Production of oil in vegetative tissues
10006039 ยท 2018-06-26
Assignee
Inventors
Cpc classification
C12N15/8218
CHEMISTRY; METALLURGY
C12Y207/07027
CHEMISTRY; METALLURGY
C12N15/8247
CHEMISTRY; METALLURGY
C12Y306/03
CHEMISTRY; METALLURGY
International classification
C12N15/82
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to compositions and methods for providing RNA interference (RNAi) vectors comprising trigalactosyldiacylglycerol (tgd) biosynthesis enzyme constructs for increasing oil content of plants. Further, the use of tgd RNAi silencing vectors in combination with co-expression of heterologous oil regulating transcription factors, such as WRINKLED1, are contemplated to overcome the reduced growth and variable levels of embryonic lethality in plants with reduced TGD protein. Additionally, plants having reduced APS1, a gene encoding a major catalytic isoform of the small subunit of AGPase (AGP() plants), co-expressing a heterologous oil modulating transcription factor are contemplated for use in combination with plants having reduced TGD. Oil harvested from vegetative tissues of plants comprising vectors and genes of the present invention is contemplated for use in biofuel and biodiesel products.
Claims
1. A double gene vector construct, comprising two expression cassettes: a first gene RNAi silencing expression cassette comprising a first constitutive promoter in operable combination with a nucleic acid segment comprising: a first trigalactosyldiacylglycerol 3 (tgd3) fragment in its sense orientation with at least 95% sequence identity to a segment of SEQ ID NO: 14, 20 or 55, and a second tgd3 fragment in antisense orientation relative to the first tqd3 fragment; and a second gene expression cassette comprising a nucleic acid encoding a WRINKLED 1 transcription factor in operable combination with a second promoter.
2. The construct of claim 1, wherein said first trigalactosyldiacylglycerol 3 (tgd3) fragment in its sense orientation has SEQ ID NO: 14.
3. The construct of claim 1, wherein said second trigalactosyldiacylglycerol 3 (tgd3) fragment in its antisense orientation has SEQ ID NO: 52.
4. The construct of claim 1, further comprising a spacer nucleic acid sequence located in between said sense fragment and said antisense fragment.
5. The construct of claim 1, wherein said RNAi silencing expression cassette comprises SEQ ID NO: 20.
6. The construct of claim 1, wherein said WRINKLED1 nucleic acid has a sequence that comprises at least 95% sequence identity to SEQ ID NO: 37 or 44.
7. The construct of claim 1, wherein said second promoter is selected from the group consisting of a 35S cauliflower mosaic virus promoter and an Oleosin (OLE) promoter.
8. A reproductively competent plant comprising said construct of claim 1, wherein said plant has accumulated lipid in its vegetative tissues.
9. The construct of claim 1, wherein said WRINKLED1 nucleic acid encodes a WRINKLED1 protein encoded by a nucleic acid with at least 95% sequence identity to SEQ ID NO: 37 or 44.
10. The plant of claim 8, wherein said plant is an oleaginous plant.
11. The plant of claim 8, wherein said plant is selected from the group consisting of rice plants, beet plants, grape plants, Arabidopsis sp. plants, Brassica sp. plants, Brassica napus plants, grass plants and algae plants.
12. A plant seed comprising said construct of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION OF THE INVENTION
(26) The present invention relates to compositions and methods for providing RNA interference (RNAi) vectors comprising trigalactosyldiacylglycerol (tgd) biosynthesis enzyme constructs for increasing oil content of plants. Specifically, plant cells, plant tissues and whole plants are provided comprising novel tgd RNAi vectors for reducing TGD protein expression for increasing oil content (including relative amounts of triacylglycerols) in cells and in vegetative tissues. In particular, vectors are provided comprising tgd RNAi constructs for silencing tgd1 and tgd4 endogenous gene translation. Further, the use of tgd RNAi silencing vectors in combination with co-expression of heterologous oil regulating transcription factors, such as WRINKLED1, are contemplated to overcome the reduced growth and variable levels of embryonic lethality in plants with reduced TGD protein. Additionally, plants having reduced APS1, a gene encoding a major catalytic isoform of the small subunit of AGPase (AGP() plants), co-expressing a heterologous oil modulating transcription factor are contemplated for use in combination with plants having reduced TGD. Oil harvested from vegetative tissues of plants comprising vectors and genes of the present invention is contemplated for use in biofuel and biodiesel products.
(27) I. Biosynthetic Oil Production.
(28) Production of alternative fuels such as biodiesel is on the rise around the world and in the U.S. due to a strong and growing desire to reduce dependency on petroleum derived diesel fuel. The acceptance of biodiesel has been slowed due to its higher cost relative to petroleum-derived diesel. The higher cost of biodiesel is directly related to the cost of feedstock used for biodiesel production, which is often derived from the same crops grown for use as human and animal food. Hence, many biodiesel producers and academic researchers are exploring technologies for alternative biodiesel feedstock, i.e. crop oils, particularly those technologies with the potential to lower feedstock costs. In some embodiments, the inventors contemplate the use of isolated vegetative lipids produced by plants and methods of the present inventions for use in biofuels. The use of these alternative plants and methods is contemplated to increase the amount of oil harvested per acreage of cultivated plant oil crop. In addition to biofuel uses for engineered oils, oils produced by engineered plants are contemplated to serve as renewable chemical feedstocks contemplated for use in replacing petroleum-based products in industrial applications (see, for examples, a review in Jaworski et al., Curr. Opin. Plant Biol. 6:178-184 (2003); Dyer et al., Seed Sci. Res. 15:255-267 (2005); and Singh et al., Curr. Opin. Plant Biol. 8:197-203 (2005), all of which are herein incorporated by reference in their entirety).
(29) However large-scale production of oils from engineered plants and natural plant species through traditional farming is often impossible because of poor agronomic traits of these engineered or natural plants producing desired oils. Furthermore, efforts to transfer genes encoding the proteins responsible for making novel fatty acids from endogenous pathways or creating unusual fatty acid biosynthesis by inserting genes from exotic plants into higher yielding plants (i.e. plants with agronomic traits for commercial use) has generally met with limited success. Often lack of success is due to much lower amounts of the desired fatty acid accumulating in the seed oils of transgenic plants (15 to 30%) compared with the seeds of native plant species (up to 90%). Thelen et al., Metab. Eng. 4:12-21 (2002), herein incorporated by reference in its entirety. Thus, engineering of existing oil seed crop species is contemplated for increasing plant oil production and for making specific engineered oils.
(30) One contemplated method for engineering existing oil seed crop plants is to increase the amount of desired oil produced and stored within a seed. However even if this was accomplished, there may be a limit on the total amount of oil a seed is capable of storing, in addition to a severe impact on the fertility of the plants. Further, plants need to be grown to maturity, using fertilizers, herbicides, and arable acreage for an entire growing season, in order to for the plant to produce fully formed seeds in order for seed oil isolation to be economically feasible. Therefore, compositions and methods involved in plant oil biosynthesis are needed for providing engineered plants producing increased amounts of industrially important oils in plants with agronomic traits suitable for commercial production.
(31) Many genes encoding enzymes affecting oil biosynthesis were isolated from plants for use in engineering plant fatty acid production with the hope of discovering novel oil compositions or increasing the amounts of oil produced per plant for commercial purposes. The majority of these genes were discovered using various methods, including natural plant mutants, artificially mutagenized plants and plants with insertions, i.e. T-DNA insertions, which changed fatty acid synthesis, primarily in seeds, often along with phenotypic changes in plant morphology when plants were grown from these seeds. Isolated genes were then used transgenically as heterologous genes to produce plants in hopes of discovering useful novel fatty acid compositions i.e. designer oils that could be successfully incorporated into commercially viable plant lines. Examples of fatty acid altering enzymes included fatty acid biosynthetic enzymes, such as acyltransferases, ketoacyl-acyl carrier protein synthetases, desaturases, and related enzymes were discovered and used for producing plants with novel fatty acid content.
(32) However the majority of the plants, or their isolated oils, are not commercially used. Many of these transgenic plants or isolated oils were not used due to lacking the type of oil compositions effective for commercial use, low production of desired oils, plants with poor agronomic traits, plants with poor fitness (in general an unhealthy plant), such as plants with reduced oil yield per plant, plants with stunted growth, infertile plants, etc.
(33) Thus, current commercial use of oils from plants engineered for designer alterations in oil production is limited to merely a few plant lines producing limited types of oils. Despite a few commercial successes with engineered plant lines having altered oil production, such as certain soybean seed oils, there are no known engineered commercial plant lines where fatty acids accumulated in vegetative tissues. Furthermore, there are no known engineered commercial plant lines where TAGs accumulated in vegetative tissues, in part due to undesirable agronomic traits when TAG production is increased in plants where at least one tgd gene is knocked out (or mutated) for undetectable or barely detectable levels of expression (see summary in Table 1 and schematic of tgd4-1, tgd4-2 and tgd4-3 insertion mutation sites in
(34) TABLE-US-00001 TABLE 1 Loss of TDG function phenotypes Loss of tgd function phenotypes, The Gene Arabidopsis Information Resource (TAIR) References tgd4-1 The tgd4-1 point mutation produced plants that Xu et al., Plant tgd4-2 were slightly pale green but otherwise Cell. 20(8): 2190-2204 tgd4-3 indistinguishable from the wild type plants. (2008). Plants carrying the tgd4-2 and tgd4-3 T-DNA insertion allele were stunted, pale yellow in color, and infertile. More lipid accumulated in tgd4-2 and tgd4-3 plants than in tgd4-1 plants. tgd3-1 No mutant plant phenotype described for tgd3-1, Lu et al., J. Biol. lipid accumulated in leaves comprising polar Chem., 282: fatty acids, polar lipid extracts of the tgd3-1 35945-53 (2007). mutant contained a new lipid staining with - naphthol additional. tgd2-1 Compared to the wild type, mutant plants Awai et al. Proc. were consistently smaller and slightly pale, as Natl. Acad. Sci. was observed for the tgd1-1 mutants. Mutant 103(28): 10817-22 plants had aberrant accumulation of (2006) oligogalactolipids and triacylglycerols. tgdl-1 Compared to the wild type, mutant plants were Awai et al. Proc. consistently smaller and slightly pale. Mutant Natl. Acad. Sci. plants had aberrant accumulation of 103(28): 10817-22 oligogalactolipids and triacylglycerols. (2006). Reduced growth, 50% of the seeds are aborted at Xu et al., Plant the embryo heart stage, the seed chloroplasts have Cell 17: 3094-3110 reduced galactolipids and leaves accumulated (2005). trigalactosyldiacylglycerol and triacylglycerol and phosphatidate
(35) Thus in one preferred embodiment, the inventors contemplate gene-silencing (knock down) methods for accumulating oil in vegetative tissues of the present inventions by reducing but not eliminating TGD protein production. However, one primary problem of several known problems encountered when engineering plants for gene silencing is target specificity. Full-length pieces of either a mutant cDNA or antisense cDNA sequence used in gene silencing constructs are more likely to bind to nontarget genes, which may or more typically may not be beneficial to desired plant oil production. Nontarget genes may have highly similar coding sequences when compared to the target gene or have high similarity in the uncoded region. Further, binding of mutant cDNA or antisense sequences expressed by silencing constructs to nontarget DNA may have adverse effects on agronomic traits necessary to commercial production. Thus in preferred embodiments, tgd sequences used in gene silencing constructs of the present inventions are fragments, i.e. partial DNA sequences for encoding mRNA of TGD proteins.
(36) Additionally, the type of regulation by the target gene affects the success of gene silencing depending on whether the gene has effects which are dominant, co-dominate, recessive, dominant negative, pleiotropic effects, variable expressivity, complete or incomplete penetrance, etc. Thus, the inventors' encountered many problems with plant growth and reproduction when reducing tgd gene expression when producing plants mutant for tgd DNA sequences, such as stunted plant growth and embryonic lethality. For example, these problems were observed when a tgd mutant gene was used for silencing one homologous tgd gene, such as tgd2-1 silencing of tgd2. The inventors discovered through the use of these mutants that tgd genes were dominant negative, such that expressing a few mutant genes produced mutant protein that displaced wild-type functional TGD proteins within the lipid transporter molecule.
(37) Thus, plants expressing mutant forms of tgd that interfered with wild-type tgd function showed a range of characteristics including a high rate of embryonic lethality, stunted growth, plants having yellow instead of green leaves and stems, and variability from plant to plant of amounts of oils stored in vegetative tissues. Therefore in preferred embodiments, the inventors contemplate the use of tgd RNAi silencing constructs for producing plants having similar levels, i.e. similar from plant to plant, of reduced TGD translation. Additionally, the use of a full-length tgd1 gene encoding the entire TGD1 protein for RNAi gene silencing in mutant tgd1-1 plants resulted in higher levels of sterile seeds or empty seed hulls and further diminished growth, Xu, et al., 2005, herein incorporated by reference in its entirety.
(38) Thus in one example, the inventors made a RNAi construct using two copies of a full-length coding region of TGD1 as a cDNA in operable combination with a constitutive promoter, S35. In other words the construct consisted of a full-length sense tgd1 sequence, a glucuronidase intron in the spacer region, and a full-length tgd1 antisense sequence. However, as described, the expression of this construct in Arabidopsis plants resulted in variable reductions of TGD protein, reduced growth and sterility like those seen, for example, in Table 1 for the tgd mutants described in
(39) In order to overcome these limitations, the inventors contemplated the use of inducible constructs for turning on the RNAi constructs after a seed germinates and begins to grow in order to reduce effects on growth.
(40) Further, the inventors contemplated overcoming the poor growth and reproductive characteristics of these tgd knock down plants by expressing additional or stacked heterologous genes, specifically, oil regulating transcription factors. Thus, the use of constructs for reducing TGD protein expression in combination with constructs overexpressing an oil regulating transcription factor can find use in the present inventions. Further, embodiments are contemplated for compositions and methods comprising RNAi constructs using fragments of tgd cDNA coding regions for use in plants expressing wild-type tgd genes for producing plants for use in establishing engineered plants having lowered TGD producing plant lines.
(41) In one embodiment, engineered plants with lowered tgd translation from the use of a tgd RNAi gene silencing construct, would be used for breeding plant lines with genetically determined lowered amounts of tgd gene translation for increasing lipid expression in vegetative tissues. In further embodiments, expression of RNAi constructs using fragments of tgd cDNA coding regions are contemplated for use in plants for reducing targeted tgd gene translation at certain levels, i.e. reduced expression of TGD proteins between 0-25%, 25-50%, 50%-70%, up to 70%-90% of wild-type protein production. In a further embodiment, a reduction of TGD protein expression is combined with overexpression of a transcription factor. In preferred embodiments, plants expressing RNAi constructs for lowering TGD expression while overexpressing an oil regulating transcription factor, such as a WRI1 cDNA, for example, deposited at GenBank (AY254038), are contemplated.
(42) In other embodiments contemplated for overcoming growth and reproductive defects observed in plants having decreased levels of either TGD proteins the inventors contemplate the use of plants having reduced expression of APS1, a gene encoding a major catalytic isoform of the small subunit of AGPase (ADP-glucose pyrophosphorylase). The inventors discovered that plants having reduced AGPase expression while concurrently ectopically expressing WRI1 increased the accumulation of TAG in Arabidopsis vegetative tissues. In fact, results showed that Arabidopsis ectopically expressing AGPRNAi-WRI1 accumulated less starch and 5.8-fold more TAG in the vegetative tissues. However the inventors contemplate that further increases in TAG content of vegetative tissues would be provided by plants having reduced APS1 along with reduced TGD expression while overexpressing WRI1.
