagTGActcgag CignGPAT9-2 SEQIDNO:113 ggtaccATGgccaccggcggccgcctgaagccctcctcctccgagctggacctggaccgcgccaacaccgaggactacctgc cctccggctcctccatcaacgagcccgtgggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccg ccggcgccatcgtggacgactcatcacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgttccccc tgtggtgatcggcgtgctgatccgctacttcatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcaccgtgatctcct catcaccgccgtgcgatcctgctgaagggccacaacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tcatcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttcctgatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctg ctgcagtccaccctgctggagtccgtgggctgcatctggttcaaccgcgccgaggccaaggaccgcgagatcgtggccaagaag ctgtgggaccacgtgcacggcgagggcaacaaccccctgctgatatccccgagggcacctgcgtgaacaaccactactccgtg atgttcaagaagggcgcatcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatatcgtggacgcatctg gaactccaagaagcactcatcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagc cccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccgacctgaag aaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaagttcgccgagtc cgtgctgcgccgcctggaggagaagTGActcgag CpalGPAT9-1 SEQIDNO:114 ggtaccATGgccaccgccggccgcctgaagccctcctcctccgagctggagctggacctggaccgcccaacatcgaggact acctgccctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccatgctgaccga ggccgccggcgccatcgtggacgactccacacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgt tccccctgtggtgatcggcgtgctgatccgctacctgatcctgaccccgcccgcgtgatcgtgctgaccgtgggctggatcaccgtg atctcctccacatcaccgtgcgcacctgctgaagggccacgactccctgcgcatcaagctggagcgcctgatcgtgcagctgttct gctcctccacgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgcccccagcaggtgtacgtggcc aaccacacctccatgatcgacttcatcatcctgaaccagatgaccgtgactccgccatcatgcagaagcaccccggctgggtggg cctgatccagtccaccatcctggagtccgtgggctgcatctggacaaccgcgccgaggccaaggaccgcgagatcgtggccaa gaagctgctggaccacgtgcacggcgagggcaacaaccccctgctgatcaccccgagggcacctgcgtgaacaaccactactc cgtgatgacaagaagggcgccacgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcacgtggacgcct tctggaactccaagaagcagtccacaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacagg agccccagaccctgaagcccggcgagacccccatcgagacgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctg aagaaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagtccacgccga gtccgtgctgcgccgcctggagaagcgcTGActcgag CpalGPATt9-2 SEQIDNO:115 ggtaccATGgccaccgccggccgcctgaagccctcctcctccgagctggagctggacctggaccgccccaacatcgaggact acctgccctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccatgctgaccga ggccgccggcgccatcgtggacgactccacacccgctgatcaagtccatcccccccgagccctggaactggaacatctacctgt tccccctgtggtgatcggcgtgctgatccgctacctgatcctgaccccgcccgcgtgatcgtgctgaccgtgggctggatcaccgtg atctcctccacatcaccgtgcgcacctgctgaagggccacgactccctgcgcatcaagctggagcgcctgatcgtgcagctgttct gctcctccacgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgcccccagcaggtgtacgtggcc aaccacacctccatgatcgacttcatcatcctgaaccagatgaccgtgactccgccatcatgcagaagcaccccggctgggtggg cctgatccagtccaccatcctggagtccgtgggctgcatctggacaaccgcgccgaggccaaggaccgcgagatcgtggccaa gaagctgctggaccacgtgcacggcgagggcaacaaccccctgctgatcaccccgagggcacctgcgtgaacaaccactactc cgtgatgacaagaagggcgccacgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcacgtggacgcct tctggaactccaagaagctgtcatcaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacagg agccccagaccctgaagcccggcgagacccccatcgagacgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctg aagaaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaccacgccg agtccgtgctgcgccgcctggaggagaagggcaacgtggtgcccaccgtgaacTGActcgag CavigDGAT1 SEQIDNO:116 ggtaccATGgccatcgccgacggcggcatcatcggcgccgccggctccatctccgccctgaccgccgacaccgaccccccct ccctgcgccgccgcaacgtgcccgccggccaggcctccgccgtgtccgccactccaccgagtccatggccaagcacctgtgcga cccctcccgcgagccctccccctcccccaagtcctccgacgacggcaaggaccccgacatcggctccgtggactccctgaacga gaagccctcctcccccgccgccggcaagggccgcctgcagcacgacctgcgcttcacctaccgcgcctcctcccccgcccaccg caaggtgaaggagtcccccctgtcctcctccaacatcacaagcagtcccacgccggcctgacaacctgtgcgtggtggtgctggt ggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggcctgctgatcaagaccggataggactcctcccgctccct gcgcgactggcccctgacatgtgctgcctgtccctgcccatcaccccctggccgccacctggtggagaagctggcccagaagaa ccgcctgcaggagcccaccgtggtgtgctgccacgtgctgatcacctccgtgtccatcctgtaccccgtgctggtgatcctgcgctg cgactccgccgtgctgtccggcgtggccctgatgctgacgcctgcatcgtgtggctgaagctggtgtcctacgcccactccaactac gacatgcgctacgtggccaagtccctggacaagggcgagcccgtggtggactccgtgatcgccgaccacccctaccgcgtgga ctacaaggacctggtgtacttcatggtggcccccaccctgtgctaccagctgtcctaccccctgaccccctgcgtgcgcaagtcctg gatcgcccgccaggtgatgaagctggtgctgacaccggcgtgatgggatcatcgtggagcagtacatcaaccccatcgtgcag aactccaagcaccccctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaacctgtacgtgtggc tgtgcatgactactgatcaccacctgtggctgaacatcctggccgagctgatctgatcggcgaccgcgagactacaaggactgg tggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgccacatctacacccct gcctgcgcaacggcatcccccgcggcgtggccgtgctgatcgccacctggtgtccgccgtgaccacgagctgtgcatcgccgtgc cctgccacgtgttcaagctgtgggcchcatcggcatcatgttccaggtgcccctggtgctggtgtccaactgcctgcagaagaagtt ccagtcctccatggccggcaacatgttatctgghcatchctgcatchcggccagcccatgtgcgtgctgctgtactaccacgacct gatgaaccgcaagggctccgcatcgacTGActcgag ChookDGAT1-1 SEQIDNO:117 ggtaccATGgccatcgccgacggcggctccgccggcgccgccggctccatctccggctccgacccctccccctccaccgcccc ctccctgcgccgccgcaacgcctccgccggccaggcchaccaccgagtccatggcccgcgacctgtgcgacccctcccgcga gccctccctgtcccccaagtcctccgacgacggcaaggaccccgccgacgacatcggcgccgccgactccgtggactccggcg gcgtgaaggacgagaagccctcctcccaggccgccgccaaggcccgcctggagcacgacctgcgatcacctaccgcgcctcc tcccccgcccaccgcaaggtgaaggagtcccccctgtcctcctccaacatchcaagcagtcccacgccggcctgttcaacctgtg cgtgggtggtgctggtggccgtgaactcccgcctgatcatcgagaacctgatgagtacggcctgctgataagaccggcttctggtt ctcctcccgctccctgcgcgactggcccctgttcatgtgctgcctgtccctgcccatcaccccctggccgcchcctggtggagaagc tggcccagaagaaccgcctgcaggagcccaccgtggtgtgctgccacgtgatcatcacctccgtgtccatcctgtaccccgtgctg gtgatcctgcgctgcgactccgccgtgctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtcctacg cccacgccaactacgacatgcgctccgtggccaagtccctggacaagggcgagaccgtggccgactccgtgatcgtggaccac ccctaccgcgtggactacaaggacctggtgtacttcatggtggcccccaccctgtgctaccagctgtcctaccccctgaccccctac gtgcgcaagtcctgggtggcccgccaggtgatgaagctggtgctgttcaccggcgtgatgggchcatcgtggagcagtacatcaa ccccatcgtgcagaactccaagcaccccctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaa cctgtacgtgtggctgtgcatgttctactgatchccacctgtggctgaacatcctggccgagctgacctgatcggcgaccgcgagt tctacaaggactggtggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgc cacatctacttcccctgcctgcgcaacggcatcccccgcggcgtggccgtgctgatcgcchcctggtgtccgccgtgttccacgag ctgtgcatcgccgtgccctgccacgtgttcaagctgtgggcchcatcggcatcatgttccaggtgcccctggtgctggtgtccaactg cctgcagaagaagttccagtcctccatggccggcaacatgttatctgghcatchctgcatchcggccagcccatgtgcgtgctgct gtactaccacgacctgatgaaccgaagggctcccgcatcgacTGActcgag CavigLPCAT SEQIDNO:118 ggtaccATGggcctggtgtccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctggccaccat ccccgtgtcchcctgtggcgcctggtgcccggccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgtcctacct gtcatcggcgcctcctccaacctgcacttcatcgtgcccatgaccctgggctacctgtccatgctgttchccgcccatctccggcct gctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcggcatcg acgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatgaactacaacgacggcctgctgaaggaggagg gcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacttcggctactgcctgtgctgcggctc ccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcatctggtcccgctcccagaagg agcccaagccctccccatcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgtacctgtacctggtgccc caccaccccctgacccgatcaccgagcccgtgtactacgagtggggcttchccgccgcctgtcctaccagtacatggccgccctg accgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggaccgagt cctccccccccaagccccgctgggaccgcgccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgcagctgc ccctggtgtggaacatccaggtgtccatctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccccggat caccagctgctggccacccagaccgtgtccgccgtgtggcacggcctgtaccccggctacatcatchatcgtgcagtccgccctg atgatcgccggctcccgcgtgatctaccgctggcagcaggcggtgcccccaagatgggcctggtgaagaacatcttcgtgttctt caacttcgcctacaccctgctggtgctgaactactccgccgtgggchcatggtgctgtccatgcacgagaccctggcctcctacgg ctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccgcccgctccaa ggcccacaaggagcagTGActcgag CpalLPCAT SEQIDNO:119 ggtaccATGgagctgggctccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctggccaccat ccccgtgtcchcctgtggcgcctggtgcccggccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgtcctacct gtccttcggcccctcctccaacctgcacttcatcgtgcccatgaccctgggctacctgtccatgctgttcttccgccccttctcggcct gctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcggcatcg acgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggaggagg gcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacatcggctactgcctgtgctgcggctc ccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcgtgtggtcccactccgagaagg agcccaagccctccccatcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgtacatgtacctggtgccc caccaccccctgtcccgatcaccgagcccgtgtactacgagtggggcacttccgccgcctgtcctaccagtacatggccggcctg accgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggaccgagt cctccccccccaagccccgctgggaccgcgccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgcagctgc ccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccccggat caccagctgctggccacccagaccgtgtccgccatctggcacggcctgtaccccggctacatcatatatcgtgcagtccgccctg atgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccccaagatgggcctggtgaagaacatcttcgtgttctt caacttcgcctacaccctgctggtgctgaactactccgccgtgggatcatggtgctgtccatgcacgagaccctggcctcctacgg ctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccgcccgctccaa ggcccacaaggagcagTGActcgag CpauLPCAT SEQIDNO:120 ggtaccATGgagctggagatcggctccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctgg ccaccatccccgtgtccttcctgtgccgcctgctgcccgcccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgt cctacctgtccttcggcccctcctccaacctgcacttcatcgtgcccatgtccctgggctacctgtccatgctgttcttccgccccttctcc ggcctgctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcgg catcgacgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggag gagggcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacttcggctactgcctgtgctgcg gctcccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcatctggtcccgctccgaga aggaccccaagccctccccatcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgcacatgtacctggt gccccaccaccccctgacccgcttcaccgagcccgtgtactacgagtggggcttcttccgccgcctgtcctaccagtacatggccg cccagaccgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggac cgagtcctccccccccaagccccgctgggacaaggccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgca gctgcccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccc cggcttcttccagctgctggccacccagaccgtgtccgccgtgtggcacggcctgtaccccggctacatcatcttcttcgtgcagtcc gccctgatgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccagaagatgggcctggtgaagaacatcttcg tgttcttcaacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcc tacggctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccacccg ctccaaggtgcacaaggagcagTGActcgag CschuLPCAT SEQIDNO:121 ggtaccATGgagctggagatggagcccctggccgccgccatcggcgtgtccgtggccgtgttccgcttcctggtgtgcttcatcg ccaccatccccgtgtccttcatctgccgcctggtgcccggcggcctgccccgccacctgttctccgccgcctccggcgccgtgctgtc ctacctgtccttcggcttctcctccaacctgcacttcctggtgcccatgaccctgggctacctgtccatgatcctgttccgccgcttctgc ggcatcctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcgg catcgacgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggag gagggcctgcgcgagtcccagaagaagaaccgcctgatccgcctgccctccctgatcgagtacttcggctactgcctgtgctgcg gctcccacttcgccggccccgtgtacgagatgaaggactacctggactggaccgagggcaagggcatctggtcccactccgaga agggccccaagccctcccccctgcgcgccgccctgcgcgccatcatccaggccggcttctgcatggccatgtacctgtacctggtg ccccactaccccctgacccgcttcaccgaccccgtgtactacgagtggggcatcctgcgccgcctgtcctaccagtacatggcctc cttcaccgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggacc gagtcctccccccccaagccccgctgggaccgcgccaagaacgtggacatcctgggcgtggagctggccaagtcctccgtgca gatccccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccc cggatcctgcagctgctggccacccagaccgtgtccgccatctggcacggcgtgtaccccggctacctgatcacttcgtgcagtcc gccctgatgatcgccggctcccgcgccatctaccgctggcagcaggccgtgccccccaagatgtccctggtgaagaacaccctg gtgacttcaacttcgcctacaccctgctggtgctgaactactccgccgtgggatcatggtgctgtccatgcacgagaccctggcctc ctacggctccgtgtactacgtgggcaccatcctgcccgtgaccctgatcctgctgggctacgtgatcaagcccggcaagtcccccc gctccaaggcctccaaggagcagTGActcgag CavigPLA2-1 SEQIDNO:122 ggtaccATGaacttcgacttcctgtccaacatcccctggttcggcgccaaggcctccgacaacgccggctcctccttcggctccg ccaccatcgtgatccagcagcccccccccgtgtcccgcggatcgacatccgccactggggctggccctggtccgtgctgtccgtg ctgccctggggcaagcccggctgcgacgagctgcgcgccccccccaccaccatcaaccgccgcctgaagcgcaacgccacct ccatgcactcctccgccgtgcgcggcaacgccgaggccgcccgcgtgcgcaccgcccctacgtgtccaaggtgccctggcaca ccggcaccgcggcctgctgtcccagctgacccccgctacggccactactgcggccccaactggtcctccggcaagaacggcgg ctcccccgtgtgggaccagcgccccatcgactggctggactactgctgctactgccacgacatcggctacgacacccacgacca ggccaagctgctggaggccgacctggccacctggagtgcctggagcgcccctcctaccccaccaagggcgacgcccacgtgg cccacatgtacaagaccatgtgcgtgaccggcctgcgcaacgtgctgatcccctaccgcacccagctgctgcgcctgaactcccg ccagcccctgatcgacttcggctggctgtccaacgccgcctggaagggctggaacgcccagaagtccTGActcgag CignPLA2-1 SEQIDNO:123 ggtaccATGaacctggacttcctgtccaagatcccctggttcgaggccaaggcctccgagaaccccggcctgaacctgggctcc accaccatcgtgatcaagcagccccgccagggatcgacatccgccactggggctggccctggtccgtgctgacctggggcaac cgcgtgaccgacgaggtgcacgccccccccaccaccatcaaccgccgcctgaagcgcaacgccaccggccccgccgtgcag ggcgacaccgaggccgcccgcctgcgcaccgcccctacgtgtccaaggtgccctggcacaccggcaccgcggcctgctgtccc agctgacccccgctacggccactactgcggccccaactggtcctccggcaagaacggcggctcccccgtgtgggaccagcgcc ccatcgactggctggactactgctgctactgccacgacatcggctacgacacccacgaccaggccaagctgctggaggccgacc tggccacctggagtgcctggagcgcccctcctaccccaccaccggcgacgcccacgtggcccacatgtacaagaccatgtgcgt gaccggcctgcgcaacgtgctgatcccctaccgcacccagctgctgcgcctgaacttccgccagcccctgatcgacttcggctggc tgtccaacgccgcctggaagggctggtccgcccagaagaccTGActcgag CuPSR23PLA2-2 SEQIDNO:124 ggtaccATGgtgcacctgccccacaccctgaagctgggcctggtgatcgccatctccatctccggcctgtgcttctcctccacccc cgcccgcgccctgaacgtgggcatccaggccgccggcgtgaccgtgtccgtgggcaagggctgctcccgcaagtgcgagtccg acactgcaaggtgccccccacctgcgctacggcaagtactgcggcctgatgtactccggctgccccggcgagaagccctgcgac ggcctggacgcctgctgcatgaagcacgacgcctgcgtgcaggccaagaacaacgactacctgtcccaggagtgctcccagaa cctgctgaactgcatggcctccaccgcatgtccggcggcaagcagacaagggctccacctgccaggtggacgaggtggtggac gtgctgaccgtggtgatggaggccgccctgctggccggccgctacctgcacaagcccTGActcgag CprocPLA2-2 SEQIDNO:125 ggtaccATGgtgcacctgccccacaccctgaagctgggcctggtgatcgccatctccatctccggcctgtgcctgtcctccacccc cgcccgcgccctgaacgtgggcatccaggccgccggcgtgaccgtgtccgtgggcaagggctgctcccgcaagtgcgagtccg acactgcaaggtgccccccacctgcgctacggcaagtactgcggcctgatgtactccggctgccccggcgagaagccctgcgac ggcctggacgcctgctgcatgaagcacgacgcctgcgtgcaggccaagaacgacgactacctgtcccaggagtgctcccagaa cctgctgaactgcatggcctccaccgcatgtccggcggcaagcagacaagggctccacctgccaggtggacgaggtggtggac gtgctgaccgtggtgatggaggccgccctgctggccgccgctacctgcacaagcccTGActcgag NucleotidesequenceoftransformingDNAcontainedinpSZ5654 PmKASII SEQIDNO:126 gtttaaacgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacgg aaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgaca cgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagc agttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcg caggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcgg aacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttgg cgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcagg catcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtg gcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactc [00253]![embedded image]()
gccactgcttcggcgcccgcctgcccaccgcctcccgccgcgccgtgcgccgcgcctggtcccgcatcgcccggggcgcgccac cgccgccgccgacgccaaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccag accatcgagcagttctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacacc accaccatcgccggcgagatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtga tcaagtacgtgtacatcgccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccgg cctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgac ccgcggcggcgtgcgcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatc ggcttcatgggccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgcc gcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaag gccctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagg gcgccggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcg ccgccacctccgacgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggag cgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccg cgccatccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccgg cgccgtggaggccgtggccgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccg gcgtggaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttc ggcggccacaactcctgcgtgatcttccgcaagtacgacgagATGGACTACAAGGACCACGACGGCGACTACAA [00254]![embedded image]()
cactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcc tcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctc gtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgca cagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggga tgggaacacaaatggagagctccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcg gcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccgttcacacacgtgccacgttggcg [00255]![embedded image]()
[00256]![embedded image]()
[00257]![embedded image]()
[00258]![embedded image]()
[00259]![embedded image]()
cacgatcgggacgctgcgcaaggccatccccgcgcactgtttcgagcgctcggcgcttcgtagcagcatgtacctggcctttg acatcgcggtcatgtccctgctctacgtcgcgtcgacgtacatcgaccctgcaccggtgcctacgtgggtcaagtacggcatc atgtggccgctctactggttcttccaggtgtgtttgagggttttggttgcccgtattgaggtcctggtggcgcgcatggaggag aaggcgcctgtcccgctgacccccccggctaccctcccggcaccttccagggcgcgtacgagaagaaccagtagagcggcca catgatgccgtacttgacccacgtaggcaccggtgcagggtcgatgtacgtcgacgcgacgtagagcagggacatgaccg cgatgtcaaaggccaggtacatgctgctacgaagcgccgagcgctcgaaacagtgcgcggggatggccttgcgcagcgtc ccgatcgtgaacggaggcttctccacaggctgcctgttcgtcttgatagccatctcgaggcagcagcagctcggatagtatcga cacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacag cctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccc tcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcg cacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgg gatgggaacacaaatggaaagctgtagagctcgatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccat gtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggca [00260]![embedded image]()
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[00263]![embedded image]()
[00264]![embedded image]()
[00265]![embedded image]()
[00266]![embedded image]()
ctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgc tggacacggccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctcc ggccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcaca acaactccttcctgttcggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggagg aggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcc cgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccctgtgcaactg gggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcggagttcacgc gccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctga acaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaac ctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaaca acctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcg cgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggc gaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaac ctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccat cctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgac acccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttct [00267]![embedded image]()
ttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgcaattaattgtgtgatgaagaaagggtggcacaaga tggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctggctcaatcttgtcgcatgtccggcgcaat gtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactgatcgcattgccatcccgtcaa ctcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaagcgtcaggaaatcg tctcggcagctggaagcgcatggaatgcggagcggagatcgaatcaggatccttagggagcgacgagtgtgcgtgcggggctggc gggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacga agaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaa gctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctc cgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgct cgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtcctgggcgaagaacgagggaatttg tgggtaaaacaagcatcgtctctcaggccccggcgcagtggccgttaaagtccaagaccgtgaccaggcagcgcagcgcgtccgt gtgcgggccctgcctggcggctcggcgtgccaggctcgagagcagctccctcaggtcgccttggacggcctctgcgaggccggtga gggcctgcaggagcgcctcgagcgtggcagtggcggtcgtatccgggtcgccggtcaccgcctgcgactcgccatccgaagagcg tttaaac NucleotidesequenceoftransformingDNAcontainedinpSZ5868 GarmFATA1(G108A) SEQIDNO:127 gaagagcgcccaatgtttaaacctcttttgctgcgtctcctcaggcttgggggcctccttgggcttgggtgccgccatgatctgcgcg catcagagaaacgttgctggtaaaaaggagcgcccggctgcgcaatatatatataggcatgccaacacagcccaacctcactcg ggagcccgtcccaccacccccaagtcgcgtgccttgacggcatactgctgcagaagcttcatgagaatgatgccgaacaagaggg gcacgaggacccaatcccggacatccttgtcgataatgatctcgtgagtccccatcgtccgcccgacgctccggggagcccgccga tgctcaagacgagagggccctcgaccaggaggggctggcccgggcgggcactggcgtcgaaggtgcgcccgtcgttcgcctgca gtcctatgccacaaaacaagtcttctgacggggtgcgtttgctcccgtgcgggcaggcaacagaggtattcaccctggtcatgggg agatcggcgatcgagctgggataagagatacggtcccgcgcaaggatcgctcatcctggtctgagccggacagtcattctggcaa gcaatgacaacttgtcaggaccggaccgtgccatatatttctcacctagcgccgcaaaacctaacaatttgggagtcactgtgcca ctgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtcagcaccgccagccggccgaggacccgagtcata [00268]![embedded image]()
ggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctc ctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgacc gaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacc atcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccacc atgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtgga gatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggt gatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcga cgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagct ggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtg acctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccg ccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccaca acggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAAGGACCACGACGGCG ACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGAatcgatggagcgacgagtgtgcgt gcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatc gagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgcca gagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaact tgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggc gagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtgagctccgcgtctcgaaca gagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttg gttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggt [00269]![embedded image]()
[00270]![embedded image]()
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[00273]![embedded image]()
[00274]![embedded image]()
tcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttcg tgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggc tggtggactggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccct ggtggtggccaaccaccgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccct ggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcct gggccaaggacgagaacaccctgaaggccggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtg gagggcacccgcttcacccaggccaagttcctggccgcccaggagtacgccgcctcccagggcctgcccatcccccgcaacgt gctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgcgctccttcgtgcccgccatctacgacatgaccgtggcc atccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctccgtggtgcacgtgcacatcaagcgctgcct gatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgacgtggagaaggacaagctgctgg acaagcacatcgccgaggacaccactccgaccagcccatgcaggacctgggccgccccatcaagtccctgctggtggtggcc tcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcgccacttc ctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtgg [00275]![embedded image]()
agggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttcccaac gccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcgtc tggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggagagc gtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcgctg ttaggctgttaatcaaggagcgtatcaataattaccgaccctatacctttatctccaacccaatcgcggcttaaggatctaagtaa gattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacg tgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagatacagcgcgagccagacacggagtg [00276]![embedded image]()
[00277]![embedded image]()
[00278]![embedded image]()
[00279]![embedded image]()
[00280]![embedded image]()
ccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgc caagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgactggggccacgccacgtccgacg acctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtg gactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccg gagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccg ccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccag gactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcacctcgg ctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccat caaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaa ccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccct gggcatcgcgtgggcctccaactgggagtactccgccacgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaag actccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagca acgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcac cggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctgg ttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaac agcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaac gacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccacc aacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgaca [00281]![embedded image]()
cgaaacaagcccctggagcatgcgtgcatgatcgtctctggcgccccgccgcgcggtttgtcgccctcgcgggcgccgcggccgcg ggggcgcattgaaattgttgcaaaccccacctgacagattgagggcccaggcaggaaggcgttgagatggaggtacaggagtcaa gtaactgaaagtttttatgataactaacaacaaagggtcgtttctggccagcgaatgacaagaacaagattccacatttccgtgtag aggcttgccatcgaatgtgagcgggcgggccgcggacccgacaaaacccttacgacgtggtaagaaaaacgtggcgggcactgtc cctgtagcctgaagaccagcaggagacgatcggaagcatcacagcacaggatcctgaggacagggtggttggctggatggggaa acgctggtcgcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtgcacacaca caacccaaccggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatcctcctccctgcc gcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacaggccccttgtgcgcagtg ctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtgtggccaagcgggctgctgggcgc gccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttctgtaagccacgcgcttgctgctttgggaagagaagg gggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttggggaggcagggagagttggaaaatgt aagtggcacgacgggcaaggagaatggtgagcatgtgcatggtgatgtcgttggtcgaggacgatcctgcacgcgtgtatctgat gtagaatacggcaatcaccctagtctacatctataccttctccgtataacgccctttccaaatgccctcccgtttctctcctattcttg atccacatgatgaccctggcactatttcaagggctggagaagagcgtttaaac NucleotidesequenceoftransformingDNAcontainedinpSZ6383 TcDGAT1andGarmFATA1(G108A) SEQIDNO:128 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00282]![embedded image]()
[00283]![embedded image]()
[00284]![embedded image]()
[00285]![embedded image]()
[00286]![embedded image]()
[00287]![embedded image]()
[00288]![embedded image]()
[00289]![embedded image]()
[00290]![embedded image]()
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[00293]![embedded image]()
ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00294]![embedded image]()
cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00295]![embedded image]()
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[00299]![embedded image]()
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cgagatcctgggctccaccgccaccgtgacctcctcctcccactccgactccgacctgaacctgctgtccatccgccgccgcacct ccaccaccgccgccgcccgcgcccccgaccgcgacgactccggcaacggcgaggccgtggacgaccgcgaccgcgtggagt ccgccaacctgatgtccaacgtggccgagaacgccaacgagatgcccaactcctccgacacccgcttcacctaccgcccccgcg tgcccgcccaccgccgcatcaaggagtcccccctgtcctccggcgccatcttcaagcagtcccacgccggcctgttcaacctgtgc atcgtggtgctggtggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggctggctgatccgctccggcttctggt tctcctcccgctccctgtccgactggcccctgttcatgtgctgcctgaccctgcccatcttccccctggccgccttcgtggtggagaa gctggtgcagcgcaactacatctccgagcccgtggtggtgttcctgcacgccatcatctccaccaccgccgtgctgtaccccgtg atcgtgaacctgcgctgcgactccgccttcctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtc ctacgcccacaccaacaacgacatgcgcgccctggccaagtccgccgagaagggcgacgtggacccctcctacgacgtgtcct tcaagtccctggcctacttcatggtggcccccaccctgtgctaccagcagtcctacccccgcacccccgccgtgcgcaagtcctgg gtggtgcgccagttcatcaagctgatcgtgttcaccggcctgatgggcttcatcatcgagcagtacatcaaccccatcgtgcag aactcccagcaccccctgaagggcaacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaacctgtacgtgtgg ctgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgctgcgcttcggcgaccgcgagttctacaagga ctggtggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgccacatctac ttcccctgcctgcgcaacggcatccccaagggcgtggccatcgtgatcgccttcctggtgtccgccgtgttccacgagctgtgcat cgccgtgccctgccacatgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctgatcaccaactacctgc aggacaagttccgctcctccatggtgggcaacatgatcttctggttcatcttctccatcctgggccagcccatgtgcgtgctgctgt [00304]![embedded image]()
gacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaac agcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttc cctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccct cgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagt gggatgggaacacaaatggacttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgta gtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggacca ggcatcgcgagatacagcgcgagccagacacggagtgccgagctatgcgcacgctccaactagatatcatgtggatgatgagcat [00305]![embedded image]()
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[00310]![embedded image]()
[00311]![embedded image]()
aatgcccgctgcggcgacctgcgtcgctcggcgggctccagggccccggcgcccagcgaggcccctccccgtgcgcggcgcgc catccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctgg ccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggc atcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctact ccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctaca agtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactgga tcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctg cagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaaca actcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctgg acatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacga gctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccg aggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaactt cctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcAT GGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAA [00312]![embedded image]()
tcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagat ccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtcca cagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattat cttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagt caatgaatggtgagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgccttgtcttttgcacgcgcgactccgt cgcttcgcgggtggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtacccccaaccacccacctgcacct ctattattggtattattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttcagctggctcccaccattgtaaatt cttgctaaaatagtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggttttcgtgctgatctcgggcacaag gcgtcgtcgacgtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcctttactccgcactccaaacgact gtcgctcgtatttttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaaaggcctcgcggccgtgctcgtgg tgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcatggtgagtgcgcatcacaagatg catgtcttgttgtctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgctttggtcattacagaaattgcacaag ggcgtcctccgggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcaggagcgcggcgcatgacgacctacc cacatgcgaagagc NucleotidesequenceoftransformingDNAcontainedinpSZ6384 TcDGAT2-andGarmFATA1(G108A) SEQIDNO:129 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00313]![embedded image]()
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[00321]![embedded image]()
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[00324]![embedded image]()
ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00325]![embedded image]()
cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00326]![embedded image]()
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[00330]![embedded image]()
[00331]![embedded image]()
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[00334]![embedded image]()
gaggagcgcaaggccaccggctaccgcgagttctccggccgccacgagttcccctccaacaccatgcacgccctgctggccat gggcatctggctgggcgccatccacttcaacgccctgctgctgctgttctccttcctgttcctgcccttctccaagttcctggtggtgt tcggcctgctgctgctgttcatgatcctgcccatcgacccctactccaagttcggccgccgcctgtcccgctacatctccaagcacg cctgctcctacttccccatcaccctgcacgtggaggacatccacgccttccaccccgaccgcgcctacgtgttcggcttcgagccc cactccgtgctgcccatcggcgtggtggccctggccgacctgaccggcttcatgcccctgcccaagatcaaggtgctggcctcct ccgccgtgttctacacccccttcctgcgccacatctggacctggctgggcctgacccccgccaccaagaagaacttctcctccctg ctggacgccggctactcctgcatcctggtgcccggcggcgtgcaggagaccttccacatggagcccggctccgagatcgccttc ctgcgcgcccgccgcggcttcgtgcgcatcgccatggagatgggctcccccctggtgcccgtgttctgcttcggccagtcccacgt gtacaagtggtggaagcccggcggcaagttctacctgcagttctcccgcgccatcaagttcacccccatcttcttctggggcatct tcggctcccccctgccctaccagcaccccatgcacgtggtggtgggcaagcccatcgacgtgaagaagaacccccagcccatc gtggaggaggtgatcgaggtgcacgaccgcttcgtggaggccctgcaggacctgttcgagcgccacaaggcccaggtgggc [00335]![embedded image]()
gctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgttt gatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcg cttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggt ttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacac aaatggacttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgt aagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagat [00336]![embedded image]()
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[00340]![embedded image]()
[00341]![embedded image]()
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gcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgca tcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcg cctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagacc gccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggctt ctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctg gtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgacta cgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtgga cgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctga agaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaacca gcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagacc atcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccga ggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgct gcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAA [00344]![embedded image]()
ggagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccacccc gcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcaca tcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacacca aaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttc ctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggt gagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgccttgtcttttgcacgcgcgactccgtcgcttcgcggg tggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtacccccaaccacccacctgcacctctattattggta ttattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttcagctggctcccaccattgtaaattcttgctaaaat agtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggttttcgtgctgatctcgggcacaaggcgtcgtcgac gtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcctttactccgcactccaaacgactgtcgctcgtatt tttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaaaggcctcgcggccgtgctcgtggtgggggccgcg agcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcatggtgagtgcgcatcacaagatgcatgtcttgttg tctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgctttggtcattacagaaattgcacaagggcgtcctccg ggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcaggagcgcggcgcatgacgacctacccacatgcgaa gagc NucleotidesequenceoftransformingDNAcontainedinpSZ6377 GarmFATA1(G108A) SEQIDNO:130 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00345]![embedded image]()
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ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00357]![embedded image]()
cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00358]![embedded image]()
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[00360]![embedded image]()
[00361]![embedded image]()
[00362]![embedded image]()
[00363]![embedded image]()
[00364]![embedded image]()
ccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggc ccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgag gccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatc gtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaac cacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgccc gcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaaga tcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatg aaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcc tggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctg gtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgcccca ggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactcc ctgacctcccccgagccctccgaggacgccgaggccgtgacaaccacaacggcaccaacggctccgccaacgtgtccgccaa cgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggc gcaagaagcccacccgcATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACA [00365]![embedded image]()
ctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgc ggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgct gccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttg caacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgc cctcgctgatcgagtgtacagtcaatgaatggtgagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgcctt gtcttttgcacgcgcgactccgtcgcttcgcgggtggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtac ccccaaccacccacctgcacctctattattggtattattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttca gctggctcccaccattgtaaattcttgctaaaatagtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggtt ttcgtgctgatctcgggcacaaggcgtcgtcgacgtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcc tttactccgcactccaaacgactgtcgctcgtatttttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaa aggcctcgcggccgtgctcgtggtgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcat ggtgagtgcgcatcacaagatgcatgtcttgttgtctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgcttt ggtcattacagaaattgcacaagggcgtcctccgggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcagg agcgcggcgcatgacgacctacccacatgcgaagagc NucleotidesequenceoftransformingDNAcontainedinpSZ6315BnOTE SEQIDNO:131 caccggcgcgctgcttcgcgtgccgggtgcagcaatcagatccaagtctgacgacttgcgcgcacgcgccggatccttcaattccaaagtgtcg tccgcgtgcgcttcttcgccttcgtcctcttgaacatccagcgacgcaagcgcagggcgctgggcggctggcgtcccgaaccggcctcggcgcac gcggctgaaattgccgatgtcggcaatgtagtgccgctccgcccacctctcaattaagtttttcagcgcgtggttgggaatgatctgcgctcatg gggcgaaagaaggggttcagaggtgctttattgttactcgactgggcgtaccagcattcgtgcatgactgattatacatacaaaagtacagctc gcttcaatgccctgcgattcctactcccgagcgagcactcctctcaccgtcgggttgcttcccacgaccacgccggtaagagggtctgtggcctc gcgcccctcgcgagcgcatattccagccacgtctgtatgattttgcgctcatacgtctggcccgtcgaccccaaaatgacgggatcctgcataa tatcgcccgaaatgggatccaggcattcgtcaggaggcgtcagccccgcgggagatgccggtcccgccgcattggaaaggtgtagagggggt [00366]![embedded image]()
[00367]![embedded image]()
[00368]![embedded image]()
[00369]![embedded image]()
[00370]![embedded image]()
[00371]![embedded image]()
gcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggacacggccgacc gcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctg gtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgc gggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacct gaagtacgacaactgctacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaa gacgggccgccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtc cggcgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgc tccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcgg cgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtga acaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtct ggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtg gcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctccaagaagctga cctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccg gcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtc [00372]![embedded image]()