(43) Thus, in even further embodiments, plants expressing RNAi constructs for lowering TGD expression in combination with plants expressing RNAi constructs for lowering AGPase expression are contemplated. In additional embodiments, plants having lowered TGD expression and AGPase expression while overexpressing an oil regulating transcription factor are contemplated. Thus plants of the present inventions co-expressing RNAi constructs with vectors for overexpressing oil regulating transcription factors are contemplated to have increased oil in vegetative tissues while overcoming the greatly reduced growth and fertility observed in other plants having reduced TGD or AGPase.
(44) A. Production of Seed Oil.
(45) For producing edible oils, industrial oils, and oil biofuel uses, an increase in seed oil content is desirable and a major goal of oilseed engineering. There are at least three major biosynthetic events involved in the production of seed oils. The first involves the synthesis of fatty acids in plastids. The second involves a modification of these fatty acids by enzymes located primarily in the endoplasmic reticulum (ER) of plastid cells. The third involves packaging of nascent fatty acids into triacylglycerols (TAGs), which subsequently accumulate in oil bodies that bud off from the Endoplasmic Reticulum within cells. Research information is currently available regarding synthesis and modification of fatty acid-containing oil body structures in seeds. For example, see, Ohlrogge et al., Plant Cell 7:957-970. (1995); and Shanklin et al., Annu Rev. Plant Physiol. Plant Mol. Biol. 49:611-641 (1998), all of which are herein incorporated by reference in their entirety.
(46) However, methods of increasing seed oil content by manipulation of oil regulating genes and transcription factors were not generally successful. One problem was that when seed oil was increased to high levels then fertility, i.e. viability, decreased. For example, several schemes were developed to modify the fatty acid composition of seed oils by genetic engineering resulting in increased nutritional and industrial value (Voelker et al., 1996, herein incorporated by reference in its entirety). However, strategies targeting single rate-limiting enzymes to increase seed oil content were moderately successful (Thelen and Ohlrogge, 2002, herein incorporated by reference in its entirety). Therefore, there remains a need for increasing oil produced on a per plant or population of plants basis with a minimum effect upon seeds whose viability and numbers are needed for agricultural and economical use.
(47) However, little is known about the interactions of enzymes and cellular mechanisms required for the selection and transfer of fatty acids into storage TAGs in other plant tissues. Even less understood was the interaction of oil production in vegetative tissues such as leaves and stems in relation to oil production in seeds.
(48) B. TGD Associated Production of Oil in Vegetative Tissues.
(49) Plants with mutations in certain genes whose translated proteins result in the reduction in synthesis of a particular fatty acid were found and named after the altered fatty acid. Thus mutated genes affecting TAG expression were identified and labeled trigalactosyldiacyl-glycerol (tgd) proteins, 1 and 2 while tgd3 and tgd4 were originally discovered under a different name and renamed due to their relation to function. Genetic analysis indicated that TGD1-3 formed a lipid transporter as a triprotein complex located in the membrane of the ER (endoplasmic reticulum).
(50) Thus, each one of the TGD1-3 proteins of Arabidopsis was required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 was contemplated to be the substrate-binding protein of a contemplated lipid transporter combined with TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins were localized in the inner chloroplast envelope membrane. TGD2 appeared to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faced the intermembrane space. Subsequent research found that one additional gene discovered under a different name, with no significant homology to TGD1-3 was also involved with TAG production. Because the encoded proteins were also involved with the same fatty acid pathway as TGD1-3 it was designated as tgd4. When plants with any one defective tgd were analyzed, the inventors discovered that TAG was increased in vegetative tissues of the plants along with other changes (alterations) to fatty acid compositions of vegetative tissues and seeds. Thus, the inventors isolated a series of Arabidopsis mutant plants for each of the genes encoding TGD 1, 2, 3, and 4 proteins whose leaves and other vegetative tissues had higher oil content, i.e. accumulated amounts of oil. These mutant genes were generally leaky with varying levels of expression from plant to plant. However when any one of the tgd genes was completely disrupted the result was detrimental to plant growth and/or plant development and fertility at levels generally associated with disruption of each one of the four genes, see mutant structures in
(51) As one example, the inventors created a dominant negative mutation for TGD2 and tested it in Arabidopsis, Awai et al. 2006, PNAS 103(28):10817-10822, herein incorporated by reference in its entirety, however in addition to the dominant negative leaky gene resulting in a range of phenotypes, those plants whose leaves accumulated oil lacked agronomic traits for commercial production, including having infertility, i.e. embryo-lethality, and stunted growth. The inventor's then attempted to completely shut off TGD1 by using an RNAi construct comprising the entire coding unit of TGD1 expressed as the sense and antisense directions separated by a spacer sequence of a glucuronidase intron, in operable combination with a constitutive promoter, Cauliflower Mosaic Virus (CaMV) 35S Promoter (i.e. S35), in plants co-expressing TGD1 mutations. The expression of this construct on the Arabidopsis background of TGD1-plants resulted in highly stunted plants with high level of embryo lethality, for example, large numbers of sterile seeds or empty seed hulls were observed (Xu, et al., Plant Cell. 2005 November; 17(11):3094-3110, herein incorporated by reference in its entirety). The ratio of aborted seeds approached 90% (409 out of 481 total for RNAi line-1 and 519 out of 583 total for RNAi line-) for the tgd1-1/TGD1-RNAi lines. Approximately 50% of the seeds of the tgd1-1 mutant were aborted. When embryos forming in siliques of the tgd1-1 mutant line were directly examined from 2 to 6 d after flowering, many were found arrested at the heart stage (see, description of heart stage, Mansfield and Briarty, Can. J. Bot. 69, 461-476 (1991), herein incorporated by reference in its entirety) just before the greening of the plastid was expected to begin.
(52) The inventors discovered that accumulation of oil in the leaves and stems was achieved by altering the function of the trigalactosyldiacylglycerol (TGD) proteins when an RNAi silencing sequence, comprising the entire coding region of tgd1 under control of a S35 constitutive promoter, was induced to express in transgenic plants. Additionally, these plants showed a range of phenotypes. Thus similar to the mutant tgd expressing plants, these plants with a full-length gene silencing construct showed undesirable agronomic traits of wide variations in capability of growth, variability in accumulation of oil in plant cells, leaves, and stem, and high levels of embryo lethality.
(53) C. Trigalactosyldiacylglycerol (tgd) Genes and TGD Proteins.
(54) Plant oil normally accumulates in seeds, which have less storage capacity than combined amounts in plant cells and plant tissues leaves and stems. Thus wild type plants with normally functioning TGD proteins do not accumulate oils, including TAGs, in a manner as described herein. However, the inventors discovered that a loss of function of any one of the tgd1-tgd4 genes caused accumulation of TAG and other plant fatty acids in leaves and stems. Specifically, the inventors discovered through the use of mutant TGD proteins and by transgenic expression of constitutive-transiently expressed mutant tgd1, 2 or 3 genes that when TGD mutant proteins were present there was a dominant-negative effect on function. Therefore, despite the presence of functional wild type TGD proteins, mutant TGD proteins were incorporated into the TGD1-3 complex causing variable levels of accumulation of oils in plant cells, plant leaf and stem tissues. However, plants comprising constitutive-transiently expressed mutant tgd genes showed leaky expression with a wide range of plant growth, embryonic lethality, and variable levels of oils accumulated in plant cells and vegetative tissues. As one example, a TGD3 inactivation line (SALK_040335) carrying a T-DNA 5 of the presumed ATG start codon of Arabidopsis gene At1g65410 showed accumulation of the oligogalactolipid. However in tgd1-3 plants, the plant accumulates oil in its plant cells and plant tissues leaves and stems at the expense of reduced plant growth and fertility.
(55) 1. Reduced Tgd4 Expression Increased Lipid Content in Leaves.
(56) Plants were also made which had insertion mutations that disrupted the normal function of TGD4 protein. The malfunctioning TGD4 protein also caused plant oil to accumulate in the leaf and stem with variable effects on plant growth and fertility, Xu, et al. 2008. The inventors further discovered that comparatively, plants with reduced TGD4 protein appeared to accumulate more oil in vegetative tissues than plants with reduced TGD1 or TGD2 protein.
(57) Specifically, in a population of ethyl methane-sulfonate mutagenized population of Arabidopsis (ecotype Columbia [Col-2] that contained plants having T-DNA insertion, 3 plant lines were found with insertions in the tgd4 gene. Thus a tgd4-1 mutant was isolated during a screen for lines accumulating oligogalactolipids. The growth and lipid phenotypes of tgd4-1 are shown in
(58) TABLE-US-00002 TABLE 2 Leaf Polar Lipid Composition of the Wild Type, tgd4-1, tgd4-2, and tgd4-3 Lipids Wild Type tgd4-1 tgd4-2 tgd4-3 MGDG 37.4 1.7 .sup.38.5 7.5 34.4.sup.a 1.0 32.6.sup.a 1.1 PtdGro 8.9 0.6 12.1.sup.a 1.2 12.3.sup.a 0.5 13.5.sup.a 0.2 DGDG 19.9 0.5 15.2.sup.a 1.6 12.3.sup.a 0.9 12.6.sup.a 0.1 SQDG 3.1 0.3 .sup.4.0 0.6 .sup.3.5 0.1 4.0.sup.a 0.2 PtdIns 1.1 0.2 2.5.sup.a 0.9 .sup.1.2 0.4 .sup.1.4 0.2 PtdEtn 13.8 0.1 10.0.sup.a,b 1.9.sup. 16.6.sup.a,b 0.3.sup. 16.8.sup.a,b 0.3.sup. PtdCho 15.8 0.3 .sup.15.8 4.1 19.6.sup.a 0.7 19.1.sup.a 0.9 Values (mol %) of three independent samples were averaged and the SD is indicated. Plants were grown on soil for 4 weeks. PtdIns, phosphatidylinositol. .sup.aValues are significantly different from wild-type values based on Student's t test (95% confidence interval). .sup.bThis lipid sample also contains TGDG at <1 mol %.
(59) The mutants have slightly reduced amounts of galactoglycerolipids and slightly increased amounts of phosphatidylglycerol (PtdGro), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho). The galactoglycerolipids are primarily found in plastids, while PtdGro and PtdCho are present in plastid and extraplastidic membranes, and PtdEtn is exclusively in extraplastidic membranes. Therefore, these lipid changes may reflect a decreased ratio of plastid-to-extraplastidic membranes in the mutants, which is consistent with the pale green color of the mutants indicative of reduced chlorophyll and photosynthetic membranes. The TGDG content of the mutants was <1 mol % of total fatty acids in leaves (e.g., 0.5 6 0.1 mol % for tgd4-3; n=3, SD is given). Viable seeds were obtained when wild-type pollen was transferred onto tgd4-2 or tgd4-3 pistils, but reciprocal transfer did not yield seeds, suggesting that the tgd4-2 and tgd4-3 mutations cause a pollen defect. Sequencing of cDNA for At3g06960 derived from tgd4-1 detected a mutation in nucleotide position 102 of the coding sequence (GenBank accession number NM_111576) in the first predicted exon of At3g06960. The mutation led to a Pro-to-Leu substitution in position 20 of the predicted protein). A wild-type cDNA derived from locus At3g06960 was fused to the open reading frame of green fluorescent protein (GFP) and when expressed in the tgd4-1 mutant under the control of the cauliflower mosaic virus (CaMV) 35S promoter and was able to restore the wild-type lipid phenotype. The point mutant allele and plants were designated tgd4-1, while two other plants having T-DNA insertion alleles affecting tgd4 expression corresponded to SAIL_760_F05, tgd4-2, and the T-DNA allele corresponding to SAIL_133_H06, tgd4-3. The use of these or other SALK lines with mutations for other tgd genes are contemplated to be problematic for use as founder plants for breeding plants expressing low tgd or for use as hosts for overexpressing other genes such as WRI1 or F-Box, as described herein. In part, due to the methods of making these lines, the plants are highly likely to have T-DNA insertions in other genes along with lacking desired agronomic traits. Further, these lines have variable leaky levels of gene expression increasing the variability of gene expression from plant to plant.
(60) 2. Reduced Expression from Certain Insertion Mutations in Each of Tgd1, Tgd2, Tgd3 and Tgd4 Genes Increased Lipid Accumulation in Plants.
(61) Analysis of neutral lipids in extracts of tgd4-1, tgd4-2 and tgd4-3 mutants revealed the accumulation of triacylglycerol (TGDG), see
(62) TABLE-US-00003 TABLE 3A Fatty Acid (FA) Composition at the sn-2 Position of the Two Major Galactoglycerolipids in the Wild Type, tgd4-1, tgd4-2, and tgd4-3 Fatty Acid Composition (Mol %).sup.a FAs 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3 MGDG WT 1.8 1.1 ND.sup.b 57.3 1.0 ND 2.7 35.9 tgd4-1 5.3 2.8 ND 51.3 1.2 ND 5.8 33.4 tgd4-2 18.3 7.3 1.5 59.7 2.4 ND 1.3 9.2 tgd4-3 16.8 6.9 0.2 64.2 1.7 ND 1.1 8.8 DGDG WT 19.0 1.2 0.4 3.8 4.2 2.0 3.0 64.4 tgd4-1 29.2 1.8 0.4 1.2 7.4 4.5 5.4 49.3 tgd4-2 65.4 1.7 0.5 0.6 8.6 4.4 2.9 14.7 tgd4-3 74.0 1.1 0.6 3.6 3.7 2.7 1.9 11.9 .sup.aThree independent measurements were averaged for all samples, except for the tgd4-2 DGDG samples, for which only two samples were available. In all cases, the SD was <10%. Plants were 4 weeks old and grown on soil. Fatty acids (FAs) are designated with number of carbon atoms:number of double bonds. .sup.bND, not detected at a limit of 0.05 mol %.
(63) Additional fatty acid analysis data for lipids in the wild type and the tgd4-2 and tgd4-3 mutants are provided in Table 3B, where the lipid abbreviations are: MGDG, monogalactosyldiacylglycerol; PtdGro, phosphatidylglycerol; DGDG, digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; PtdIns, phosphatidylinositol; PtdEtn, phosphatidylethanolamine; PtdCho, phosphatidylcholine. MGDG (sn-2) and DGDG (sn-2) refer to the fatty acids in the sn-2 position only of the respective lipid.
(64) TABLE-US-00004 TABLE 3B Complete fatty acid composition data sets for wild type (WT), tgd4-2 and tgd4-3 mutants Fatty Acid (mol %) 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3 WT MGDG 3.9 1.4 0.4 0.0 0.1 0.1 23.8 2.1 4.0 4.2 0.0 0.0 4.6 1.0 63.3 2.7 MGDG (sn-2) 1.8 0.1 1.1 0.1 0.0 0.0 57.3 0.0 1.0 0.2 0.0 0.0 2.7 0.3 35.9 0.2 PtdGro 25.1 1.5 13.6 2.3 0.4 0.1 2.6 0.1 9.4 6.2 4.7 0.8 9.5 1.8 34.7 0.1 DGDG 12.6 0.3 2.1 0.3 0.1 0.0 1.9 0.0 2.3 0.8 1.7 0.0 4.6 0.2 74.7 1.1 DGDG (sn-2) 19.0 1.2 1.2 0.0 0.3 0.0 3.8 3.0 4.2 0.4 2.0 0.0 3.0 0.2 64.4 3.6 SQDG 31.0 1.5 1.2 0.2 0.3 0.0 0.7 1.0 13.0 6.5 2.4 0.1 4.8 0.5 46.5 3.1 PtdIns 27.2 0.2 1.5 0.3 0.7 0.6 0.0 0.0 29.2 4.9 2.3 0.6 8.6 1.2 30.4 4.2 PtdEtn 27.5 0.3 0.4 0.0 0.2 0.0 0.0 0.0 5.2 1.1 3.6 0.0 37.2 0.9 25.8 0.0 PtdCho 18.5 0.1 0.5 0.0 0.2 0.0 0.0 0.0 4.3 0.2 6.9 0.0 36.1 0.1 33.6 0.1 tgd4-2 MGDG 7.2 0.1 2.6 0.0 0.1 0.0 24.6 0.0 1.6 0.1 9.0 0.6 6.7 0.4 48.2 0.3 MGDG (sn-2) 18.3 1.2 7.3 0.4 1.4 0.1 59.7 2.4 2.4 0.2 0.0 0.0 1.3 0.1 9.2 0.9 PtdGro 23.7 0.3 19.3 0.1 0.0 0.0 2.2 0.3 4.3 0.7 11.7 0.1 9.2 0.5 29.6 0.6 DGDG 37.2 1.3 1.5 0.5 0.3 0.1 4.7 0.1 4.7 0.8 7.7 0.1 10.8 0.0 33.1 0.2 DGDG (sn-2) 65.4 5.1 1.7 0.1 0.5 0.0 0.6 0.1 8.6 0.7 4.4 0.4 2.9 0.1 14.7 1.3 SQDG 50.4 0.6 1.3 0.0 0.2 0.0 1.0 1.4 10.3 1.0 4.9 0.3 4.2 0.1 27.8 1.1 PtdIns 27.8 2.1 1.0 1.4 0.4 0.4 0.0 0.0 27.7 0.2 6.8 0.2 20.0 3.3 16.5 0.5 PtdEtn 26.2 0.4 0.7 0.0 0.2 0.0 2.3 0.0 4.7 0.2 14.1 0.0 38.8 0.5 13.1 0.1 PtdCho 14.1 0.1 0.8 0.1 0.1 0.0 2.1 0.0 4.2 0.0 27.9 0.3 32.6 0.0 18.3 0.2 tgd4-3 MGDG 8.0 0.3 3.4 0.1 0.1 0.0 22.6 0.0 1.2 0.0 11.6 0.2 6.2 0.1 46.9 0.1 MGDG (sn-2) 16.8 0.2 6.9 0.5 0.2 0.0 64.2 1.2 1.7 0.1 0.0 0.0 1.1 0.1 8.8 0.7 PtdGro 23.9 0.5 19.6 0.6 0.0 0.0 2.0 0.4 3.4 0.1 14.1 0.4 8.9 0.3 28.1 0.8 DGDG 37.7 0.7 1.5 0.2 0.2 0.0 4.3 0.0 3.2 0.2 9.0 0.0 9.9 0.1 34.2 1.0 DGDG (sn-2) 74.0 3.4 1.1 0.0 0.6 0.0 3.6 0.3 3.7 0.3 2.7 0.2 1.9 0.0 11.9 0.0 SQDG 50.6 0.0 1.2 0.0 0.3 0.2 1.6 0.2 5.3 0.4 6.1 0.4 5.3 0.2 29.7 0.3 PtdIns 30.0 0.4 2.1 0.4 0.7 0.0 0.0 0.0 18.5 0.2 10.9 0.6 21.4 3.1 16.3 2.7 PtdEtn 25.6 0.0 0.9 0.0 0.2 0.0 2.3 0.0 3.3 0.1 19.4 0.0 35.3 0.2 13.1 0.2 Ptdcho 12.7 0.2 1.0 0.0 0.1 0.0 1.9 0.0 2.7 0.0 36.2 0.0 28.6 0.1 16.9 0.1 TGDG 12.6 1.2 5.7 1.4 8.9 3.7 28.2 3.9 6.3 1.4 7.6 0.2 10.2 0.3 20.5 2.8 TAG 41.7 9.4 5.5 4.0 11.7 11.0 2.7 1.9 17.9 7.0 5.9 1.3 10.3 7.3 4.4 5.5
(65) It was apparent that 16-carbon fatty acids were highly enriched in the sn-2 position in the mutants consistent with an overabundance of molecular species derived from the plastid pathway. In other words, similar to the tgd1, tgd2, and tgd3 mutants, the ER pathway of galactoglycerolipid biosynthesis appeared to be disrupted in the tgd4 mutants.
(66) Thus, the inventors discovered that when the expression of one of these tgd genes was lowered through a mutation in one of tgd1-1 dgd1, or reduced tgd1, tgd2-1, and tgd3-1, and tgd4 genes, various amounts of oils accumulated in leaves and stems of these plants. Three tgd mutant alleles (tgd1-1, tgd2-1, tgd3-1) analyzed and discussed here were leaky, i.e. varying levels of expression in different plants, leading to attenuated phenotypes with up to 40% lethality, i.e. tgd1-1, tgd2-1. More severe impairment of the system as previously shown for TGD1 causes embryo-lethality making fully gene-disrupted mutants currently inaccessible to analysis. Therefore, leaf lipid extracts from the tgd1, tgd2 and the tgd3 lines were compared by thin-layer chromatography of neutral (
(67) Further confirmation of the identities of TGDG and TAG lipids was provided by LC/MS analyses of leaf extracts of Col-2 wild type and tgd3 mutants (
(68) Analysis of the fatty acid composition of mono- and digalactosyldiacylglycerol revealed distinct changes in the fatty acid profiles in tgd3 as compared to the wild type (
(69) Accordingly, the 16-carbon-to-18-carbon ratio for the digalactolipid increased from 0.29 to 0.76 in the tgd3 mutant (
(70) Overall, tgd3 has a complex lipid phenotype very similar to tgd1 and tgd2 that was consistent with an impairment of the endoplasmic reticulum-pathway of galactolipid biosynthesis. This similarity in phenotypes also indicated that the TGD1, TGD2, and TGD3 proteins are involved in the same biochemical process.
(71) In summary, when expression of a trigalactosyldiacylglycerol (tgd) gene was disrupted by a T-DNA insertion or when a plant was induced to produce a malfunctioning protein that disrupted the TGD1-3 complex or TGD4 function, the presence of undesirable traits of the plants reduced the economic value of any plant lines developed from the mutant lines or use of mutant TGD genes.
(72) Therefore, the inventors contemplated several methods as described herein in order to increase the value of plant lines having reduced TGD production with increased oil accumulation in nonseed parts. Thus in some embodiments, the inventors contemplated disruption of the expression of tgd gene translation after any one of the following developmental phases, i.e. after the plant germinated, early during seedling growth, later during seedling growth, after senescence, etc., by using an inducible RNAi construct or a construct whose expression was under control of a developmentally regulated promoter, such as a leaf promoter, a senescence promoter, i.e. a promoter turned on by the plant during or after seed formation, etc. In other embodiments, the inventors contemplated the use of a construct whose expression was limited to specific tissues, such as green tissues through the use of a ribusco promoter. In preferred embodiments, the technology described herein is contemplated for use by reduction (i.e. silencing) tgd gene translation for increasing the capacity of engineered plants to store oil while retaining desirable (or regaining by stacking heterologous gene expression) agronomic traits causing plant oils to accumulate in the leaf and stem structures of the plant.
(73) D. Brassica Trigalactosyldiacylglycerol Genes and Encoded Proteins for Use in the Methods of the Present Inventions.
(74) In one embodiment, the present invention contemplates making and using recombinant silencing constructs comprising gene fragments of Brassicaceae genes encoding trigalactosyldiacylglycerol (tgd) mRNA for translation into TGD proteins, i.e. TGD1, TGD2, TGD3, and TGD4. Brassicaceae genes from Arabidopsis thaliana plants are described herein. It is not meant to limit the inventions to the use of Arabidopsis thaliana tgd genes, such that tgd genes are contemplated for use from other Brassicaceae plants, such as rapeseed (canola). Even further, tgd gene sequences from other plants are contemplated for use, such as soybean, safflower, grape, rice, etc. Information on the biochemical characterization of mutant tgd genes and plants expressing these genes that can be used in the present inventions including information on tgd1-1 (including double tgd1-1 and dgd1) and tgd-2 (tgd2-1) mutant genes, and their role in oil biosynthesis, i.e. tgd1-1 dgd1 can be found in Xu et al., (2003) EMBO J. 22:2370-2379, Xu et al., The Plant Cell, Vol. 17, 3094-3110, (2005), tgd-2 Awai PNAS Jul. 11, 2006, 103(28):10817-10822, U.S. patent application Ser. No. 12/506,633, all of which are herein incorporated by reference in their entirety. Mutant genes for tgd3 (tgd3-1) and tgd4 (tgd4-1) and mutant plants were identified with their respective encoded proteins and plant phenotypes, Lu et al., 2007, The Journal of Biological Chemistry, 282:35945-35953, and Xu et al. The Plant Cell, 20:2190-2204, 2008, respectively, both of which are herein incorporated by reference in their entirety.
(75) In one embodiment, expression of tgd silencing constructs in a plant cell, tissue or whole plant can be used for accumulation biosynthetic oil in plant cells, plant tissues, plant leaves, and plant stems. Thus in a preferred embodiment, the inventors contemplate accumulating consistently high levels of vegetative oils in plants of a plant line comprising a silenced tgd gene and agronomic traits for commercial production (e.g., SEQ ID NOs: 1, 3, 4, 6, 8, 9, 11) for reducing expression of their encoded proteins (e.g., SEQ ID NOs: 2, 5, 7, 10). In other preferred embodiments, tgd1 and tgd4 and their encoded proteins provide novel targets for engineering approaches with the goal of optimizing vegetative oil producing plant lines while reducing the loss of agronomic desirable traits. In one embodiment, the accumulated oil comprises triacylglycerols.
(76) TABLE-US-00005 TABLE 5 Exemplary Trigalactosyldiacylglycerol sequences as RNAi targets for lowering amounts of translated protein in plant cells. Examples of gene polymorphism are provided as exemplary additional targets. Trigalactosyldiacylglycerol Genes; Polymorphisms: proteins in Arabidopsis Example of gene plants mRNA or cDNA Protein SEQ ID NO: polymorphism TGD4 Sequence: Protein: 94% identity PDE320 (PIGMENT AT3G06960.1; AT3G06960.1; TAIR AT3G06960.2 DEFECTIVE 320) TAIR Accession Accession: AA (splice variant) 9 Locus: 20775621 Sequence: 10091186935 TGD3 Sequence: Protein: NA ATNAP11 AT1G65410.1; AT1G65410.1; (ARABIDOPSIS TAIR Accession TAIR THALIANA NON- Locus: Accession INTRINSIC ABC 2206275 AA Sequence: PROTEIN 11) 10091049196 TGD2 Sequence: Protein: 83% identity overall; AT3G20320.1; AT3G20320.1; 88% to 3 area TAIR Accession TAIR AT3G20320.2 Locus: Accession: (splice variant) 2092354 AA Sequence: 10091205107 TGD1 Sequence: Protein: 99% identity AT1G19800.2; AT1G19800.2; AT1G19800.3 TAIR Accession TAIR (splice variant) Locus: 2013099 Accession: 98% identity AA Sequence: AT1G19800.1 10091205108
(77) For example, an Arabidopsis thaliana pigment defective 320 Trigalactosyldiacylglycerol 4(tgd4) cDNA sequence is available with accession number AT3G06960.1 from the Arabidopsis information website at arabidopsis.org/servlets/TairObject?type=sequence&id=2002982503. This tgd4 cDNA has the following sequence (SEQ ID NO:1).
(78) TABLE-US-00006 1 AGCTGGGTGTAGAAATCGAGCGACGGCGGCGGAGACGACG 41 GAGATGAACAGAATGAGATGGGTCGGAGAGGGAGACATCT 81 GGGACCTCGATATGTCAACTCCGGTGACGCTCGAGGGCAC 121 CGCACGAGCTGTTCCTGACGATCCTCTTCCTCTAGGTCTC 161 TCTAGAGGCACTCGTCTATCTCGCCCTAAGCAAGTTGAGT 201 TCTTCCACCGCTTCATGGCCTCACCTCTCATCCCTTCCTT 241 CTCCCCTATCCGTCCCAACACCGGAGATGGAGGCGGTGGT 281 GGATTCTCTCTTCAAAGAGTCCTCACTCTTCCTTTCTCCA 321 ACAACTGGCTTGTGTCTCTTCTGGGCCAATTCGATGTTCA 361 GAGATTCGTAACGGAGATAGATAAGACTAAAGCTTTTGGT 401 CGAGGGTCTTCGTCTACAGTAGCTTCTCGTTTAAACACAA 441 TTGGCAAGCATTTGAAGGATAAATCTTTGTACGCATTGGG 481 TTTTTGTTCTGAGTTTTTGTTATCACCAGATGATACTTTG 521 CTTCTTAGCTATGATGCTTACAAAGGTGATCTCGATAAGA 561 ATCCTAGAGCTAAGGCTATCTTCAATCACGAGTTTCCGCT 601 TCACAATCTGACAGCAGAAGCGGTTTGGCCTGGACTTTTT 641 GTGGATAAACATGGTGAATATTGGGATGTGCCACTCTCAA 681 TGGCTATTGATCTAGCATCTCTTCCTGCTGAATCTGGTCC 721 AAGTTACCATTTATGTTTACACCATAACAGCGGATCACCC 761 AAGAAGTTACATTCTGATACTATGGAAGTGCCTCCACCGT 801 CACTGCTTCCTGGTTTGTCTCTGAAATCTGCAGTCTCTTA 841 TAGGACAAACATGGATCTCTGGAGGGGTACCACTCCAAAG 881 CTCGAAACTTGCAAGCCCTATGATGTCTTCCTCAGTAGTC 921 CTCATGTCGCAGTATCTGGGATTATCGGCTCTGTGATGAC 961 CGCAGCATTTGGTGAAAATTCAATCAGATCAAAATTTGAA 1001 AATGATTCTGAGGGTGTTGGAGGGTTCTCTCTTCATTTTC 1041 CATCTGTAAATTCCGGATTCATGGCTGATGCCTTAGGGCG 1081 GGCATCACTCACAGCTCAATATGGAAACTTCCAGAAATTC 1121 TTCTTTGATCTCACCCGTTTCCATGCTAGATTAGACTTTC 1161 CGCATGGTTTGAGGTTTCTTACCGGTGCCACTAGCGTCGC 1201 ACAAGATCTTTTAAATTCTCGGCAGCCTAGTTTAGAAGCA 1241 TTTCAGAAAATCTGCCCTGAAGTATTAGTTTCTCTACAGC 1281 AACAGATTGTTGGACCGTTTAGTTTCAAAGTGGAGTCTGG 1321 AATTGAGATCGATCTGAGGAACGGAGCTAACCCTGTGACT 1361 GTAGATAAGACAGTATTTGCTATTGAATATGCTCTTCAAG 1401 TGCTTCTTTCTGCCAAGGCTGTTGTTTCGTACTCCCCAAA 1441 ACAGCAGAAGTTCATGGTTGAGCTTCGTTTCTTTGAGACA 1481 TAGTATCAGGATTTTCCACTCAAAATGTCAAGCTTGATCC 1521 TGTGAAGATTGTAGTCTTGCAGAGAAGTAAATACTAAATA 1561 GACAATGTTCTAATTGTTCAGTTTCTTATGTCAAACAGAA 1601 GAATGTTTCAATAGAAGGGAAGTTTACATTTTGTTATAGT 1641 GTGATGTCTACCAG
(79) The TGD4 protein sequence encoded by the SEQ ID NO:1 cDNA has the following sequence (SEQ ID NO:2).