accggcgctgatgtggcgcggacgccgtcgtactctttcagactttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgc aattaattgtgtgatgaagaaagggtggcacaagatggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctg gctcaatcttgtcgcatgtccggcgcaatgtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactg atcgcattgccatcccgtcaactcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaag cgtcaggaaatcgtctcggcagctggaagcgcatggaatgcggagcggagatcgaatcaggatcccgcgtctcgaacagagcgcgcagagga acgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgt [00373]![embedded image]()
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[00375]![embedded image]()
[00376]![embedded image]()
[00377]![embedded image]()
[00378]![embedded image]()
[00379]![embedded image]()
[00380]![embedded image]()
[00381]![embedded image]()
[00382]![embedded image]()
[00383]![embedded image]()
[00384]![embedded image]()
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[00386]![embedded image]()
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[00390]![embedded image]()
[00391]![embedded image]()
[00392]![embedded image]()
[00393]![embedded image]()
tcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcgg tggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaaca cttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcacta ttatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaat gaatggtgagctccgcgcctgcgcgaggacgcagaacaacgctgccgccgtgtcttttgcacgcgcgactccggcgcttcgctggtggcacccc cataaagaaaccctcaattctgtttgtggaagacacggtgtacccccacccacccacctgcacctctattattggtattattgacgcgggagtgg gcgttgtaccctacaacgtagcttctctagttttcagctggctcccaccattgtaaattcatgctagaatagtgcgtggttatgtgagaggtatag tgtgtctgagcagacggggcgggatgcatgtcgtggtggtgatctttggctcaaggcgtcgtcgacgtgacgtgcccgatcatgagagcaatac cgcgctcaaagccgacgcatagcctttactccgcaatccaaacgactgtcgctcgtatttfttggatatctattttaaagagcgagcacagcgcc gggcatgggcctgaaaggcctcgcggccgtgctcgtggtgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggag gaacgcatggtgcgtgcgcaatataagatacatgtattgttgtcctgcagg NucleotidesequenceofBnOTE(D124A)inpSZ6316: SEQIDNO:132 [00394]![embedded image]()
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[00398]![embedded image]()
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[00407]![embedded image]()
NucleotidesequenceofBnOTE(D209A)inpSZ6317: SEQIDNO:133 [00408]![embedded image]()
[00409]![embedded image]()
[00410]![embedded image]()
[00411]![embedded image]()
[00412]![embedded image]()
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[00418]![embedded image]()
[00419]![embedded image]()
[00420]![embedded image]()
[00421]![embedded image]()
NucleotidesequenceofBnOTE(D124A,D209A)inpSZ6318 SEQIDNO:134 [00422]![embedded image]()
[00423]![embedded image]()
[00424]![embedded image]()
[00425]![embedded image]()
[00426]![embedded image]()
[00427]![embedded image]()
[00428]![embedded image]()
[00429]![embedded image]()
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NucleotidesequenceoftransformingDNAcontainedinpSZ5083 GarmGATA1 SEQIDNO:135 ccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttg gcacggctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcg tccagagacggcaagtcgtccatcctctgcgtgtgtggcgcgacgctgcagcagtccctctg cagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttctta aagcaaatgactgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagat agcgaccatgttgctgcgtctgaaaatctggattccgaattcgaccctggcgctccatccat gcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccactta gattcccgatcacccacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctc aagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgccgctggctgcaggg tctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggt cacttgccttccagattgccaataaactcaattatggcctctgtcatgccatccatgggtct gatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccctgccccgccca ccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaGGTACCCTTTCTTGCGCTA TGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACAC CGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCA GGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAA GCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCA CTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTCTAGAATATC Aatgatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcg gctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcc cagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcagga cgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacg tggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctg tcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgca caccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgccc gcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctg gcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggt gacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttca tcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgac atcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgc ccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttctgaCAATTGACGC CCGCGCGGCGCACCTGACCTGTTCTCTCGAGGGCGCCTGTTCTGCCTTGCGAAACAAGCCCC TGGAGCATGCGTGCATGATCGTCTCTGGCGCCCCGCCGCGCGGTTTGTCGCCCTCGCGGGCG CCGCGGCCGCGGGGGCGCATTGAAATTGTTGCAAACCCCACCTGACAGATTGAGGGCCCAGG CAGGAAGGCGTTGAGATGGAGGTACAGGAGTCAAGTAACTGAAAGTTTTTATGATAACTAAC AACAAAGGGTCGTTTCTGGCCAGCGAATGACAAGAACAAGATTCCACATTTCCGTGTAGAGG CTTGCCATCGAATGTGAGCGGGCGGGCCGCGGACCCGACAAAACCCTTACGACGTGGTAAGA AAAACGTGGCGGGCACTGTCCCTGTAGCCTGAAGACCAGCAGGAGACGATCGGAAGCATCAC AGCACAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTC GCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCAT TAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGT GGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGTGAAAACTCGCTCGACCGCCCGC GTCCCGCAGGCAGCGATGACGTGTGCGTGACCTGGGTGTTTCGTCGAAAGGCCAGCAACCCC AAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGCTTGGACCAGATCCCCCACGATGC GGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATTGGTGTCC GATACCTTGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGC TACCAGGGTTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACCGGCGCAGAGGGTGAGT TGATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGTGTGTCTGTTTTCGGCTGCAC AATTTCAATAGTCGGATGGGCGACGGTAGAATTGGGTGTTGCGCTCGCGTGCATGCCTCGCC CCGTCGGGTGTCATGACCGGGACTGGAATCCCCCCTCGCGACCCTCCTGCTAACGCTCCCGA CTCTCCCGCCCGCGCGCAGGATAGACTCTAGTTCAACCAATCGACAACTAGTatggccaccg catccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccggg ccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgt ggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcc tggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttc atcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacct gctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctcca ccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatc tacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaa gatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcg ccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggac gtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaa caactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcc tggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggc tgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccct ggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccct ccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgcc aacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagat caaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacg gcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaATCGATgcagca gcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccaca cttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgat cttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccaccccca gcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctg ctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctc cgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaag tagtgggatgggaacacaaatggaAAGCTTGAGCTCcagcgccatgccacgccctttgatgg cttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaata atacacttgatttcttgctcacggcaatctcggcttgtccgcaggttcaaccccatttcgga gtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgagg tgagctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagc acccaagcaaaatgtcaagtgcgttccgatttgcgtccgcaggtcgatgttgtgatcgtcgg cgccggatccgccggtctgtcctgcgcttacgagctgaccaagcaccctgacgtccgggtac gcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcg cgaaactgtgctcgattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgag cagggcgttgctcccggcggcggcgcctggctggggggacagctgttctcggccatgtgtgt acgtagaaggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttca gactattgttgaatgtttttagatttcttaggatgcatgatttgtctgcatgcgact AminoacidsequenceofGmFATAwild-typeparentalgene;D3997, pSZ5083. SEQIDNO:136 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAS111A,V193Amutantgene;D3998,pSZ5084. SEQIDNO:137 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFaTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAS111V,V193Amutantgene;D3999,pSZ5085. SEQIDNO:138 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFvTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG96Amutantgene;D4000, pSZ5086. SEQIDNO:139 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVaCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG96Tmutantgene;D4001, pSZ5087. SEQIDNO:140 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVtCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG96Vmutantgene;D4002, pSZ5088. SEQIDNO:141 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVvCNHAQSVGYSTGGFSTT YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL SKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG108Amutantgene;D4003, pSZ5089. SEQIDNO:142 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTaGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAL91Fmutantgene;D4004, pSZ5090. SEQIDNO:143 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANfLQEVGCDCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAL91Kmutantgene;D4005, pSZ5091. SEQIDNO:144 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANkLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK Figure10.AminoacidsequenceofGmFATAL91Smutantgene; D4006,pSZ5092.