(80) TABLE-US-00007 1 MNRMRWVGEGDIWDLDMSTPVTLEGTARAVPDDPLPLGLS 41 RGTRLSRPKQVEFFHRFMASPLIPSFSPIRPNTGDGGGGG 81 FSLQRVLTLPFSNNWLVSLLGQFDVQRFVTEIDKTKAFGR 121 GSSSTVASRLNTIGKHLKDKSLYALGFCSEFLLSPDDTLL 161 LSYDAYKGDLDKNPRAKAIFNHEFPLHNLTAEAVWPGLFV 201 DKHGEYWDVPLSMAIDLASLPAESGPSYHLCLHHNSGSPK 241 KLHSDTMEVPPPSLLPGLSLKSAVSYRTNMDLWRGTTPKL 281 ETCKPYDVFLSSPHVAVSGIIGSVMTAAFGENSIRSKFEN 321 DSEGVGGFSLHFPSVNSGFMADALGRASLTAQYGNFQKFF 361 FDLTRFHARLDFPHGLRFLTGATSVAQDLLNSRQPSLEAF 401 QKICPEVLVSLQQQIVGPFSFKVESGIEIDLRNGANPVTV 441 DKTVFAIEYALQVLLSAKAVVSYSPKQNEFMVELRFFET
(81) A variant of the Arabidopsis thaliana pigment defective 320 Trigalactosyldiacylglycerol 4(tgd4) cDNA sequence with 94% identity to the SEQ ID NO:1 sequence is available from the Arabidopsis database as accession number AT3G06960.2, which has the following sequence (SEQ ID NO:3).
(82) TABLE-US-00008 1 ATGAACAGAATGAGATGGGTCGGAGAGGGAGACATCTGGG 41 ACCTCGATATGTCAACTCCGGTGACGCTCGAGGGCACCGC 81 ACGAGCTGTTCCTGACGATCCTCTTCCTCTAGGTCTCTCT 121 AGAGGCACTCGTCTATCTCGCCCTAAGCAAGTTGAGTTCT 161 TCCACCGCTTCATGGCCTCACCTCTCATCCCTTCCTTCTC 201 CCCTATCCGTCCCAACACCGGAGATGGAGGCGGAGAGGGA 241 TTCTCTCTTCAAAGAGTCCTCACTCTTCCTTTCTCCAACA 281 ACTGGCTTGTGTCTCTTCTGGGCCAATTCGATGTTCAGAG 321 ATTCGTAACGGAGATAGATAAGACTAAAGCTTTTGGTCGA 361 GGGTCTTCGTCTACAGTAGCTTCTCGTTTAAACACAATTG 401 GCAAGCATTTGAAGGATAAATCTTTGTACGCATTGGGTTT 441 TTGTTCTGAGTTTTTGTTATCACCAGATGATACTTTGCTT 481 CTTAGCTATGATGCTTACAAAGGTGATCTCGATAAGAATC 521 CTAGAGCTAAGGCTATCTTCAATCACGAGTTTCCGCTTCA 561 CAATCTGACAGCAGAAGCGGTTTGGCCTGGACTTTTTGTG 601 GATAAACATGGTGAATATTGGGATGTGCCACTCTCAATGG 641 CTATTGATCTAGCATCTCTTCCTGCTGAATCTGGTCCAAG 681 TTACCATTTATGTTTACACCATAACAGCGGATCACCCAAG 721 AAGTTACATTCTGATACTATGGAAGTGCCTCCACCGTCAC 761 TGCTTCCTGGTTTGTCTCTGAAATCTGCAGTCTCTTATAG 801 GACAAACATGGATCTCTGGAGGGGTACCACTCCAAAGCTC 841 GAAACTTGCAAGCCCTATGATGTCTTCCTCAGTAGTCCTC 881 ATGTCGCAGTATCTGGGATTATCGGTATGATAAGTTTCTT 921 CAACTTATTTCAGAAGCATTTTATTGTCAAGACTGAAAGT 961 TTTGTGATTTCTCTAATAAGTTTTGTTCAACTCTTATCAT 1001 TTGAGTTCTCCAATTCCAATATTTGA
(83) An Arabidopsis thaliana Trigalactosyldiacylglycerol 3 (tgd3) cDNA sequence is available with accession number AT1G65410.1, and is shown below and identified herein as SEQ ID NO:4.
(84) TABLE-US-00009 1 CTTTGTTTCTGGGTTTCTCCTAAATCATCCAAATTGGTAT 41 CGAATTTGCCCTTCTCCGATTCAATTTCTTCACGATCTCA 81 AAACCCAGAAGAAAGAATCATGCTTTCGTTATCATGCTCT 121 TCTTCTTCTTCTTCGTTGCTTCCTCCGAGTTTACACTACC 161 ACGGTTCTTCTTCTGTTCAGTCCATCGTTGTACCAAGAAG 201 GAGTCTTATCTCGTTTCGTCGGAAAGTCTCTTGCTGTTGC 241 ATAGCTCCACCTCAGAACTTGGACAACGATGCCACCAAAT 281 TCGATAGTCTTACCAAGTCTGGAGGAGGTATGTTTAAAGA 321 GCGAGGGCTTAAGAATAATTCTGATGTTCTTATTGAATGT 361 AGAGATGTCTATAAATCGTTTGGGGAGAAACATATCTTGA 401 AAGGTGTTAGCTTTAAGATTAGACATGGTGAAGCTGTTGG 441 GGTGATTGGTCCTTCTGGAACTGGAAAATCAACAATTTTA 481 AAGATTATGGCTGGTCTTCTTGCTCCAGACAAGGGAGAAG 521 TTTATATACGAGGAAAAAAACGAGCTGGTTTGATAAGTGA 561 TGAGGAAATATCAGGACTTCGTATTGGCCTGGTATTTCAG 601 AGTGCAGCTCTCTTTGATTCACTATCAGTTCGTGAAAATG 641 TTGGTTTTCTACTTTATGAAAGGAAAAAAATGTCCGAGAA 681 TCAAATATCTTAGCTTCTAACACAAACCTTGGCAGCTGTT 721 GGTTTGAAGGGGGTTGAGAATCGATTACCTTCTGAGCTAT 761 CTGGTGGAATGAAGAAAAGGGTTGCTTTAGCTCGTTCACT 801 AATTTTTGATACAACAAAAGAGGTCATAGAGCCAGAGGTG 841 CTTTTGTACGATGAGCCAACTGCTGAACTTGATCCAATTG 881 CATCAACTGTAGTTGAAGATCTTATACGGTCTGTTAACAT 921 AACAGACGAAGATGCAGTTGGAAAACCTGGAAAAATTGCG 961 TCTTATCTTGTTGTTACCCATCAACATAGCACCATTCAAA 1001 GAGCTGTAGACAGGTTATTGTTTCTGTATGAAGGAAAGAT 1041 CGTTTGGCAAGGAATGATACATGTATTCACAACCTCAACT 1081 AATCCAATAGTTCAACAGTTTGCTACAGGCAGCCTCGATG 1121 GACCAATCAGATACTAGGGGAGGCAAACCGAGCCTAAAGA 1161 GGGACACTAACCGATAATAGGGAACGCAAACAAGTAATGG 1201 CTGACATACACCACATGGCTGGATCAATTGGTTCAATACG 1241 ATGCTACTTGTAAACACTATTTTTTCTTAGATGCATAGAT 1281 CAGAAAAGCATTGTCAGTTG
(85) The TGD3 protein sequence encoded by the SEQ ID NO:4 cDNA has the following sequence (SEQ ID NO:5).
(86) TABLE-US-00010 1 MLSLSCSSSSSSLLPPSLHYHGSSSVQSIVVPRRSLISFR 41 RKVSCCCIAPPQNLDNDATKFDSLTKSGGGMCKERGLEND 81 SDVLIECRDVYKSFGEKHILKGVSFKIRHGEAVGVIGPSG 121 TGKSTILKIMAGLLAPDKGEVYIRGKKRAGLISDEEISGL 161 RIGLVFQSAALFDSLSVRENVGFLLYERSKMSENQISELV 201 TQTLAAVGLKGVENRLPSELSGGMKKRVALARSLIFDTTK 241 EVIEPEVLLYDEPTAGLDPIASTVVEDLIRSVHMTDEDAV 281 GKPGKIASYLVVTHQHSTIQRAVDRLLFLYEGKIVWQGMT 321 HEFTTSTNPIVQQFATGSLDGPIRY
(87) An Arabidopsis thaliana Trigalactosyldiacylglycerol 2 (tgd2) cDNA sequence is available with accession number AT3G20320.1, and is shown below and identified herein as SEQ ID NO:6.
(88) TABLE-US-00011 1 TATTCTCAGATTCACGACACCAGTTCGTCACAAGCTTCGA 41 GCCCAGCTCGGAAAACAAAATTGGAACTTGCTGCATAAAG 81 TTTAGTTTTTTTAATTGAATTTGGAAGGATGATTGGGAAT 121 CCAGTAATTCAAGTTCCATCATCACTAATGCCATCATCCT 161 CCATGATTGCTTGTCCTCGAGTTTCACCCAATGGGGTTCC 201 TTATCTTCCACCAAAACCTAGAACTAGGCATTTAGTGGTC 241 AGAGCTGCATCCAATTCCGATGCTGCTCATGGTCAACCAT 281 CGTCTGATGGGGGGAAGAATCCTCTCACCGTTGTTTTGGA 321 TGTGCCCAGGAATATATGGAGACAGACTTTAAAACCTTTG 361 AGTGATTTTGGGTTTGGTAAGAGAAGTATTTGGGAAGGTG 401 GTGTTGGTTTGTTTATTGTCTCTGGAGCTACTCTTCTTGC 441 TCTTAGCTGGGCTTGGTTGCGAGGTTTTCAAATGCGGTCG 481 AAGTTTAGGAAATATCAGACTGTGTTTGAGCTTAGTCATG 521 CTTCTGGTATTTGCACGGGAACACCGGTTAGGATCCGTGG 561 GGTTACTGTTGGTACGATTATCCGTGTTAATCCTTCCTTG 601 AAGAATATTGAAGCTGTTGCTGAGATAGAAGATGATAAGA 641 TTATTATCCCGAGGAATTCATTGGTTGAGGTGAATCAGTC 681 TGGTCTTCTAATGGAAACTATGATCGACATTATGCCTAGG 721 AATCCGATACCAGAACCTTCAGTAGGACCTCTGCATCCTG 761 AATGTGGTAAGGAAGGTCTGATCGTTTGTGATAGGCAGAC 801 AATAAAAGGAGTGCAAGGAGTTAGTTTAGATGAATTAGTT 841 GGAATTTTCACTCGTATTGGACGCGAAGTTGAGGCCATTG 881 GTGTTGCCAATACGTATTCGCTTGCTGAGAGAGCTGCTTC 921 GGTTATTGAGGAAGCAAGGCCATTGCTCAAAAAGATTCAA 961 GCCATGGCTGAAGATGCTCAACCTTTGCTCTCTGAGTTTC 1001 GTGATAGCGGCTTGCTCAAGGAAGTTGAGTGTCTTACTCG 1041 AAGCCTTACCCAAGCTTCTGACGATTTGAGAAAGGTTAAT 1081 TCGTCAATTATGACTCCTGAGAATACAGAACTCATACAGA 1121 AGTCAATCTACACTCTGGTTTATACTTTGAAGAACGTCGA 1161 GAGTATAAGCTCAGATATTCTGGGATTCACAGGAGATGAA 1201 GCCACAAGAAAAAACCTTAAACTACTCATCAAATCCCTAA 1241 GCAGGCTACTATGATCAGCCTGTAGATTTTAGACTGGATA 1281 AATAAAATCCAGAATTTTTATGGTAAGCAAGTTTTAAAAA 1321 TTCGAAAAATGTGTTGTTTCTTCTTTAGAGTTATTTTTGT 1361 TTTCGTTTTGTGTTCTGAGATTGGGGTTTAATGGAGAGAC 1401 ATAATTCAGTTTTTATAAGAACAAAAATGTTTGTTT
(89) The TGD2 protein sequence encoded by the SEQ ID NO:6 cDNA has the following sequence (SEQ ID NO:7).
(90) TABLE-US-00012 1 MIGNPVIQVPSSLMPSSSMIACPRVSPNGVPYLPPKPRTR 41 HLVVRAASNSDAAHGQPSSDGGKNPLTVVLDVPRNIWRQT 81 LKPLSDFGFGKRSIWEGGVGLFIVSGATLLALSWAWLRGF 121 QMRSKFRKYQTVFELSHASGICTGTPVRIRGVTVGTIIRV 161 NPSLKNIEAVAEIEDDKIIIPRNSLVEVNQSGLLMETMID 201 IMPRNPIPEPSVGPLHPECGKEGLIVCDRQTIKGVQGVSL 241 DELVGIFTRIGREVEAIGVANTYSLAERAASVIEEARPLL 281 KKIQAMAEDAQPLLSEFRDSGLLKEVECLTRSLTQASDDL 321 RKVNSSIMTPENTELIQKSIYTLVYTLKNVESISSDILGF 351 TGDEATRKNLKLLIKSLSRLL
(91) A variant of the Arabidopsis thaliana tgd2 cDNA sequence with 83% identity to the SEQ ID NO:6 sequence is available from the Arabidopsis database as accession number AT3G20320.2, which has the following sequence (SEQ ID NO:8).
(92) TABLE-US-00013 1 CAGTTCGTCACAAGCTTCGAGCCCAGGTATTCTCTCTTTC 41 GCTCAAAAACCCTAATCTCGACTTATAATTCGATCGATAA 81 AGTAGAAGCTTCACGCAATTCACATGTTCTCTATCTTCTT 121 TCTAACTACAGCTCGGAAAACAAAATTGGAACTTGCTGCA 161 TAAAGTTTAGTTTTTTTATTGAATTTGGAAGGATGATTGG 201 GAATCCAGTAATTCAAGTTCCATCATCACTAATGCCATCA 241 TCCTCCATGATTGCTTGTCCTCGAGTTTCACCCAATGGGG 281 TTCCTTATCTTCCACCAAAACCTAGAACTAGGCATTTAGT 321 GGTCAGAGCTGCATCCAATTCCGATGCTGCTCATGGTCAA 361 CCATCGTCTGATGGGGGGAAGAATCCTCTCACCGTTGTTT 401 TGGATGTGCCCAGGAATATATGGAGACAGACTTTAAAACC 441 TTTGAGTGATTTTGGGTTTGGTAAGAGAAGTATTTGGGAA 481 GGTGGTGTTGGTTTGTTTATTGTCTCTGGAGCTACTCTTC 521 TTGCTCTTAGCTGGGCTTGGTTGCGAGGTTTTCAAATGCG 561 GTCGAAGTTTAGGAAATATCAGACTGTGTTTGAGCTTAGT 601 CATGCTTCTGGTATTTGCACGGGAACACCGGTTAGGATCC 641 GTGGGGTTACTGTTGGTACGATTATCCGTGTTAATCCTTC 681 CTTGAAGAATATTGAAGCTGTTGCTGAGATAGAAGATGAT 721 AAGATTATTATCCCGAGGAATTCATTGGTTGAGGTGAATC 761 AGTCTGGTCTTCTAATGGAAACTATGATCGACATTATGCC 801 TAGGAATCCGATACCAGAACCTTCAGTAGGACCTCTGCAT 841 CCTGAATGTGGTAAGGAAGGTCTGATCGTTTGTGATAGGC 881 AGACAATAAAAGGAGTGCAAGGAGTTAGTTTAGATGAATT 921 AGTTGGAATTTTCACTCGTATTGGACGCGAAGTTGAGGCC 961 ATTGGTGTTGCCAATACGTATTCGCTTGCTGAGAGAGCTG 1001 CTTCGGTTATTGAGGAAGCAAGGCCATTGCTCAAAAAGTG 1041 ATGTCACAGATTCAAGCCATGGCTGAAGATGCTCAACCTT 1081 TGCTCTCTGAGTTTCGTGATAGCGGCTTGCTCAAGGAAGT 1121 TGAGTGTCTTACTCGAAGCCTTACCCAAGCTTCTGACGAT 1161 TTGAGAAAGGTTAATTCGTCAATTATGACTCCTGAGAATA 1201 CAGAACTCATACAGAAGTCAATCTACACTCTGGTTTATAC 1241 TTTGAAGAACGTCGAGAGTATAAGCTCAGATATTCTGGGA 1281 TTCACAGGAGATGAAGCCACAAGAAAAAACCTTAAACTAC 1321 TCATCAAATCCCTAAGCAGGCTACTATGATCAGCCTGTAG 1361 ATTTTAGACTGGATAAATAAAATCCAGAATTTTATGGTAA 1401 GCAAGTTTTAAAAATTCGAAAAATGTGTTGTTTCTTCTTT 1441 AGAGTTATTTTTGTTTTCGTTTTGTGTTCTGAGATTGGGG 1481 TTTAATGGAGAGACATAATTCAGTTTTTATAAGAACAAAA 1521 ATGTTTGTTTCTC
(93) An Arabidopsis thaliana Trigalactosyldiacylglycerol 1 (tgd1) cDNA sequence is available with accession number AT1G19800.2, and is shown below and identified herein as SEQ ID NO:9.
(94) TABLE-US-00014 1 TGTGTGTTGTTGTTGTTGGCACTGTGCCACTTTCTCTCTC 41 GATGAACCCTCTCAAGCAAGCTTCTTCGATCTTCCGAGCT 81 TAGTTTCGTTTCTAAATTAGAGATTTCACCTAGATTGGTC 121 CGTACATATCTTATACTGGGATTCGAATTTGGCTGCCTCA 161 GAGTCAGAGATTGATTAATTGATCAGATTCAGCTGTTGAA 201 ATCGTGCTTATTGCTACAAATTGAGAGGCACTAAATCAGT 241 GAGGTCGTAAAGAAGAAGGCAACCACAATGATGCAGACTT 281 GTTGTATCCATCAATCGTTTTGTTTCCCTCATAGAGTCTT 321 TCCACGGTTTGATGCTTCGATTGGTATTAAGCCCCCAAAG 361 CTTTGTCAAGTTGGTTTCATTGGAAAGACTCAATCTTATG 401 GGATTTCAAGTCCGATACGGCAAAGAAGATTATATGTGAA 441 TTTGAATGCTAATGATGGTCACCCATCCATGTCTATGTTG 481 GAAGAAGAAACCTCTACTGAAAACAACGCACCCAGTCAAG 521 AAGCCGAGCTTCCGTTCAGCAAATGGTCACCTTCTAAGTA 561 CATATGGAGAGGTTTATCAGTTCCTATTATAGCAGGACAA 601 GTCGTTCTCCGGATTTTAAAGGGTAAGATTCACTGGAGAA 641 ACACTCTTCAACAGCTGGAGAGAACCGGACCGAAATCTCT 681 AGGAGTTTGTCTTCTGACTTCTACATTTGTTGGTATGGCT 721 TTCACAATCCAGTTCGTTAGAGAATTCACTAGACTAGGTC 761 TAAACAGATCCATTGGAGGTGTCTTGGCTTTAGCCTTCTC 801 TAGAGAGCTAAGTCCAGTCATCACATCGATTGTTGTTGCT 841 GGACGAATGGGAAGTGCATTTGCAGCTGAACTAGGGACAA 881 TGCAAGTCTCAGAGCAAACTGATACACTCCGTGTTTTAGG 921 AGCTGACCCAATTGATTATCTAATCACTCCAAGAGTCATC 961 GCCTCGTGTTTGGCTCTACCGTTTCTGACACTCATGTGTT 1001 TCACTGTTGGTATGGCTTCAAGCGCTCTGCTCTCTGATGC 1041 AGTTTACGGGATCAGCATTAACATAATCATGGACTCGGCT 1081 CACCGAGCACTTAGACCATGGGACATTGTGAGTGCCATGA 1121 TTAAATCTCAAGTCTTTGGAGCTATAATATCGGTAATTAG 1161 TTGTTCTTGGGGAGTAACCACTACTGGAGGTGCTAAAGGT 1201 GTTGGAGAATCTACAACTTCTGCTGTCGTCATGTCTCTTG 1241 TCGGAATCTTCATCGCGGACTTTGTGCTTTCTTCCTTCTT 1281 CTTTCAAGGTGCTGGAGATTCTTTGAAGAACTGTGTTTGA 1321 CATATTATTTTCTGTCTTCTTTTGTTGTGGTTTAGATGGG 1361 TTTATGTAAATCAGTTGTCTTAAATTGAGAAAGTAACATC 1401 ATTTTAGAAAGAACAGAAAGATTGCTATATTTCTATTCCA 1441 ATAATGATACACATTGAATAAT
(95) The TGD1 protein sequence encoded by the SEQ ID NO:9 cDNA has the following sequence (SEQ ID NO:10).
(96) TABLE-US-00015 1 MMQTCCIHQSFCFPHRVFPRFDASIGIKPPKLCQVGFIGK 41 TQSYGISSPIRQRRLYVNLNANDGHPSMSMLEEETSTENN 81 APSQEAELPFSKWSPSKYIWRGLSVPIIAGQVVLRILKGK 121 IHWRNTLQQLERTGPKSLGVCLLTSTFVGMAFTIQFVREF 161 TRLGLNRSIGGVLALAFSRELSPVITSIVVAGRMGSAFAA 201 ELGTMQVSEQTDTLRVLGADPIDYLITPRVIASCLALPFL 241 TLMCFTVGMASSALLSDAVYGISINIIMDSAHRALRPWDI 281 VSAMIKSQVFGAIISVISCSWGVTTTGGAKGVGESTTSAV 321 VMSLVGIFIADFVLSSFFFQGAGDSLKNCV
(97) A variant of the Arabidopsis thaliana tgd1 cDNA sequence with 99% identity to the SEQ ID NO:9 sequence is available from the Arabidopsis database as accession number AT1G19800.3, which has the following sequence (SEQ ID NO:11).
(98) TABLE-US-00016 1 TGTGTGTGTTGTTGTTGTTGGCACTGTGCCACTTTCTCTC 41 TCGATGAACCCTCTCAAGCAAGCTTCTTCGATCTTCCGAG 81 CTTAGTTTCGTTTCTAAATAGATTTCACCTAGATTGGTCC 121 GTACATATCTTATACTGGGATTCGAATTTGGCTGCCTCAG 161 AGTCAGAGATTGATTAATTGATCAGATTCAGCTGTTGAAA 201 TCGTGCTTATTGCTACAAATTGAGAGGCACTAAATCAGTG 241 AGGTCGTAAAGAAGAAGGCAACCACAATGATGCAGACTTG 281 TTGTATCCATCAATCGTTTTGTTTCCCTCATAGAGTCTTT 321 CCACGGTTTGATGCTTCGATTGGTATTAAGCCCCCAAAGC 361 TTTGTCAAGTTGGTTTCATTGGAAAGACTCAATCTTATGG 401 GATTTCAAGTCCGATACGGCAAAGAAGATTATATGTGAAT 441 TTGAATGCTAATGATGGTCACCCATCCATGTCTATGTTGG 481 AAGAAGAAACCTCTACTGAAAACAACGCACCCAGTCAAGA 521 AGCCGAGCTTCCGTTCAGCAAATGGTCACCTTCTAAGTAC 561 ATATGGAGAGGTTTATCAGTTCCTATTATAGCAGGACAAG 601 TCGTTCTCCGGATTTTAAAGGGTAAGATTCACTGGAGAAA 641 CACTCTTCAACAGCTGGAGAGAACCGGACCGAAATCTCTA 681 GGAGTTTGTCTTCTGACTTCTACATTTGTTGGTATGGCTT 721 TCACAATCCAGTTCGTTAGAGAATTCACTAGACTAGGTCT 761 AAACAGATCCATTGGAGGTGTCTTGGCTTTAGCCTTCTCT 801 AGAGAGCTAAGTCCAGTCATCACATCGATTGTTGTTGCTG 841 GACGAATGGGAAGTGCATTTGCAGCTGAACTAGGGACAAT 881 GCAAGTCTCAGAGCAAACTGATACACTCCGTGTTTTAGGA 921 GCTGACCCAATTGATTATCTAATCACTCCAAGAGTCATCG 961 CCTCGTGTTTGGCTCTACCGTTTCTGACACTCATGTGTTT 1001 CACTGTTGGTATGGCTTCAAGCGCTCTGCTCTCTGATGCA 1041 GTTTACGGGATCAGCATTAACATAATCATGGAACCGGACC 1081 ACCGAGCACTTAGACCATGGGACATTGTGAGTGCCATGAT 1121 TAAATCTCAAGTCTTTGGAGCTATAATATCGGTAATTAGT 1161 TGTTCTTGGGGAGTAACCACTACTGGAGGTGCTAAAGGTG 1201 TTGGAGAATCTACAACTTCTGCTGTCGTCATGTCTCTTGT 1241 CGGAATCTTCATCGCGGACTTTGTGCTTTCTTCCTTCTTC 1281 TTTCAAGGTGCTGGAGATTCTTTGAAGAACTGTGTTTGAC 1321 ATATTATTTTCTGTCTTCTTTTGTTGTGGTTTAGATGGGT 1361 TTATGTAAATCAGTTGTCTTAAATTGAGAAAGTAACATCA 1401 TTTTAGAAAGAACAGAAAGATTGCTATATTTCTATTCCAA 1441 TAATGATACACATTGAATAAT
(99) The methods of the present invention contemplate the use of at least one heterologous gene encoding a tgd RNAi of the present invention for use in a gene-silencing construct. Therefore, the inventors created exemplary gene constructs and methods comprising these gene constructs, as described herein, contemplated for increasing the amount of oils produced and stored in leaves and stems without stunting the growth of host plants. Even further the inventors contemplated that plants of the present inventions would have high fertility levels (at least greater than 60%) for increasing the amount of oil produced per plant in addition to having agronomical desirable traits.
(100) Therefore, in one experiment designed to avoid stunted plant growth and embryo lethality while obtaining plants with a more consistent phenotype of accumulated, i.e. increased, oil in vegetative tissues, the inventors created an TGD3 RNAi construct using two copies of a fragment of the tgd3 gene, one in the sense direction and the other in an antisense direction, separated by a spacer sequence of a tgd3 intron, in operable combination with an inducible promoter.
(101) The tgd3 sense sequence of the RNAi construct containing 5 untranslated and 5 translated (underlined) regions of the cDNA is shown in
(102) TABLE-US-00017 1 AAAAATGGCAATGTGACTCACTCAATCGGTGACTCGCTAT 41 AGTCTGTGAAGAAAGGCCAATTTCGCCATAAAGTTCACAC 81 CTTTGATCTCCTTTGTTTCTGGGTTTCTCCTAAATCATCC 121 AAATTGGTATCGAATTTGCCCTTCTCCGATTCAATTTCTT 161 CACGATCTCAAAACCCAGAAGAAAGAATCATGCTTTCGTT 201 ATCATGCTCTTCTTCTTCTTCTTCGTTGCTTCCTCCGAGT 241 TTACACTACCACGGTTCTTCTTCTGTTCAGTCCATCGTTG 281 TACCAAGAAGGAGTCTTATCTCGTTTCGTCGGAAAGTCTC 321 TTGCTGTTGCATAGCTCCACCTCAGAACTTGGACAACGAT 361 GCCACCAAATTCGATT
(103) This tgd3 RNAi construct was used to transform wild-type Arabidopsis plants (i.e. with functional TGD1, 2, 3 and 4). After germination, the roots of established control and RNAi construct-containing seedlings (around 3 weeks after sprouting) were soaked in the inducing chemical. Growing plant leaves of seedlings exposed to the inducing chemical began accumulating oil in their leaves. Thus, plants were able to grow normally before oil began accumulating in vegetative tissues. Exemplary analysis of these oils is shown in
(104) Thus accumulation of the oil in the leaf and stem was achieved by altering the function of the trigalactosyldiacylglycerol (TGD) proteins after the plant is established (i.e. as a young seedling). The inventors were surprised that the resulting seedling plants accumulated substantial amounts of oils in leaves and stems with the use of either promoter. Also surprisingly, unlike plants engineered for a reduction in TGD1 and TGD2, separately, the use of a constitutive promoter for a construct reducing the production of TGD3 was not lethal in these TGD3 knock down plants.
(105) Thus, in one embodiment, the inventors contemplate increasing the amount of oil produced and stored within a plant using either a constitutive promoter or an inducible promoter in operable combination with a gene silencing construct, i.e. a RNAi tgd gene fragment construct, comprising a fragment of a tgd gene as described herein. Therefore, in some embodiments, tgd silencing constructs of under control of constitutive promoters. In other embodiments, tgd silencing constructs are under control of inducible promoters. In yet other embodiments, tgd silencing constructs are under control of developmental promoters. In yet further embodiments, tgd silencing constructs are under control of tissue specific promoters. The inventors additionally contemplate using other types of inducible RNAi tgd gene constructs for silencing tgd genes, such as an ethanol-inducible promoter, Peebles, et al., Biotechnology Progress, 23(5):1258-1260 (2007), herein incorporated by reference in its entirety, nonendogenous glucocorticoid promoter, and the like. In addition to using constructs with constitutive promoters for silencing tgd genes using RNAi constructs with DNA sequences, for examples, using tgd SEQ ID NOs:12-15.
(106) E. Engineering Plants for Modifying Oil Content in Vegetative Tissues while Maintaining Plant Viability for Breeding Agronomically Desired Commercial Varieties Through the Use of Oil Regulating Transcription Factors.
(107) Engineering the accumulation of triacylglycerols (TAGs) and other lipids in vegetative tissues is contemplated to support efforts for increasing yield of biofuels and/or edible lipids in relation to resources needed for crop growth. However as described, when oil production is manipulated in plants there is frequently a concurrent lowering of plant viability and seed fertility, such as shown when tgd expression is lowered. Thus, plants making larger amounts of oil having vigorous growth characteristics along with high fertility levels are needed. In one contemplated embodiment, the use of oil regulating transcription factors (TF) is contemplated to overcome these problems. As one example which succeeded in part, plants overexpressing WRINKLED1 having reduced amounts of AGPase proteins have increased amounts of TAGs in vegetative tissues. However, although the co-expression of WRINKLED1 increased oil levels in these plants also have not possessed reduced growth rates or fertility.
(108) 1. Use of Oil Regulating Transcription Factors for Increasing Oil Production in Vegetative Tissues of Plants.