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheL91Sresidueislower-casecold SEQIDNO:145 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANsLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG108Vmutantgene;D4007,pSZ5093.Thealgal transitpeptideisunderlined,theFLAGepitopetagisuppercaseboldandtheG108Vresidueis lower-casebold. SEQIDNO:146 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTvGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Fmutantgene;D4008, pSZ5094.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheT156Fresidueislower-casebold. SEQIDNO:147 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGfRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Amutantgene;D4009, pSZ5095.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheT156Aresidueislower-casebold. SEQIDNO:148 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGaRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Kmutantgene;D4010,pSZ5096.The algaltransitpeptideisunderlined,theFLAGepitopetagisuppercaseboldandtheT156Kresidue islower-casebold. SEQIDNO:149 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGkRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Vmutantgene;D4011,pSZ5097.The algaltransitpeptideisunderlined,theFLAGepitopetagisuppercaseboldandtheT156Vresidue islower-casebold. SEQIDNO:150 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGvRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK NucleotidesequenceoftheGmFATAS111A,V193Amutantgene(D3998, pSZ5084).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ5083. SEQIDNO:151 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAs111V,V193Amutantgene(D3999, pSZ5085).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ5083. SEQIDNO:152 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttcgtcaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG96Amutantgene(D4000,pSZ5086).The promoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ5083 SEQIDNO:153 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG96Tmutantgene(D4001,pSZ5087).The promoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ5083 SEQIDNO:154 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgacgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG96Vmutantgene(D4002,pSZ5088).The promoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ5083 SEQIDNO:155 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtggtgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG108Amutantgene(D4003,pSZ5089).The promoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ50836. SEQIDNO:156 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgcc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAL91Fmutantgene(D4004,pSZ5090).The promoter,3UTR,selectionmarkerandtargetingarmsarethesameaspSZ5083 SEQIDNO:157 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacttcctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAL91Kmutantgene(D4005,pSZ5091). SEQIDNO:158 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacaagctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAL91Smutantgene(D4006,pSZ5092). SEQIDNO:159 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaactcgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG108Vmutantgene(D4007,pSZ5093). SEQIDNO:160 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgtc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Fmutantgene(D4008,pSZ5094). SEQIDNO:161 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcttccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Amutantgene(D4009,pSZ5095) SEQIDNO:162 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcgcgcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Kmutantgene(D4010,pSZ5096). SEQIDNO:163 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcaagcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Vmutantgene(D4011,pSZ5097). SEQIDNO:164 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcgtgcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga AminoacidsequenceofwildtypeBnOTEinpSZ6315(SeeSEQIDNO:131) SEQIDNO:165 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASQLRKPALDPLRAVISADQGSISPVNSCTPADRLRAGR LMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGFSTDGFATTLTMRKLHLIWVTARMHIEIYKY PAWSDVVEIETWCQSEGRIGTRRDWILRDSATNEVIGRATSKWVMMNQDTRRLQRVTDEVRDEYLVFCPREPRL AFPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTYIGWVLESIPQEIIDTHELQVITLDYRRECQQD DIVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNESQFLHMLRLSENGQEINRGRTQWRKKSSRMDYKDHDGDYK DHDIDYKDDDDK AminoAcidsequenceofBnOTE(D124A)inpSZ6316(SeeSEQIDNO:132) SEQIDNO:166 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASQLRKPALDPLRAVISADQGSISPVNSCTPADRLRAGR LMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGFSTAGFATTLTMRKLHLIWVTARMHIEIYKYP AWSDVVEIETWCQSEGRIGTRRDWILRDSATNEVIGRATSKWVMMNQDTRRLQRVTDEVRDEYLVFCPREPRLA FPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTYIGWVLESIPQEIIDTHELQVITLDYRRECQQDD IVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNESQFLHMLRLSENGQEINRGRTQWRKKSSRMDYKDHDGDYKD HDIDYKDDDDK AminoAcidsequenceofBnOTE(D209A)inpSZ6317(SeeSEQIDNO:133) SEQIDNO:167 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASQLRKPALDPLRAVISADQGSISPVNSCTPADRLRAGR LMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGFSTDGFATTLTMRKLHLIWVTARMHIEIYKY PAWSDVVEIETWCQSEGRIGTRRDWILRDSATNEVIGRATSKWVMMNQDTRRLQRVTAEVRDEYLVFCPREPRL AFPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTYIGWVLESIPQEIIDTHELQVITLDYRRECQQD DIVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNESQFLHMLRLSENGQEINRGRTQWRKKSSRMDYKDHDGDYK DHDIDYKDDDDK AminoacidsequenceofBnOTE(D124A,D209A)inpSZ6318(SeeSEQIDNO:134) SEQIDNO:168 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRASQLRKPALDPLRAVISADQGSISPVNSCTPADRLRAGR LMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGFSTAGFATTLTMRKLHLIWVTARMHIEIYKYP AWSDVVEIETWCQSEGRIGTRRDWILRDSATNEVIGRATSKWVMMNQDTRRLQRVTAEVRDEYLVFCPREPRLA FPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTYIGWVLESIPQEIIDTHELQVITLDYRRECQQDD IVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNESQFLHMLRLSENGQEINRGRTQWRKKSSRMDYKDHDGDYKD HDIDYKDDDDK CpauLPAAT SEQIDNO:169 MAIPAAAVIFLFGLLFFTSGLIINLFQALCFVLVWPLSKNAYRRINRVFAELLLS ELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMLGWVMGQHLGC LGSILSVAKKSTKFLPVLGWSMWFSEYLYIERSWAKDRTTLKSHIERLTDYPLPF WMVIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPA VYDVTVAFPKTSPPPTLLNLFEGQSIVLHVHIKRHAMKDLPESDDAVAQWCRDKF VEKDALLDKHNAEDTFSGQEVHRTGSRPIKSLLVVISWVVVITFGALKFLQWSSW KGKAFSVIGLGIVTLLMHMLILSSQAERSSNPAKVAQAKLKTELSISKKATDKEN CprocLPAAT1i SEQIDNO:170 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVFAELLLS ELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGC LGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKDKSTLKSHIERLKDYPLPF WLVIFAEGTRFTQTKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPA VYDLTVAFPKTSPPPTLLNLFEGQSVVLHVHIKRHAMKDLPESDDEVAQWCRDKF VEKDALLDKHNAEDTFSGQELQHTGRRPIKSLLVVISWVVVIAFGALKFLQWSSW KGKAFSVIGLGIVTLLMHMLILSSQAERSKPAKVAQAKLKTELSISKTVTDKEN CpcuLPAAT1 SEQIDNO:171 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVFAELLPL EFLWLFHWCAGAKLKLFTDPETFRLMGKEHALVIINHKIELDWMVGWVLGQHLGC LGSISVAKKSTKFLPVFGWSLWFSGYLFLERSWAKDKITLKSHIESLKDYPLPF WLIIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPA IYDVTVAFPKTSPPPTMLKLFEGQSVELHVHIKRHAMKDLPESDDAVAQWCRDKF VEKDALLDKHNSEDTFSGQEVHHVGRPIKALLVVISWVVVIIFGALKFLLWSSLL SSWKGKAFSVIGLGIVAGIVTLLMHILILSSQAEGSNPVKAAPAKLKTELSSSKK VTNKEN ChookLPAAT1 SEQIDNO:172 MAIPSAAVVFLFGLLFFTSGLINLFQAFCFVLISPLSKNAYRRINRVFAELLPL EFLWLFHWCAGAKLKLFTDPETFRLMGKEHALVIINHKIELDWMVGWVLGQHLGC LGSILSVAKKSTKFLPVFGWSLWFSEYLFLERSWAKDKITLKSHIESLKDYPLPF WLIIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPA IYDVTVAFPKTSPPPTMLKLFEGQSVELHVHIKRHAMKDLPESDDAVAQWCRDKF VEKDALLDKHNSEDTFSGQEVHHVGRPIKALLVVISWVVVIIFGALKFLLWSSLL SSWKGKAFSVIGLGIVAGIVTLLMHILILSSQAEGSNPVKAAPAKLKTELSSSKK VTNKEN CignLPAAT1 SEQIDNO:173 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVFAELLLM DLLCLFHWWAGAKIKLFTDPETFRLMGMEHALVIMNHKIDLDWMVGWILGQHLGC LGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKDKSTLKSHMEKLKDYPLPF WLVIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSCVSNMRSFVPA VYDVTVAFPKSSPPPTMLKLFEGQSIVLHVHIKRHALKDLPESDDAVAQWCRDKF VEKDALLDKHNAEDTFSGQEVHHIGRPIKSLLVVIAWVVVIIFGALKFLQWSSLL STWKGKAFSVIGLGIATLLMHMLILSSQAERSNPAKVAK CavigLPAAT1 SEQIDNO:174 MTIASAAVVFLFGILLFTSGLIINLFQAFCSVLVWPLSKNAYRRINRVFAEFLPL EFLWLFHWWAGAKLKLFTDPETFRLMGKEHALVIINHKIELDWMVGWVLGQHLGC LGSILSVAKKSTKFLPVFGWSLWFSEYLFLERNWAKDKKTLKSHIERLKDYPLPF WLIIFVEGTRFTRTKLLAAQQYAASAGLPVPRNVLIPHTKGFVSSVSHMRSFVPA IYDVTVAFPKTSPPPTMLKLFEGHFVELHVHIKRHAMKDLPESEDAVAQWCRDKF VEKDALLDKHNAEDTFSGQEVHHVGRPIKSLLVVISWVVVIIFGALKFLQWSSLL SSWKGIAFSVIGLGTVALLMQILILSSQAERSIPAKETPANLKTELSSSKKVTNK EN CavigLPAAT2 SEQIDNO:175 MAIAAAAVIVPVSLLFFVSGLIVNLVQAVCFVLIRPLFKNTYRRINRVVAELWL ELVWLIDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGC LGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPF WLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPA IYDVTVAIPKTSPPPTLLRMFKGQSSVLHVHLKRHQMNDLPESDDAVAQWCRDIF VEKDALLDKHNAEDTFSGQELQDTGRPIKSLLIVISWAVLVVFGAVKFLQWSSLL SSWKGLAFSGIGLGVITLLMHILILFSQSERSTPAKVAPAKPKIEGESSKTEMEK EH CpalLPAAT1 SEQIDNO:176 MAIAAAAVIVPLGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWL ELVWLIDWWAGVKIKVFTDHETLSLMGKEHALVICNHKSDIDWLVGWVLAQRSGC LGSTLAVMKKSSKFLPVIGWSMWFSEYLPESDDAVAQWCRDIFVEKDALLDKHNA EDTFSGQELQDTGRPIKSLLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGVG LGIITLLMHILILFSQSERSTPAKVAPAKPKKDGESSKTEIEKENVPGALLGQGR EHPEVRPEPPEGLPPALLAGPVRGGHPLHPRQAAGRPAVRHLLRPARAPQRADPP HQGLRVLRVPHALLRARHLRRDRGHPQDLPPPHHAAHVQGPVLRAARAPEAPPDE GP CuPSR23LPAAT2 SEQIDNO:177 MAIAAAAVIFLFGLIFFASGLIINLFQALCFVLIRPLSKNAYRRINRVFAELLLS ELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGC LGSIISVAKKSTKFLPVLGWSMWFSEYLYLERSWAKDKSTLKSHIERLIDYPLPF WLVIFVEGTRFTRTKLLAAQQYAVSSGLPVPRNVLIPRTKGFVSCVSHMRSFVPA VYDVTVAFPKTSPPPTLLNLFEGQSIMLHVHIKRHAMKDLPESDDAVAEWCRDKF