(109) Triacylglycerols (TAGs) derived from seed oils such as canola, soybean and palm are contemplated for use as a feedstock for biodiesel production (Durrett et al., 2008, herein incorporated by reference in its entirety) in addition to other types of uses. However, the current supply of these energy-rich compounds is limited because of low crop yield and arable land availability. For example, it has been estimated that 50%-75% increase in canola oil production will be required to meet the growing seed oil demand in Canada alone during the next 7-10 years (Weselake et al., 2009, herein incorporated by reference in its entirety). One contemplated approach to increase biodiesel feedstocks is to synthesize TAGs in vegetative tissues such as leaves, stems, roots and storage organs, using a metabolic engineering approach. For comparison, certain plants are naturally able to accumulate oils in nonseed tissues to various degrees, for instance, avocado mesocarp (Takenaga et al., 2008, herein incorporated by reference in its entirety), nut sedge roots (Stoller and Weber, 1975, herein incorporated by reference in its entirety) and stems of the oil firewood Tetraena mongolica Maxim (Wang et al., 2007, herein incorporated by reference in its entirety). However these plants are not suitable for commercial agriculture nor do they produce the type of oils needed for commercial use.
(110) The biochemistry of fatty acids (FA) and TAG synthesis in plants is complex, regulated by different factors in different cellular compartments, and each intermediate biochemical reaction was found to be catalyzed by specialized enzymes (Ohlrogge and Jaworski, 1997; Durrett et al., 2008, The Plant J, 54:593-607, all of which are herein incorporated by reference in their entirety). Photosynthate in the form of sugar from the source tissues of the parent plant was discovered as an import into developing seeds and converted via glycolysis into precursors of fatty acid biosynthesis (Durrett et al., 2008; Baud and Lepiniec, 2010, all of which are herein incorporated by reference in its entirety). Genetic studies in Arabidopsis revealed some of the factors that control the seed oil biosynthesis pathway. For example, a wri1-1 mutant, which was deficient in a transcription factor, had an 80% reduction in seed TAG content and required an external carbon source for germination (Focks and Benning, 1998, herein incorporated by reference in its entirety).
(111) Similarly, several additional transcription factors such as LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3) were identified in Arabidopsis and discovered to play a role in seed maturation and oil biosynthesis (Baumlein et al., 1994; Meinke et al., 1994; Stone et al., 2001, all of which are herein incorporated by reference in their entirety). Thus earning the designation of oil regulating transcription factors. WRI1 in particular, a member of the APETALA2/ETHYLENE-RESPONSIVE ELEMENT BINDING (AP2/EREB) transcription factor family was discovered to have a role in oil regulation in both seeds and seedlings (Cernac and Benning, 2004; Cernac et al., 2006, all of which are herein incorporated by reference in their entirety). Analysis of gene expression in wri1-1 mutants and transgenic plants that expressed WRI1 cDNA or in a WRI1 enhancer-driven overexpression line showed targets of this transcription factor, including many genes encoding enzymes of the fatty acid biosynthesis pathway (Ruuska et al., 2002; Baud et al., 2007; Maeo et al., 2009, all of which are herein incorporated by reference in their entirety). A combination of molecular and biochemical approaches identified contemplated WRI1-binding motifs in some target genes (Baud et al., 2009; Maeo et al., 2009, all of which are herein incorporated by reference in their entirety). Overexpression of Brassica napus WRI1 in Arabidopsis and maize WRI1 in a low oil maize genetic (mutant) background enhanced oil accumulation in maize seeds (Liu et al., 2010; Shen et al., 2010, all of which are herein incorporated by reference in their entirety). However the mutant background was required. These results demonstrated that WRI1 regulated seed oil biosynthesis in both dicotyledonous and monocotyledonous plants. However, the numerous attempts to metabolically engineer TAG accumulation in seeds by targeting the expression of single genes or a combination of genes had moderate success (Thelen and Ohlrogge, 2002; Abbadi et al., 2004; Taylor et al., 2009, all of which are herein incorporated by reference in their entirety).
(112) Another contemplated method for using an oil seed regulating transcription factor for overcoming the lack of success for increasing oil seed biosynthesis was the overexpression of LEC1 in Arabidopsis and LEC2 in the fatty acid break down mutant comatose (cts2), which led to the accumulation of oil in vegetative tissues (Mu et al., 2008; Slocombe et al., 2009, all of which are herein incorporated by reference in their entirety). In another test, transgenic tobacco engineered with DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and a transcription factor LEC2 showed accumulated oil surprisingly in the leaves of plants (Andrianov et al., 2010, herein incorporated by reference in its entirety).
(113) In the case of developing seeds, carbon derived from photosynthesis was partitioned into different storage compounds. Therefore that carbon flux into oil was contemplated to be in competition with the use of carbon for other metabolic pathways (Vigeolas et al., 2004; Ekman et al., 2008, all of which are herein incorporated by reference in their entirety). One of these competing pathways is the biosynthesis of starch, which serves as a major carbon reserve in plants with up to 50% of photosynthate stored as starch. There are two types of starch, storage and transitory (Ballicora et al., 2004; Smith, 2008, all of which are herein incorporated by reference in their entirety). The synthesis and accumulation of starch in storage tissues is largely controlled by growth and development (Smith, 2008, herein incorporated by reference in its entirety). For example, seeds of Arabidopsis and B. napus accumulate starch early in development and later shift from synthesizing starch to oil (Focks and Benning, 1998; Vigeolas et al., 2004, all of which are herein incorporated by reference in their entirety).
(114) Transitory starch is stored in the chloroplasts of leaves and is built up during the day. Its synthesis can consume up to half of the carbon fixed by photosynthesis. This starch is then broken down at night to provide a continuous source of carbon to the plant when CO.sub.2 cannot be fixed by photosynthesis (Weise et al., 2004, herein incorporated by reference in its entirety). Adenosine Diphospho (ADP)-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch biosynthesis and converts glucose-1-phosphate and ATP to ADP-glucose and inorganic pyrophosphate. ADP-glucose is subsequently turned into starch by multiple isoforms of starch synthase and branching enzyme (Ballicora et al., 2004; Smith, 2008, all of which are herein incorporated by reference in their entirety). Inactivation of AGPase in maize (Zea mays), rape seed (B. napus) and pea (Pisum sativum) led to considerable reduction in seed starch content (Vigeolas et al., 2004; Weigelt et al., 2009, all of which are herein incorporated by reference in their entirety). Therefore, several attempts were made to increase starch content through the genetic engineering of AGPase activity in crops (Stark et al., 1992; Smidansky et al., 2002; Ihemere et al., 2006, all of which are herein incorporated by reference in their entirety). However these plants had enhanced starch content not enhanced oil. This was due to the finding that starch and oil are competing pathways for use of carbon so when carbon is used for starch production it is less available for oil production.
(115) Further, it was discovered that starch biosynthesis was partially blocked by suppression of the expression of APS1 while concurrently ectopically expressing WRI1 resulted in increased accumulation of TAG in Arabidopsis vegetative tissues. Arabidopsis plants that ectopically expressed AGPRNAi-WRI1 accumulated less starch and 5.8-fold more TAG in the vegetative tissues. Therefore it is contemplated that the amount of oil accumulated in plants would be improved further with the use of RNAi TGD (i.e. reduced translation of TGD) also having reduced APS1 while concurrently overexpressing WRI1. Hence a strategy was developed to divert carbon from starch to oil by silencing the expression of one of the AGPase gene. Thus resultant plants were contemplated to accumulate less starch and then have more available sugar for conversion into oil by concomitant expression of sugar sensitive WRI1 transcription factor.
(116) In fact, in the AGPRNAi-WRI1 line 28, a 5.8-fold increased oil accumulation along with a 50% reduction in starch relative to wild type was observed in the vegetative tissues, compared to 2.6-2.8-fold oil accumulation in the transgenic lines expressing WRI1 cDNA alone. From data shown in
(117) In most plants, TAGs are stored in oil droplets in the seeds, which support seedling growth after germination (Jolivet et al., distinct oil droplets in the leaf and root tissues as revealed by Nile red staining) These observations were consistent with increased oil accumulation in the AGPRNAi-WRI1 lines. Intriguingly, the presence of long-chain fatty acid containing TAG species typically found restricted to seeds in Arabidopsis were detected in the vegetative tissue of the AGPRNAi-WRI1 lines (
(118) The main cause for the reduction of starch levels in the 35S-AGPRNAi and AGPRNAi-WRI1 lines was the introduction of the RNAi cassette specifically targeting an AGPase small subunit that led to the down-regulation of APS1 expression (
(119) In addition to serving as an energy source, sugars play a role as signaling molecules in controlling gene expression in plants (Rolland et al., 2006, herein incorporated by reference in its entirety). In particular, the expression of WRI1 was regulated by various sugars. For example, glucose and fructose were necessary for seedlings ectopically overexpressing WRI1 to produce TAGs (Cernac and Benning, 2004, herein incorporated by reference in its entirety). It was demonstrated that WRI1 expression was enhanced by sucrose in Arabidopsis leaves (Masaki et al., 2005, herein incorporated by reference in its entirety). Consistent with these results, the expression of WRI1 in seedlings of 35S-AGPRNAi lines was increased 2.5-11-fold compared to wild-type plants (
(120) However, the largest increase in WRI1 transcript levels in the AGPRNAi-WRI1 lines compared to wild-type lines was because of the expression of the 35S-WRI1 cassette (construct). Surprisingly, the relative expression level of SUS2 was considerably elevated in the AGPRNAi-WRI1 line (
(121) The relative expression of plastidic proteins involved in de novo fatty acid biosynthesis BCCP2 and ACP1 was enhanced in the AGPRNAi-WRI1 line. Previous studies showed that BCCP2 and ACP1 were WRI1 targets (Baud et al., 2007; Maeo et al., 2009, all of which are herein incorporated by reference in their entirety). Surprisingly, results described herein indicated moderate changes in the relative expression of some known WRI1 targets involved in the glycolysis and later stages of oil synthesis, for example PI-PKb1, 3OAR, PDHE1a and TAG1. It may be possible that in vegetative versus embryonic tissues, the expression of these genes or the activity of the encoded enzymes is differentially regulated based on the source/sink or sugar state of the cells. For example, recent work has suggested that the activity of Nasturtium DGAT1 is regulated by its phosphorylation state (Xu et al., 2008, herein incorporated by reference in its entirety). Given the relatively high accumulation of TAG in these AGPRNAi-WRI1 lines, it is tempting to speculate that sugar signaling might be involved in such post-translational regulation of the activity of enzymes involved in TAG biosynthesis. Future detailed studies are needed to understand the effect of sugars on the WRI1 regulatory networks in vegetative tissues.
(122) In conclusion, altering the carbon partitioning in the vegetative tissues in combination with up-regulation of TAG biosynthesis led to an enhanced accumulation of TAGs in transgenic seedlings, in principle enhancing the energy density of the biomass. Ultimately, TAGs need to be synthesized in the vegetative tissues of mature plants; these promising results are a first step towards that goal. However, further studies are necessary to understand possible factors which limit WRI1 expression, TAG synthesis and accumulation in the vegetative tissues to reach the amount needed to make an economically viable product. Following oil extraction, the remaining sugars and lignocellulose are still available for biofuel production. However, the greater energy density and compatibility with existing engine technology make TAG containing oils desirable such that the larger the TAG fraction in biomass, the higher is the crop value.
(123) However, to be economically useful, changes in oil production must not come at the expense of overall seed yield, fertility, or at the loss of other high-value components, nor at a loss of agronomic traits desirable for commercial plant lines. For example, soybean is the largest source of vegetable oil, comprising 30% of the world market, and now constitutes over 80% of all dietary vegetable oils in the United States. Although termed an oilseed, soybean contains 18-22% oil on a seed dry-weight basis and is grown principally as a high-protein meal for animal feeds. Thus, increasing oil in soybean will in most cases not be useful if it comes at the expense of high-value soy protein that drives the crop's economics. By comparison, other oilseed crops (except cotton) are grown primarily for their oil and produce seeds with 40-60% oil. The wide range of seed oil percentage observed in nature suggests that this pathway might be amenable to metabolic engineering, particularly in low-oil oilseeds, provided mechanisms which control oil content are identified. Even further, the inventors contemplate that economically valuable increases in oil production in vegetative tissues combined with increases in seed oil content would provide plants and oil crops that would overcome current limitations.
(124) 2. WRINKLED1 Overexpression Increased Seed Oil Content.
(125) A WRI1 gene encodes an APETALA2/ethylene-responsive element-binding protein transcription factor involved in the control of metabolism, particularly glycolysis, in the developing seeds. WRI1 encodes a putative transcription factor of the AP2/EREBP class (Jofuku et al., 1994; Riechmann et al., 2000; Weigel, 1995, all of which are herein incorporated by reference in its entirety). Examples of wild type Arabidopsis plants overexpressing a heterologous WRI1 gene showed lipid accumulation in seedlings, see, Cernac and Benning, The Plant Journal 40: 575-585 (2004), herein incorporated by reference in its entirety).
(126) In the absence of sugar in the medium, seedlings of most of the transgenic plants lines were generally indistinguishable from wild type and developed into Arabidopsis plants which did not form ectopic embryos or show other obvious abnormalities. The requirement for sugar to bring about the aberrant phenotype may be simply explained by the fact that carbon skeletons and reducing power are needed to synthesize the large amounts of oil in these compromised seedlings. Plants grown from abnormal seedlings grown in the presence of sugar were often stunted in growth and yellowish suggesting reduced photosynthetic capability. They were often sterile as well. However, in the presence of sugar, oil-accumulated in seedlings in plant lines, which ectopically expressed WRI1. Essentially, the phenotype of these abnormal seedlings was consistent with an extension or reinitiation of the embryonic state of developing seeds during oil accumulation. However, ectopic expression of WRI1 did not lead to ectopic embryo formation as observed for LEC1 (Lotan et al., 1998, herein incorporated by reference in its entirety) in Arabidopsis or BABY BOOM in Brassica (Boutilier et al., 2002, herein incorporated by reference in its entirety).
(127) Thus, ectopic expression of the WRI1 cDNA caused a sugar-inducible accumulation of seed-like oil in the transgenic seedlings. The general appearance of the transgenic seedlings suggested a resumption of embryonic development following germination. The overall conclusion was that the WRI1 gene product was involved in controlling seed development, in particular the phase of embryo maturation in which TAG accumulates, with sugar providing one part of the process (Cernac and Benning, 2004, herein incorporated by reference in its entirety). Moreover, the ectopic expression of WRI1 under the control of a CAMV 35S promoter led to an increase in WRI1 mRNA abundance in developing seedlings and concomitantly to an increase in the abundance of mRNA predicted to encode a plastidic isoform of the glycolytic enzyme pyruvate kinase. The expression of the WRI1 gene was observed in developing siliques and seeds isolated from the siliques in addition to roots and to a lower extent in young seedlings and flowers. Expression in strictly photosynthetic tissues was not detected by RNA/DNA hybridization. Specifically, a wild-type WRI1 cDNA (wri1) under the control of the CAMV 35S promoter was introduced into Arabidopsis wild type plants and wri1-1 mutant plants. Thus, transgenic plants expressing the WRI1 cDNA under the control of the CAMV 35S promoter (wri1-cDNA, WT-cDNA) were made. In the case of the transgenic lines, 10 T2 seeds from 104 and 107 independently isolated lines were analyzed (mutant and wild-type background respectively). For wild type 10 seeds of 32 independent plants each and for the wri1 mutant 30 samples of 10 seeds each from a pooled seed batch were analyzed.