VEKDALLDKHNAEDTFSGQEVCHSGSRQLKSLLVVISWVVVTTFGALKFLQWSSW KGKAFSAIGLGIVTLLMHVLILSSQAERSNPAEVAQAKLKTGLSISKKVTDKEN CkoeLPAAT1 SEQIDNO:178 MAIPAAVAVIPIGLLFIISGLIVNLIQAVVYVLIRPLSKNLHRKINKPIAELLWL ELIWLVDWWAGIKVEVYADSQTLELMGKEHALLICNHRSDIDWLVGWVLAQRARC LGSALAIMKKSAKFLPVIGWSMWFSDYIFLDRTWAKDEKTLKSGFERLADFPMPF WLALFVEGTRFTKAKLLAAQEYAASRGLPVPQNVLIPRTKGFVTAVTHMRSYVPA IYDCTVDISKAHPAPSILRLIRGQSSVVKVQITRHSMQELPETADGISQWCMDLF VTKDGFLEKYHSKDIFGSLPVQNIGRPVKSLIVVLCWYCLMAFGLFKFFMWSSLL SSWEGILSLGLILLAVAIVMQILIQSTESERSTPVKSIQKDPSKETLLQN CkoeLPAAT2 SEQIDNO:179 MHVLLEMVTFRFSSFFVFDNVQALCFVLIWPLSKSAYRKINRVFAELLLSELLCL FDWWAGAKLKLFTDPETFRLMGKEHALVITNHKIDLDWMIGWILGQHFGCLGSVI SIAKKSTKFLPIFGWSLWFSEYLFLERNWAKDKRTLKSHIERMKDYPLPLWLILF VEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTKGFVSSVSHMRSFVPAVYDVT VAFPKTSPPPTMLSLFEGQSVVLHVHIKRHAMKDLPDSDDAVAQWCRDKFVEKDA LLDKHNAEDTFSGQEVHHVGRTIKSLLVVISWMVVIIFGALKFLQWSSLLSSWKG KAFSAIGLGIATLLMHVLVVFSQADRSNPAKVPPAKLNTELSSSKKVTNKEN CprocLPAAT2 SEQIDNO:180 MAIPAAVAVIPIGLLFIISGLIVNLIQAVVYVLIRPLSKNLYRKINKPIAELLWL ELIWLVDWWAGIKVEVYADSETLESMGKEHALLICNHRSDIDWLVGWVLAQRARC LGSALAIMKKSAKFLPVIGWSMWFSDYIFLDRTWEKDEKTLKSGFERLADFPMPF WLALFVEGTRFTKAKLLAAQEFAASRGLPVPQNVLIPRTKGFVTAVTHMRSYVPA IYDCTVDISKAHPAPSILRLIRGQSSVVKVQITRHSMQELPETPDGISQWCMDLF VTKDAFLEKYHSKDIFGSLPVHDIGRPVKSLIVVLCWYSLMAFGFYKFFMWSSLL SSWEGILSLGLVLIVIAIVMQILIQSSESERSTPVKSVQKDPSKETLLQN CavigGPAT9 SEQIDNO:181 MATGGSLKPSSSDLDLDHPNIEDYLPSGSSINEPAGKLRLRDLLDISPTLTEAAG AIVDDSFTRCFKSIPREPWNWNLYLFPLWCIGVLIRYFILFPGRVIVLTMGWITV ISSFIAVRVLLKGHDALQIKLERLIVQLLCSSFVASWTGVVKYHGPRPSIRPKQV YVANHTSMIDFFILDQMTVFSVIMQKHPGWVGLLQSTLLESVGCIWFDRAEAKDR GIVAKKLWDHVHGEGNNPLLIFPEGTCVNNNYSVMFKKGAFELGCTVCPVAIKYN KIFVDAFWNSKKQSFTRHLLQLMTSWAVVCDVWYLEPQTLKPGETPIEFAERVRD IISARAGLKKVPWDGYLKYSRPSPKHRERKQQTFAESVLQRLEE ChookGPAT9-1 SEQIDNO:182 MATAGSLKPSRSELDFDRPNIEDYLPSGSSIIEPAGKLRLRDLLDISPTLTEAAG AIVDDSFTRCFKSNPPEPWNWNIYLFPLWCFGVLIRYLILFPARVIVLTIGWIIF LSSFIPVHLLLKGHDALRIKLERLLVELICSFFVASWTGVVKYHGPRPSIRPKQV YVANHTSMIDFFILDQMTVFSVIMQKHPGWVGLLQSTLLESVGCIWFDRAEAKDR GIVAKKLWDHVHGEGNNPLLIFPEGTCVNNNYSVMFKKGAFELGCTVCPVAIKYN KIFVDAFWNSKKQSFTRHLLQLMTSWAVVCDVWYLEPQTLKPGETPIEFAERVRD IISVRAGLKKVPWDGYLKYSRPSPKHTERKQQNFAESVLQRLEKK CignGPAT9-1 SEQIDNO:183 MATGGRLKPSSSELDLDRANTEDYLPSGSSINEPVGKLRLRDLLDISPTLTEAAG AIVDDSFTRCFKSIPPEPWNWNIYLFPLWCFGVLIRYFILFPARVIVLTIGWITV ISSFTAVRFLLKGHNALQIKLERLIVQLLCSSFVASWTGVVKYHGPRPSIRPKQV YVANHTSMIDFLILDQMTVFSVIMQKHPGWVGLLQSTLLESVGCIWFNRAEAKDR EIVAKKLWDHVHGEGNNPLLIFPEGTCVNNHYSVMFKKGAFELGCTVCPVAIKYN KIFVDAFWNSRKQSFTMHLLQLMTSWAVVCDVWYLEPQTLKPGETAIEFAERVRD IIVRAGLKKVPWDGYLKYSRPSPKHRESKQQSFAESVLRRLEEK CignGPAT9-2 SEQIDNO:184 MATGGRLKPSSSELDLDRANTEDYLPSGSSINEPVGKLRLRDLLDISPTLTEAAG AIVDDSFTRCFKSIPPEPWNWNIYLFPLWCFGVLIRYFILFPARVIVLTIGWITV ISSFTAVRFLLKGHNALQIKLERLIVQLLCSSFVASWTGVVKYHGPRPSIRPKQV YVANHTSMIDFLILDQMTVFSVIMQKHPGWVGLLQSTLLESVGCIWFNRAEAKDR EIVAKKLWDHVHGEGNNPLLIFPEGTCVNNHYSVMFKKGAFELGCTVCPVAIKYN KIFVDAFWNSKKHSFTRHLLQLMTSWAVVCDVWYLEPQTLKPGETPIEFAERVRD IISVRADLKKVPWDGYLKYSRPSPKHRERKQQKFAESVLRRLEEK CpalGPAT9-1 SEQIDNO:185 MATAGRLKPSSSELELDLDRPNIEDYLPSGSSINEPAGKLRLRDLLDISPMLTEA AGAIVDDSFTRCFKSIPPEPWNWNIYLFPLWCFGVLIRYLILFPARVIVLTVGWI TVISSFITVRFLLKGHDSLRIKLERLIVQLFCSSFVASWTGVVKYHGPRPSIRPQ QVYVANHTSMIDFIILNQMTVFSAIMQKHPGWVGLIQSTILESVGCIWFNRAEAK DREIVAKKLLDHVHGEGNNPLLIFPEGTCVNNHYSVMFKKGAFELGCTVCPVAIK YNKIFVDAFWNSKKQSFTMHLLQLMTSWAVVCDVWYLEPQTLKPGETPIEFAERV RDIISVRAGLKKVPWDGYLKYSRPSPKHRERKQQSFAESVLRRLEKR CpalGPATt9-2 SEQIDNO:186 MATAGRLKPSSSELELDLDRPNIEDYLPSGSSINEPAGKLRLRDLLDISPMLTEA AGAIVDDSFTRCFKSIPPEPWNWNIYLFPLWCFGVLIRYLILFPARVIVLTVGWI TVISSFITVRFLLKGHDSLRIKLERLIVQLFCSSFVASWTGVVKYHGPRPSIRPQ QVYVANHTSMIDFIILNQMTVFSAIMQKHPGWVGLIQSTILESVGCIWFNRAEAK DREIVAKKLLDHVHGEGNNPLLIFPEGTCVNNHYSVMFKKGAFELGCTVCPVAIK YNKIFVDAFWNSKKLSFTMHLLQLMTSWAVVCDVWYLEPQTLKPGETPIEFAERV RDIISVRAGLKKVPWDGYLKYSRPSPKHRERKQQTFAESVLRRLEEKGNVVPTVN CavigDGAT1 SEQIDNO:187 MAIADGGIIGAAGSISALTADTDPPSLRRRNVPAGQASAVSAFSTESMAKHLCDP SREPSPSPKSSDDGKDPDIGSVDSLNEKPSSPAAGKGRLQHDLRFTYRASSPAHR KVKESPLSSSNIFKQSHAGLFNLCVVVLVAVNSRLIIENLMKYGLLIKTGFWFSS RSLRDWPLFMCCLSLPIFPLAAFLVEKLAQKNRLQEPTVVCCHVLITSVSILYPV LVILRCDSAVLSGVALMLFACIVWLKLVSYAHSNYDMRYVAKSLDKGEPVVDSVI ADHPYRVDYKDLVYFMVAPTLCYQLSYPLTPCVRKSWIARQVMKLVLFTGVMGFI VEQYINPIVQNSKHPLKGDLLYAIERVLKLSVPNLYVWLCMFYCFFHLWLNILAE LICFGDREFYKDWWNAKTVEEYWRMWNMPVHKWMVRHIYFPCLRNGIPRGVAVLI AFLVSAVFHELCIAVPCHVFKLWAFIGIMFQVPLVLVSNCLQKKFQSSMAGNMFF WFIFCIFGQPMCVLLYYHDLMNRKGSRID ChookDGAT1-1 SEQIDNO:188 MAIADGGSAGAAGSISGSDPSPSTAPSLRRRNASAGQAFSTESMARDLCDPSREP SLSPKSSDDGKDPADDIGAADSVDSGGVKDEKPSSQAAAKARLEHDLRFTYRASS PAHRKVKESPLSSSNIFKQSHAGLFNLCVVVLVAVNSRLIIENLMKYGLLIKTGF WFSSRSLRDWPLFMCCLSLPIFPLAAFLVEKLAQKNRLQEPTVVCCHVIITSVSI LYPVLVILRCDSAVLSGVALMLFACIVWLKLVSYAHANYDMRSVAKSLDKGETVA DSVIVDHPYRVDYKDLVYFMVAPTLCYQLSYPLTPYVRKSWVARQVMKLVLFTGV MGFIVEQYINPIVQNSKHPLKGDLLYAIERVLKLSVPNLYVWLCMFYCFFHLWLN ILAELTCFGDREFYKDWWNAKTVEEYWRMWNMPVHKWMVRHIYFPCLRNGIPRGV AVLIAFLVSAVFHELCIAVPCHVFKLWAFIGIMFQVPLVLVSNCLQKKFQSSMAG NMFFWFIFCIFGQPMCVLLYYHDLMNRKGSRID CavigLPCAT SEQIDNO:189 MGLVSVAAAIGVSVPVARFLLCFLATIPVSFLWRLVPGRLPKHLYSAASGAILSY LSFGASSNLHFIVPMTLGYLSMLFFRPFSGLLTFFLGFGYLIGCHVYYMSGDAWK EGGIDATGALMVLTLKVISCSMNYNDGLLKEEGLRESQKKNRLTKMPSLIEYFGY CLCCGSHFAGPVYEMKDYLEWTEGKGIWSRSQKEPKPSPFGGALRAIIQAAVCMA MYLYLVPHHPLTRFTEPVYYEWGFFRRLSYQYMAALTARWKYYFIWSISEASLII SGLGFSGWTESSPPKPRWDRAKNVDIIGVEFAKSSVQLPLVWNIQVSIWLRHYVY DRLVQNGKRPGFFQLLATQTVSAVWHGLYPGYIIFFVQSALMIAGSRVIYRWQQA VPPKMGLVKNIFVFFNFAYTLLVLNYSAVGFMVLSMHETLASYGSVYYIGTILPI TLILLSYVIKPGKPARSKAHKEQ CpalLPCAT SEQIDNO:190 MELGSVAAAIGVSVPVARFLLCFLATIPVSFLWRLVPGRLPKHLYSAASGAILSY LSFGPSSNLHFIVPMTLGYLSMLFFRFSGLLTFFLGFGYLIGCHVYYMSGDAWK EGGIDATGALMVLTLKVISCSINYNDGLLKEEGLRESQKKNRLTKMPSLIEYIGY CLCCGSHFAGPVYEMKDYLEWTEGKGVWSHSEKEPKPSPFGGALRAIIQAAVCMA MYMYLVPHHPLSRFTEPVYYEWGFFRRLSYQYMAGLTARWKYYFIWSISEASLII SGLGFSGWTESSPPKPRWDRAKNVDIIGVEFAKSSVQLPLVWNIQVSTWLRHYVY DRLVQNGKRPGFFQLLATQTVSAIWHGLYPGYIIFFVQSALMIAGSRVIYRWQQA VPPKMGLVKNIFVFFNFAYTLLVLNYSAVGFMVLSMHETLASYGSVYYIGTILPI TLILLSYVIKPGKPARSKAHKEQ CpauLPCAT SEQIDNO:191 MELEIGSVAAAIGVSVPVARFLLCFLATIPVSFLCRLLPARLPKHLYSAASGAIL SYLSFGPSSNLHFIVPMSLGYLSMLFFRPFSGLLTFFLGFGYLIGCHVYYMSGDA WKEGGIDATGALMVLTLKVISCSINYNDGLLKEEGLRESQKKNRLTKMPSLIEYF GYCLCCGSHFAGPVYEMKDYLEWTEGKGIWSRSEKDPKPSPFGGALRAIIQAAVC MAMHMYLVPHHPLTRFTEPVYYEWGFFRRLSYQYMAAQTARWKYYFIWSISEASL IISGLGFSGWTESSPPKPRWDKAKNVDIIGVEFAKSSVQLPLVWNIQVSTWLRHY VYDRLVQNGKRPGFFQLLATQTVSAVWHGLYPGYIIFFVQSALMIAGSRVIYRWQ QAVPQKMGLVKNIFVFFNFAYTLLVLNYSAVGFMVLSMHETLASYGSVYYIGTIL PITLILLSYVIKPGKPTRSKVHKEQ CschuLPCAT SEQIDNO:192 MELEMEPLAAAIGVSVAVFRFLVCFIATIPVSFICRLVPGGLPRHLFSAASGAVL SYLSFGFSSNLHFLVPMTLGYLSMILFRRFCGILTFFLGFGYLIGCHVYYMSGDA WKEGGIDATGALMVLTLKVISCSINYNDGLLKEEGLRESQKKNRLIRLPSLIEYF GYCLCCGSHFAGPVYEMKDYLDWTEGKGIWSHSEKGPKPSPLRAALRAIIQAGFC MAMYLYLVPHYPLTRFTDPVYYEWGILRRLSYQYMASFTARWKYYFIWSISEASL IISGLGFSGWTESSPPKPRWDRAKNVDILGVELAKSSVQIPLVWNIQVSTWLRHY VYDRLVQNGKRPGFLQLLATQTVSAIWHGVYPGYLIFFVQSALMIAGSRAIYRWQ QAVPPKMSLVKNTLVFFNFAYTLLVLNYSAVGFMVLSMHETLASYGSVYYVGTIL PVTLILLGYVIKPGKSPRSKASKEQ CawgPLA2-1 SEQIDNO:193 MNFDFLSNIPWFGAKASDNAGSSFGSATIVIQQPPPVSRGFDIRHWGWPWSVLSV LPWGKPGCDELRAPPTTINRRLKRNATSMHSSAVRGNAEAARVRFRPYVSKVPWH TGFRGLLSQLFPRYGHYCGPNWSSGKNGGSPVWDQRPIDWLDYCCYCHDIGYDTH DQAKLLEADLAFLECLERPSYPTKGDAHVAHMYKTMCVTGLRNVLIPYRTQLLRL NSRQPLIDFGWLSNAAWKGWNAQKS CignPLA2-1 SEQIDNO:194 MNLDFLSKIPWFEAKASENPGLNLGSTTIVIKQPRQGFDIRHWGWPWSVLTWGNR VTDEVHAPPTTINRRLKRNATGPAVQGDTEAARLRFRPYVSKVPWHTGFRGLLSQ LFPRYGHYCGPNWSSGKNGGSPVWDQRPIDWLDYCCYCHDIGYDTHDQAKLLEAD LAFLECLERPSYPTTGDAHVAHMYKTMCVTGLRNVLIPYRTQLLRLNFRQPLIDF GWLSNAAWKGWSAQKT CuPSR23PLA2-2 SEQIDNO:195 MVHLPHTLKLGLVIAISISGLCFSSTPARALNVGIQAAGVTVSVGKGCSRKCESD FCKVPPFLRYGKYCGLMYSGCPGEKPCDGLDACCMKHDACVQAKNNDYLSQECSQ NLLNCMASFRMSGGKQFKGSTCQVDEVVDVLTVVMEAALLAGRYLHKP CprocPLA2-2 SEQIDNO:196 MVHLPHTLKLGLVIAISISGLCLSSTPARALNVGIQAAGVTVSVGKGCSRKCESD FCKVPPFLRYGKYCGLMYSGCPGEKPCDGLDACCMKHDACVQAKNDDYLSQECSQ NLLNCMASFRMSGGKQFKGSTCQVDEVVDVLTVVMEAALLAGRYLHKP pSZ4329 SEQIDNO:197 agcggaagagcgcccaatgtttaaacccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttggcacgg ctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcgtccagagacggcaagtcgtccatcctctgcgtgtgtggc gcgacgctgcagcagtccctctgcagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttcttaaagcaaatga ctgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagatagcgaccatgttgctgcgtctgaaaatctggattccgaattcg accctggcgctccatccatgcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccacttagattcccgatcacc cacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctcaagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgc cgctggctgcagggtctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggtcacttgccttccagattg ccaataaactcaattatggcctctgtcatgccatccatgggtctgatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccct gccccgcccaccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaggtaccctttcttgcgctatgacacttccagcaaaaggtag ggcgggctgcgagacggcttcccggcgctgcatgcaacaccgatgatgcttcgaccccccgaagctccttcggggctgcatgggcgctccgatg ccgctccagggcgagcgctgtttaaatagccaggcccccgattgcaaagacattatagcgagctaccaaagccatattcaaacacctagatca ctaccacttctacacaggccactcgagcttgtgatcgcactccgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaactctaga atatcaatgctgctgcaggccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccct ggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagt acaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgcc atcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacc cgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttca ccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatca tgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagg gcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatc aaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcg tggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaa ctgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacc cggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttg acgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgat ctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcct ccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttca agagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtcca ccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccagg tgcgcgaggtcaagtgacaattgacgcccgcgcggcgcacctgacctgttctctcgagggcgcctgttctgccttgcgaaacaagcccctggag catgcgtgcatgatcgtctctggcgccccgccgcgcggtttgtcgccctcgcgggcgccgcggccgcgggggcgcattgaaattgttgcaaacc ccacctgacagattgagggcccaggcaggaaggcgttgagatggaggtacaggagtcaagtaactgaaagtttttatgataactaacaaca aagggtcgtttctggccagcgaatgacaagaacaagattccacatttccgtgtagaggcttgccatcgaatgtgagcgggcgggccgcggacc cgacaaaacccttacgacgtggtaagaaaaacgtggcgggcactgtccctgtagcctgaagaccagcaggagacgatcggaagcatcacag cacaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataacca cctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgg agctgatggtcgaaacgttcacagcctagggatatcgcctgctcaagcgggcgctcaacatgcagagcgtcagcgagacgggctgtggcgat cgcgagacggacgaggccgcctctgccctgtttgaactgagcgtcagcgctggctaaggggagggagactcatccccaggctcgcgccaggg ctctgatcccgtctcgggcggtgatcggcgcgcatgactacgacccaacgacgtacgagactgatgtcggtcccgacgaggagcgccgcgagg cactcccgggccaccgaccatgtttacaccgaccgaaagcactcgctcgtatccattccgtgcgcccgcacatgcatcatcttttggtaccgactt cggtcttgttttacccctacgacctgccttccaaggtgtgagcaactcgcccggacatgaccgagggtgatcatccggatccccaggccccagc agcccctgccagaatggctcgcgctttccagcctgcaggcccgtctcccaggtcgacgcaacctacatgaccaccccaatctgtcccagacccc aaacaccctccttccctgcttctctgtgatcgctgatcagcaacaactagtaacaatggccaccgcctccaccttctccgccttcaacgcccgctg cggcgacctgcgccgctccgccggctccggcccccgccgccccgcccgccccctgcccgtgcgcgccgccatcaacgactccgcccaccccaag gccaacggctccgccgtgagcctgaagagcggcagcctgaacacccaggaggacacctcctccagcccccccccccgcaccttcctgcaccag ctgcccgactggagccgcctgctgaccgccatcaccaccgtgttcgtgaagtccaagcgccccgacatgcacgaccgcaagtccaagcgcccc gacatgctggtggacagcttcggcctggagtccaccgtgcaggacggcctggtgttccgccagtccttctccatccgctcctacgagatcggcac cgaccgcaccgccagcatcgagaccctgatgaaccacctgcaggagacctccctgaaccactgcaagagcaccggcatcctgctggacggctt cggccgcaccctggagatgtgcaagcgcgacctgatctgggtggtgatcaagatgcagatcaaggtgaaccgctaccccgcctggggcgaca ccgtggagatcaacacccgcttcagccgcctgggcaagatcggcatgggccgcgactggctgatctccgactgcaacaccggcgagatcctgg tgcgcgccaccagcgcctacgccatgatgaaccagaagacccgccgcctgtccaagctgccctacgaggtgcaccaggagatcgtgcccctgt tcgtggacagccccgtgatcgaggactccgacctgaaggtgcacaagttcaaggtgaagaccggcgacagcatccagaagggcctgaccccc ggctggaacgacctggacgtgaaccagcacgtgtccaacgtgaagtacatcggctggatcctggagagcatgcccaccgaggtgctggagac ccaggagctgtgctccctggccctggagtaccgccgcgagtgcggccgcgactccgtgctggagagcgtgaccgccatggaccccagcaaggt gggcgtgcgctcccagtaccagcacctgctgcgcctggaggacggcaccgccatcgtgaacggcgccaccgagtggcgccccaagaacgccg gcgccaacggcgccatctccaccggcaagaccagcaacggcaactccgtgtccatggactacaaggaccacgacggcgactacaaggacca cgacatcgactacaaggacgacgacgacaagtgactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatg gactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttg cgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatttacgc tgtcctgctatccctcagcgctgctcctgctcctgctcactgccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctg taaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttgagctccagcgccatgccacgccctttga tggcttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaataatacacttgatttcttgctcacggcaatctcg gcttgtccgcaggttcaaccccatttcggagtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgaggtg agctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagcacccaagcaaaatgtcaagtgcgttccgatttgcg tccgcaggtcgatgttgtgatcgtcggcgccggatccgccggtctgtcctgcgcttacgagctgaccaagcaccctgacgtccgggtacgcgag ctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcgcgaaactgtgctcgattgcaagaaattgatcgtcctc cactccgcaggtcgccatcatcgagcagggcgttgctcccggcggcggcgcctggctggggggacagctgttctcggccatgtgtgtacgtaga aggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttcagactattgttgaatgtttttagatttcttaggatgcatgattt gtctgcatgcgactgaagagcgttt pSZ5078 SEQIDNO:198 agcggaagagcgcccaatgtttaaacccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttggcacgg ctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcgtccagagacggcaagtcgtccatcctctgcgtgtgtggc gcgacgctgcagcagtccctctgcagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttcttaaagcaaatga ctgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagatagcgaccatgttgctgcgtctgaaaatctggattccgaattcg accctggcgctccatccatgcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccacttagattcccgatcacc cacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctcaagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgc cgctggctgcagggtctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggtcacttgccttccagattg ccaataaactcaattatggcctctgtcatgccatccatgggtctgatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccct gccccgcccaccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaggtaccctttcttgcgctatgacacttccagcaaaaggtag ggcgggctgcgagacggcttcccggcgctgcatgcaacaccgatgatgcttcgaccccccgaagctccttcggggctgcatgggcgctccgatg ccgctccagggcgagcgctgtttaaatagccaggcccccgattgcaaagacattatagcgagctaccaaagccatattcaaacacctagatca ctaccacttctacacaggccactcgagcttgtgatcgcactccgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaactctaga atatcaatgctgctgcaggccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccct ggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagt acaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgcc atcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacc cgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttca ccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatca tgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagg gcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatc aaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcg tggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaa ctgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacc cggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttg acgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgat ctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcct ccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttca agagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtcca ccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccagg tgcgcgaggtcaagtgacaattgacgcccgcgcggcgcacctgacctgttctctcgagggcgcctgttctgccttgcgaaacaagcccctggag catgcgtgcatgatcgtctctggcgccccgccgcgcggtttgtcgccctcgcgggcgccgcggccgcgggggcgcattgaaattgttgcaaacc ccacctgacagattgagggcccaggcaggaaggcgttgagatggaggtacaggagtcaagtaactgaaagtttttatgataactaacaaca aagggtcgtttctggccagcgaatgacaagaacaagattccacatttccgtgtagaggcttgccatcgaatgtgagcgggcgggccgcggacc cgacaaaacccttacgacgtggtaagaaaaacgtggcgggcactgtccctgtagcctgaagaccagcaggagacgatcggaagcatcacag cacaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataacca cctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgg agctgatggtcgaaacgttcacagcctagggatatcgaattcggccgacaggacgcgcgtcaaaggtgctggtcgtgtatgccctggccggca ggtcgttgctgctgctggttagtgattccgcaaccctgattttggcgtcttattttggcgtggcaaacgctggcgcccgcgagccgggccggcggc gatgcggtgccccacggctgccggaatccaagggaggcaagagcgcccgggtcagttgaagggctttacgcgcaaggtacagccgctcctgc aaggctgcgtggtggaattggacgtgcaggtcctgctgaagttcctccaccgcctcaccagcggacaaagcaccggtgtatcaggtccgtgtca tccactctaaagagctcgactacgacctactgatggccctagattcttcatcaaaaacgcctgagacacttgcccaggattgaaactccctgaa gggaccaccaggggccctgagttgttccttccccccgtggcgagctgccagccaggctgtacctgtgatcgaggctggcgggaaaataggcttc gtgtgctcaggtcatgggaggtgcaggacagctcatgaaacgccaacaatcgcacaattcatgtcaagctaatcagctatttcctcttcacgag ctgtaattgtcccaaaattctggtctaccgggggtgatccttcgtgtacgggcccttccctcaaccctaggtatgcgcgcatgcggtcgccgcgca actcgcgcgagggccgagggtttgggacgggccgtcccgaaatgcagttgcacccggatgcgtggcacctiftttgcgataatttatgcaatgg actgctctgcaaaattctggctctgtcgccaaccctaggatcagcggcgtaggatttcgtaatcattcgtcctgatggggagctaccgactaccc taatatcagcccgactgcctgacgccagcgtccacttttgtgcacacattccattcgtgcccaagacatttcattgtggtgcgaagcgtccccagt tacgctcacctgtttcccgacctccttactgttctgtcgacagagcgggcccacaggccggtcgcagccactagtatggccaccgcctccaccttc tccgccttcaacgcccgctgcggcgacctgcgccgctccgccggctccggcccccgccgccccgcccgccccctgcccgtgcgcgccgccatcaa ctcccgcgcccaccccaaggccaacggctccgccgtgtccctgaagtccggctccctgaacacccaggaggacacctcctcctcccccccccccc gcaccttcctgcaccagctgcccgactggtcccgcctgctgaccgccatcaccaccgtgttcgtgaagtccaagcgccccgacatgcacgaccg caagtccaagcgccccgacatgctgatggactccttcggcctggagtccatcgtgcaggagggcctggagttccgccagtccttctccatccgct cctacgagatcggcaccgaccgcaccgcctccatcgagaccctgatgaactacctgcaggagacctccctgaaccactgcaagtccaccggca tcctgctggacggcttcggccgcacccccgagatgtgcaagcgcgacctgatctgggtggtgaccaagatgaagatcaaggtgaaccgctacc ccgcctggggcgacaccgtggagatcaacacctggttctcccgcctgggcaagatcggcaagggccgcgactggctgatctccgactgcaaca ccggcgagatcctgatccgcgccacctccgcctacgccaccatgaaccagaagacccgccgcctgtccaagctgccctacgaggtgcaccagg agatcgcccccctgttcgtggactccccccccgtgatcgaggacaacgacctgaagctgcacaagttcgaggtgaagaccggcgactccatcc acaagggcctgacccccggctggaacgacctggacgtgaaccagcacgtgtccaacgtgaagtacatcggctggatcctggagtccatgccc accgaggtgctggagacccaggagctgtgctccctggccctggagtaccgccgcgagtgcggccgcgactccgtgctggagtccgtgaccgcc atggaccccaccaaggtgggcggccgctcccagtaccagcacctgctgcgcctggaggacggcaccgacatcgtgaagtgccgcaccgagtg gcgccccaagaaccccggcgccaacggcgccatctccaccggcaagacctccaacggcaactccgtgtccatggactacaaggaccacgacg gcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgattaattaactcgaggcagcagcagctcggatagtatcgaca cactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgt gtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttg catcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgc ctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttgagc tccagcgccatgccacgccctttgatggcttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaataatacac ttgatttcttgctcacggcaatctcggcttgtccgcaggttcaaccccatttcggagtctcaggtcagccgcgcaatgaccagccgctacttcaag gacttgcacgacaacgccgaggtgagctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagcacccaagca aaatgtcaagtgcgttccgatttgcgtccgcaggtcgatgttgtgatcgtcggcgccggatccgccggtctgtcctgcgcttacgagctgaccaa gcaccctgacgtccgggtacgcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcgcgaaactgtgc tcgattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgagcagggcgttgctcccggcggcggcgcctggctggggggacagc tgttctcggccatgtgtgtacgtagaaggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttcagactattgttgaatgtt tttagatttcttaggatgcatgatttgtctgcatgcgactgaagcgtttaaaccgcct