(128) The transgenic lines were grown in parallel in 16 h light, 8 h dark, at 22 C. in the same chamber, while the untransformed lines were grown under identical conditions but in a different chamber to avoid cross-pollination between transgenic and untransformed lines. Individual plants were grouped in 1 g intervals according to fatty acid content per seed. For better comparison, a broken line was centered through the peak of the wild type seed distribution,
(129) However, contrary to line 107 a small fraction of the plants of line 106 showed an abnormal morphological phenotype in the absence of sucrose. To be able to distinguish between the endogenous mutant wri1-1 RNA and the transgene-derived wild-type mRNA, semi-quantitative PCR was employed using diagnostic primers flanking the intron that was not removed in the mutant (
(130) 3. Transgenic Seedlings Accumulated Triacylglycerols in the Presence of Sucrose.
(131) The abnormal seedlings of lines 106 and 107 and other transgenic lines were reminiscent of oil-accumulating embryos that had outgrown their seed coat. Indeed, seed oil was present in seedlings of these lines when grown on sucrose (
(132) TABLE-US-00018 TABLE 4 Sugar Specificity for Ectopic Oil Accumulation in 35S-WRI1 cDNA Transgenic Lines Long Chain Fatty Acid Content (g/seedling) Sugar type Line 106 Line 107 No sugar 0.1 0 nd Glucose 5.1 0.4 0.6 0.2 Fructose 1.8 0.4 nd Sucrose 5.3 0.3 0.9 0.1 Glucose/Fructose 3.7 0.3 0.9 0.2 Maltose 2.9 0.1 nd Sorbitol 0.4 0 nd Galactose 0.5 0.1 nd
(133) In another embodiment, the engineered plants of the present inventions are contemplated for combining overexpression of WRINKLED1 with plants having reduced TGD proteins.
(134) The inventors contemplated several methods for making inventive engineered plants with stacked genes for alterations in protein expression, i.e. one protein overexpressed while another protein has reduced expression. In one embodiment, plants engineered to overexpress a heterologous WRINKLED1 gene are used as breeding stock for introducing this heterologous gene into another transgenic engineered breeding stock having reduced TGD proteins. In a further embodiment, a heterologous WRINKLED1 gene is under control of a promoter, such as a constitutive promoter or an induced promoter or a tissue specific promoter, etc. As merely one example, the WRINKLED1 gene might be under the control of a constitutive promoter while as another example, the WRINKLED1 gene might be under the control of a ribusco promoter, etc. In a preferred embodiment, these overexpressing engineered plants are bred to engineered plants having reduced expression of a TGD protein resulting from the use of a TGD RNAi vector of the present inventions. The order of plant breeding strategy is not limited to one particular scheme. In other words the order of which plants are made and used is not important so long as the resulting engineered plant has increased expression of a WRINKLED1 gene along with reduced TGD protein for increasing lipid accumulation in vegetative tissues while retaining or regaining desirable agronomic traits. In an additional embodiment, aspects of the present inventions comprise overexpression of a heterologous WRINKLED1 gene in combination with reduced expression of a TGD protein for modifying and/or increasing lipid production in a plant or plant line. In one embodiment, TAG are increased in the vegetative tissues of engineered plants of the present inventions.
(135) Accordingly, methods of making these engineered plants with increased TAG amounts in vegetative tissues comprises breeding WRINKLED1 overexpressing plants with engineered plants having reduced expression of a TGD gene as the result of transformation of the host plant with a TGD RNAi gene silencing vector of the present inventions. In a preferred embodiment, these methods of breeding further comprise using breeding stock with desired agronomic traits, such as large seed sizes, a particular oil composition, strong growth characteristics, growth characteristics for a particular hardiness zone, high levels of fertility, etc., and/or desirable economic traits, such as roundup ready varieties or other plant lines having glyphosate-resistance.
(136) Thus methods of the present inventions further comprise transforming plants producing seed oil, in particular oil seed plants for expressing oil in vegetative tissues. Optimized agronomic traits of transgenic plants of the present inventions may be obtained by screening candidate plants for persistent expression of the transgene along with reduced TGD target proteins through multiple generations of breeding or rounds of vegetative propagation.
(137) Therefore, with the discovery of the genes described herein and their effects upon oil production, described herein, the inventors' contemplated increasing the amount of oil per plant by increasing oil production in vegetative tissues. Further, the inventors contemplated that increasing the amount of oil per plant would be accomplished by increasing oil production in both vegetative tissues and seeds. Specifically, the inventors contemplated that by lowering tgd translation in plants through the use of tgd RNAi silencing constructs in specific amounts along with the overexpression of an oil regulating transcription factor, then a balance might be achieved between increased oil production and negative impacts on plant growth and fertility.
(138) Additionally a AP2/EREB WRI1 protein described herein and in Cernac, 2004, herein incorporated by reference in its entirety, modified oil content in developing oil seeds and other tissues although it was discovered to control metabolic processes primarily for seed oil accumulation. The ectopic expression of WRI1 under the control of the CAMV 35S promoter was originally directed at the investigation of WRI1 function and not at engineering of oil content. However, even this crude expression system led to increases in seed oil content as well as the ectopic production of triacylglycerols in developing seedlings. Refined approaches toward the tissue-specific engineering of WRI1 RNA abundance as described herein, may well provide the means to control triacylglycerol biosynthesis in desirable tissues or at extended times of the life cycle of the plant.
(139) F. F-Box Protein.
(140) Further, an F-Box transcription protein was discovered whose overexpression in plants induced an increased production of plant oil. In fact, some of the inventors discovered that overexpression of F-box protein in transgenic Arabidopsis lines increased total oil content of plants by more than 10%. Therefore, the inventors contemplated that in some embodiments, by increasing the expression of the WRI1 gene and/or the F-Box gene in plants also having decreased production of TGD4, TGD3, TGD2 OR TGD1, then the resulting plants would have more desirable growth characteristics and fertility, i.e. in addition to other agronomic traits, than plants expressing normal amounts of any one of these genes or plants having lowered TGD production or overexpressing WRI1 or F-Box. In other words, engineered plants of the present inventions are contemplated to achieve a balance of highly increased lipid production in vegetative tissues and seeds balanced with desirable agronomic and commercial traits for use in producing commercially viable plant lines having significantly increased amounts of oil per plant. In one embodiment, TAG production is highly increased on a per plant and/or per acre basis.
(141) Plants were engineered for lipid modification and/or increased lipid production in seeds using f-box genes. Indeed, this increase was confirmed over at least two generations of breeding engineered plants and the analysis was validated with two different approaches (i.e. total FAMEs and C:N ratio analysis). In particular, the overexpression of the Arabidopsis thaliana F-box protein gene (GenBank Accession Nos. NM_111499 (cDNA) and NP_566277 (protein) using the seed specific promoter phaseolin, produced seeds with a higher oil content phenotype (see
(142) In a further embodiment, the genetic constructs of the invention comprises a sequence that encodes the plant F-box protein has a polypeptide sequence that is at least 75% identical to the polypeptide sequence of an F-box/kelch-repeat protein from Arabidopsis thaliana (also disclosed as SEQ ID NO: 1 of WO 2007 038345, specifically incorporated by reference herein in its entirety), which is provided below as SEQ ID NO:56.
(143) TABLE-US-00019 1 MKAIQLLWEAIMEATKRERRREDDDGEKASPESLVLPPEI 41 ITEILLRLPAKSIGRFRCVSKLFCTLSSDPGFAKIHLDLI 81 LRNESVRSLHRKLIVSSHNLYSLDFNSIGDGIRDLAAVEH 121 NYPLKDDPSIFSEMIRNYVGDHLYDDRRVMLKLNAKSYRR 161 NWVEIVGSSNGLVCISPGEGAVFLYNPTTGDSKRLPENFR 201 PKSVEYERDNFQTYGFGFDGLTDDYKLVKLVATSEDILDA 241 SVYSLKADSWRRICNLNYEHNDGSYTSGVHFNGAIHWVFT 281 ESRHNQRVVVAFDIQTEEFREMPVPDEAEDCSHRFSNFVV 321 GSLNGRLCVVNSCYDVHDDIWVMSEYGEAKSWSRIRINLL 361 YRSMKPLCSTKNDEEVLLELDGDLVLYNFETNASSNLGIC 401 GVKLSDGFEANTYVESLISPNSYGIES
(144) In other embodiments, the plant F-box protein has a polypeptide sequence that is at least 90% identical to the polypeptide sequence of SEQ ID NO:56. In still further useful embodiments, the genetic constructs of the invention comprise a plant F-box protein having the polypeptide sequence of SEQ ID NO:56. In particular embodiments, the plant F-box protein is encoded by the nucleic acid sequence with NCBI accession number NM_111499, shown below and herein identified as SEQ ID NO:57
(145) TABLE-US-00020 1 TTTTTCAAATCAAATCAGAATACATTGATTCTGTATATCT 41 TATTGAAAAATCCATCAATTTACATCAACAATTTTATATC 81 TAATAATTAATTTAAAGAGAAAATTTATAAAAGTTTATTA 121 GAGAGAAAGACTCAAACTCGGATTTTATAGTCGTTATGAC 161 CCGGTTTGACTATTGAACCGTTTAACCGAGAAATTGGGAC 201 TCAATTAAGACAACCGAAACTAGACCCGGATCCAGTGTTA 241 GCGGGCTAGATTAAGGTGTCGGGTCATAGCGGAGAAGCAA 281 CCAGACGCCAACAATGAAAGCGATCCAGTTGCTGTGGGAA 321 GCGATAATGGAGGCGACGAAGAGAGAAAGACGGAGAGAAG 361 ATGACGACGGCGAAAAAGCTTCACCGGAATCACTCGTTCT 400 TCCACCAGAGATCATTACAGAAATTCTTCTCCGATTACCA 441 GCCAAATCGATCGGGCGATTCAGGTGCGTATCAAAGCTCT 481 TTTGCACTTTATCGTCAGATCCAGGGTTCGCGAAGATTCA 521 CCTCGATCTGATCCTTCGAAACGAATCCGTAAGATCGCTC 561 CACCGTAAGCTCATTGTGTCTTCACATAATCTGTACTCGT 601 TAGATTTCAATTCGATCGGTGACGGAATTAGGGATTTAGC 641 GGCTGTGGAACACAATTATCCTCTTAAAGACGATCCAAGC 681 ATTTTCTCTGAGATGATTAGGAATTACGTGGGGGACCATC 721 TGTACGATGATCGTCGCGTGATGCTTAAGCTGAATGCGAA 761 ATCGTATCGAAGAAACTGGGTTGAGATCGTTGGATCTTCC 801 AATGGTTTAGTGTGTATCTCTCCTGGTGAAGGAGCTGTTT 841 TCTTGTATAATCCAACTACCGGAGATTCCAAGAGATTACC 881 TGAAAATTTTCGTCCCAAATCTGTAGAATACGAAAGAGAT 921 AATTTCCAAACTTATGGATTTGGTTTCGATGGTCTCACTG 961 ATGATTACAAATTGGTGAAGCTTGTTGCTACCAGTGAAGA 1001 TATTCTCGATGCTAGTGTCTATTCCTTGAAGGCTGACTCA 1041 TGGAGACGGATCTGCAATTTGAATTATGAGCACAACGATG 1081 GCTCCTACACGTCCGGTGTGCATTTCAACGGTGCGATTCA 1121 CTGGGTGTTCACAGAGAGTAGGCACAACCAAAGAGTGGTT 1161 GTAGCATTTGATATTCAAACCGAGGAGTTTCGAGAGATGC 1201 CAGTGCCTGATGAAGCTGAAGATTGTTCCCATAGGTTTAG 1241 CAACTTTGTGGTCGGAAGTCTCAATGGACGTCTCTGTGTG 1281 GTCAATAGTTGCTACGATGTGCATGATGATATATGGGTGA 1321 TGAGTGAGTACGGTGAAGCTAAATCCTGGAGCAGAATTCG 1361 AATCAACTTGTTGTATAGGTCGATGAAACCGCTCTGTTCG 1401 ACTAAGAACGATGAAGAGGTTCTTCTGGAGCTTGATGGAG 1441 ACCTGGTGTTGTACAACTTTGAAACCAATGCATCGAGTAA 1481 TCTAGGAATTTGTGGGGTTAAGCTCAGTGACGGGTTCGAG 1521 GCAAATACATACGTAGAGAGCCTCATATCACCCAACTCTT 1561 ATGGTATAGAGAGCTGAGGAAGTCTGCTTTTTGCTAAGAT 1601 ATAATAAACCAACATTCGGATTAGAAATGTTTTAGAAACA 1641 TAATCATGTAATATGTATCATGTAATTAACAACGAATGGT 1681 CAATGGGTATTTTAAGTTTCTTTCTCCT
(146) In further embodiments, the F-box protein is encoded by a nucleic acid that hybridizes to the nucleic acid sequence of SEQ ID NO: 57. In particularly useful embodiments, the F-box protein is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:57.
(147) In a further aspect, the invention provides a recombinant Arabidopsis thaliana plant cell having a heterologous genetic construct that includes a gene promoter sequence functionally linked to a plant F-box encoding sequence. In certain particularly useful embodiments, the F-box sequence encoded corresponds to the plant F-box polypeptide of SEQ ID NO: 56.
(148) In yet another aspect, the invention provides a plant having a transgenic plant cell of the invention, as described above, or a part, propagule or progeny thereof comprising one of the genetic constructs of the invention.
(149) In still another aspect, the invention provides a method of modifying oil production in a plant by, first, stably incorporating into the genome of the plant a genetic construct of the invention, as described above, to provide a transformed plant, and, then, regenerating the transformed plant so that expression of the incorporated genetic construct modifies oil production in the plant. In particular embodiments, the plant modified is Arabidopsis thaliana. In other embodiments, the plant is an agricultural crop plant, such as corn or wheat. In further embodiments, the plant is an oil-producing agricultural crop such as soybean, palm, rapeseed or sunflower. In particular,
(150) Evaluation of F-box gene at T3 generation: T2 plants from F-box2 and F-box3 lines (see
(151) G. Plants Engineered for Lipid Modification and/or Increased Lipid Production in Vegetative Tissues Using at Least Two Oil Regulating Transcription Factors, for Example, a WRINKLED1 Gene and an f-Box Gene.
(152) In another embodiment, the engineered plants of the present inventions are contemplated for combining overexpression of at least two heterologous transcriptional control genes, WRINKLED1 and f-box in combination with reduced TGD proteins. The inventors' contemplated several methods for making engineered plants of the present inventions. In one embodiment, plants engineered to overexpress an F-box protein gene are used as breeding stock for introducing this heterologous gene into another transgenic engineered breeding stock capable of overexpressing a heterologous WRINKLED1 gene such that the resulting plant overexpresses both the f-box gene and the WRINKLED1 gene. In a further embodiment, one or both heterologous genes are each under control of separate promoters, such as a constitutive promoter or an induced promoter or a tissue specific promoter, etc. As merely one example, the WRINKLED1 gene might be under the control of a constitutive promoter while the f-box gene might be under the control of a phaseolin promoter while as another example, the WRINKLED1 gene might be under the control of a ribusco promoter while the f-box gene might be under the control of a constitutive promoter, etc. In a further embodiment, these duel overexpressing engineered plants are bred to engineered plants having reduced expression of a TGD protein resulting from the use of a TGD RNAi vector of the present inventions. The order of plants used in a breeding strategy is not limited to one particular scheme. In other words the order of which plants are used in breeding is not important so long as the resulting engineered plant has increased expression of the f-box and WRINKLED1 genes along with reduced TGD protein. In an additional embodiment, aspects of the present inventions comprise overexpression of both a heterologous f-box protein gene and a WRINKLED1 gene in combination with reduced expression of a TGD protein for modifying and/or increasing lipid production in a plant or plant line. In one embodiment, TAG is increased in the vegetative tissues of engineered plants of the present inventions. In another embodiment, a vector construct is contemplated as comprising both a f-box gene and a WRINKLED1 gene for transformation and overexpression in a host plant. Further, each of these heterologous genes may be under the control of their own promoter, or both of these genes may be under control of a single promoter for making plants capable of overexpressing both of these heterologous genes. Regardless of how these new duel overexpressing engineered plants are made, the inventors' contemplated their use in making engineered plants having reduced expression of a TGD gene for substantially increasing TAG amounts in vegetative tissues with or without substantially increasing TAG amounts in seeds of these engineered plants.
(153) Accordingly, methods of making these engineered plants with increased TAG amounts comprises breeding these f-box and WRINKLED1 overexpressing plants with engineered plants having reduced expression of a TGD gene as the result of transformation of the host plant with a TGD RNAi gene silencing vector of the present inventions. In a preferred embodiment, these methods of breeding further comprise using breeding stock with desired agronomic traits, such as large seed sizes, a particular oil composition, strong growth characteristics, growth characteristics for a particular hardiness zone, high levels of fertility, etc., and/or desirable economic traits, such as roundup ready varieties or other plant lines having glyphosate-resistance.
(154) Thus, in one preferred embodiment, an overexpression construct of the present inventions is provided as a duel vector for overexpressing both an f-box protein gene and a WRINKLED1 gene in a host plant for increasing lipid production without significantly impacting host plant viability and fertility. Thus methods of the present inventions further comprise transforming plants, in particular oil seed plants, with a duel overexpression vector that when expressed increases oil production in the vegetative tissues of a plant. Such methods further include two primary means for making transgenic plants which further have reduced TGD expression, wherein a transgenic plant further comprises an RNAi vector of the present inventions for silencing a TGD gene. In another embodiment, a vector construct is contemplated as comprising both a f-box gene and a WRINKLED1 gene for transformation and overexpression in a host plant. Further, each of these heterologous genes may be under the control of their own promoter, or both of these genes may be under control of a single promoter for making plants capable of overexpressing both of these heterologous genes. Optimized transgenic plants of the invention may be obtained by screening candidate plants for persistent expression of the transgene through multiple generations of breeding or multiple rounds of vegetative propagation.
(155) III. Use of Vectors and Plants Having Reduced APS1/AGPase while Overexpressing an Oil Regulating Transcription Factor, WRINKLED1 in Compositions and Methods of the Present Inventions.
(156) Arabidopsis thaliana were engineered to ectopically overproduce the transcription factor WRINKLED1 (WRI1) involved in the regulation of seed oil biosynthesis. Furthermore, the misexpression (expression of a protein with reduced function compared to wild-type) and/or reduced expression of APS1 encoding a major catalytic isoform of the small subunit of ADP-glucose pyrophosphorylase involved in starch biosynthesis was reduced using an RNAi approach. The resulting AGPRNAi-WRI1 lines accumulated less starch and more hexoses. In addition, these lines produced 5.8-fold more oil in vegetative tissues than plants with WRI1 or AGPRNAi alone. Abundant oil droplets were visible in vegetative tissues. TAG molecular species contained long-chain fatty acids, similar to those found in seed oils. In AGPRNAi-WRI1 lines, the relative expression level of sucrose synthase 2 was considerably elevated and correlated with the level of sugars. The relative expression of the genes encoding plastidic proteins involved in de novo fatty acid synthesis, biotin carboxyl carrier protein isoform 2 and acyl carrier protein 1, was also elevated. The relative contribution of TAG compared to starch to the overall energy density increased 9.5-fold in one AGPRNAi-WRI1 transgenic line consistent with altered carbon partitioning from starch to oil.
(157) IV. Gene Silencing for Knock Down of TGD Production.
(158) Gene silencing (i.e. Gene knockdown) may or may not entirely eliminate the expression or translation of the target gene. Several methods can be used to silence a gene, in other words to reduce translations of a gene. In one method, antisense inhibition of protein production may be achieved by the use of an expression vector that overexpresses one copy of cDNA, orientated in the sense or antisense direction, which causes changes in encoded levels of proteins. However this process suffers from lack of specificity for the target sequence, incomplete efficacy, and inconsistent results. For example, expressing antisense sequences or sequences intended for co-suppression (examples of post-transcriptional gene silencing) was frequently observed in transgenic plants (Baulcombe, Plant Mol Biol 32:79-82 (1996); Jorgensen, Trends Genet. 1999; 15:11-2, all of which are herein incorporated by reference in their entirety). However, in some cases, partial functions were suppressed by antisense or co-suppression.
(159) In contrast, methods of RNA silencing by RNAi inhibition may provide more consistent results and be amendable to controlling the amount of inhibition, such as desired in the present inventions where some translation of the targeted gene, i.e. tgd, is desired. Post-Transcriptional Gene Silencing (PTGS) is a RNA silencing pathway triggered by double stranded RNA (dsRNA). It includes antisense-mediated gene silencing, co-suppression, RNA interference (RNAi) and virus-induced gene silencing. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a knockdown, to distinguish it from knockout procedures in which expression of a gene is entirely eliminated. In preferred embodiments, tgd gene translation is knock down not knock out. Post-transcriptional silencing may result in a strong reduction of mRNA accumulation in the cytoplasm without significant changes in the rate of transcriptional initiation in the nucleus. It can affect the expression of transgenes and homologous host genes, a phenomenon referred to as co-suppression (Chalcone synthase, van Blokland et al. 1994; glucanase, de Carvalho Niebel et al. 1995; chitinase, Kunz et al. 1996; nitrate reductase, Vaucheret et al. 1997). Thus, RNA molecules containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. Although, RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Therefore, sequence identity for use in RNAi constructs may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is also preferred.
(160) Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. The length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases. There is no upper limit on the length of the dsRNA that can be used. For example, the dsRNA can range from about 21 base pairs (bp) of the gene to the full length of the gene or more. In one embodiment, the dsRNA used in the methods of the present invention is about 1000 bp in length. In another embodiment, the dsRNA is about 500 bp in length. In yet another embodiment, the dsRNA is about 22 bp in length. In some preferred embodiments, the sequences that mediate RNAi are from about 100 to about 200 nucleotides. The double stranded RNA of the present invention should be sufficiently similar to natural RNA that it has the ability to mediate RNAi for the target RNA.
(161) Accordingly, in some embodiments, the present invention provides isolated RNA molecules (double-stranded or single-stranded) that are complementary to sequences required for oil biosynthesis. In some embodiments, the RNA molecules utilized mediate RNAi for one or more of the oil biosynthesis enzymes identified in
(162) The use of inducible RNAi constructs and silencing gene constructs of the present inventions comprising inducible promoters is contemplated to avoid problems with abnormalities caused by a knocked down or knocked out gene in early developmental stages of plants. Thus in one embodiment, plants comprising silencing gene constructs of the present inventions having inducible promoters are exposed to the inducing compound after seed germination. In one embodiment, plants comprising silencing genes of the present inventions with inducible promoters are exposed to the inducing compound as seedling plants. In some embodiments, seedlings and immature plants are used to isolate oils from vegetative tissues instead of using mature plants.
(163) There are several methods of using RNA sequences for controlling gene expression, especially for gene silencing, including RNA interference (RNAi), small interfering RNA (siRNA), small hairpin RNA (shRNA), small temporal RNAs (stRNA), microRNA (miRNA), and the like. In general, the term RNA interference (RNAi) describes the use of double-stranded RNA, comprising a sense and antisense nucleic acid for targeting specific endogenous mRNAs for degradation, thereby silencing their expression, in other words silencing translation. A dsRNA (RNAi, siRNA, shRNA, and the like) triggers destruction of a homologous mRNA that has the same sequence as one of the dsRNA strands.
(164) V. Constructs.
(165) The present invention relates to gene-specific silencing of genes involved in oil biosynthesis through RNA interference or other methods, and in particular, to vectors for expressing RNAi molecules that inhibit the expression of trigalactosyldiacylglycerol (tgd) protein genes in the oil biosynthesis pathway. In some embodiments, the present invention provides compositions and methods for inducible or constitutive expression of RNAi molecules, and/or for long-term expression of RNAi molecules. Hence the compositions and methods described herein are suitable for regulatable and/or sustained gene-specific silencing in cells, and further for silencing genes altering oil biosynthesis. In one embodiment, the lowering of tgd genes available for translation results in accumulation of a biosynthetic oil, in vegetative leaves and stems. In one embodiment, the biosynthetic oil comprises large amounts of triacylglycerol.
(166) In some embodiments, the present invention provides methods and gene constructs for silencing otherwise known as down regulating one or more of the oil biosynthesis tgd enzymes. In preferred embodiments, corn-specific gene constructs encoding RNAi's specific for targeting one or more of the tgd enzymes are introduced into oil seed plants, including Brassica oil seed plants, preferably elite oil seed plants comprising agronomic traits. In some embodiments, oil content of plant parts, comprising leaves, stems, roots and seeds, of each plant are isolated and compared with control non-transgenic plants of the same age. In some preferred embodiments, oil content of plant parts, comprising leaves, stems, roots and seeds, of each plant are isolated and used for producing commercial oil products.
(167) A. RNAi Constructs.
(168) The present invention contemplates the use of RNA interference (RNAi) constructs to downregulate the translation of tgd genes. In some embodiments, the dsRNA used to initiate RNAi, may be isolated from native source or produced by known means, e.g., transcribed from DNA. The promoters and vectors described in more detail below are suitable for producing dsRNA. RNA is synthesized either in vivo or in vitro. In some embodiments, endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. In other embodiments, the RNA is provided transcription from a transgene in vivo or an expression construct. In some embodiments, the RNA strands are polyadenylated; in other embodiments, the RNA strands are capable of being translated into a polypeptide by a cell's translational apparatus. In still other embodiments, the RNA is chemically or enzymatically synthesized by manual or automated reactions. In further embodiments, the RNA is synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA may be used with little or a minimum purification in order to avoid losses due to sample processing. In some embodiments, the RNA was dried for storage or dissolved in an aqueous solution. In other embodiments, the solution contains buffers or salts to promote annealing, and/or stabilization of the duplex strands. In some embodiments, the dsRNA is transcribed from the vector as two separate stands. When the dsRNA is thus-produced, the DNA sequence to be transcribed is flanked by two promoters, one controlling the transcription of one of the strands, and the other that of the complementary strand. These two promoters may be identical or different. In some embodiments, a DNA duplex provided at each end with a promoter sequence can directly generate RNAs of defined length, and which can join in pairs to form a dsRNA. See, e.g., U.S. Pat. No. 5,795,715, incorporated herein by reference. In some embodiments, the dsRNA is transcribed from the vector as one strand which forms a secondary structure which causes inhibition of translation. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for inhibition of translation of the target gene. RNA molecules containing a nucleotide sequence identical to or nearly identical to a portion of the target gene are preferred for inhibition.
(169) Accordingly, in some embodiments, the present invention provides isolated RNA molecules (double-stranded or single-stranded) that are complementary to sequences required for oil biosynthesis. In some embodiments, the RNA molecules utilized mediate RNAi for one or more of the oil biosynthesis enzymes identified in
(170) In some embodiments, the present invention provides transgenic plants that express dsRNA molecules that correspond to target tgd RNA. A heterologous gene encoding at least a portion of the RNAi of the present invention, which includes variants of the RNAi gene, includes any suitable sequence that encodes a double stranded molecule specific for a tgd target RNA. Preferably, the heterologous gene is provided within an expression vector such that transformation with the vector results in expression of the double stranded RNA molecule; suitable vectors are described below in the Examples.
(171) B. Spacer Sequences for RNAi Constructs.
(172) In preparing a construct comprising a nucleic acid sequence encoding a RNAi sequence of the present invention, various DNA fragments can be used to separate the 2 copies of the sequences for use in gene silencing. This sequence is known as a spacer sequence. A spacer sequence can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation. Hirai, et al. The effects of spacer sequences on silencing efficiency of plant RNAi vectors, Plant Cell Reports Volume 26, Number 5, 651-659, herein incorporated by reference in its entirety. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. A broad range of the RNAi-induced silencing phenotypes can be generated by replacing the spacer sequence of RNAi construct tgd RNAi construct. In addition, in some embodiments, constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Calais et al. (1987) Genes Develop. 1:1183; herein incorporated by reference in its entirety). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
(173) C. Constructs for Expression of Stacked Genes.
(174) Heterologous genes, such as WRINKLED1 alone or in combination with AGPRNAi and F-Box, intended for expression in plants as stacked genes along with reduced TGD protein production, i.e. tgd RNAi constructs, are assembled in expression cassettes comprising a promoter. Methods which are well known to those skilled in the art may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are widely described in the art (See e.g., Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., all of which are herein incorporated by reference in their entirety). More specific examples of constructs for overexpressing heterologous genes, i.e. WRIL1 and F-box, and plants engineered for expressing these constructs are provided as described herein, and in Cernac and Benning, The Plant J, 40:575-585 (2004) and WO 2007 038345 respectively, herein incorporated by reference in their entirety.
(175) In some embodiments, WRIL1 and/or F-box genes are expressed constitutively throughout the whole plant, for example, the CaMV 35S promoter is contemplated for use.
(176) D. Promoters.
(177) The methods of the present invention contemplate the use of at least one heterologous gene encoding a tgd gene silencing sequence of the present invention, or other heterologous sequence, operably linked to a promoter. Heterologous genes intended for expression in plant cells are operably connected to a promoter before or during ligation into expression constructs and cassettes. Many methods may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are widely described in the art (See for example, Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.).
(178) Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmentally specific promoters, and inducible promoters. Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase (LAP, Chao et al. (1999) Plant Physiol 120: 979-992; herein incorporated by reference); a chemically inducible promoter from tobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267; herein incorporated by reference in its entirety); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422; herein incorporated by reference); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053; herein incorporated by reference in its entirety). In some preferred embodiments, the promoter is a phaseolin promoter.
(179) In one embodiment, the promoter is a constitutive promoter such that the cell continuously expresses the genes in operable combination with the promoter. In another embodiment, the promoter is part of an inducible promoter system, for examples, see, Xu, et al. EMBO J., 2003, 22(10):2370-9; Lu et al., 2007, The Journal of Biological Chemistry, 282:35945-35953; and Benning Progress in Lipid Research 47, Issue 5, 2008, 381-389, all of which are herein incorporated by reference in their entirety. In yet other embodiments of the present invention, a transgenic plant comprises a heterologous gene encoding a RNAi sequence of the present invention operably linked to an inducible promoter, and is grown either in the presence of the an inducing agent, or is grown and then exposed to an inducing agent. In still other embodiments of the present invention, a transgenic plant comprises a heterologous gene encoding a RNAi sequence of the present invention operably linked to a promoter which is either tissue specific or developmentally specific, and is grown to the point at which the tissue is developed or the developmental stage at which the developmentally-specific promoter is activated. Such promoters include seed and root specific promoters. In still other embodiments of the present invention, the transgenic plant comprises an RNAi sequence of the present invention operably linked to constitutive promoter. In further embodiments, the transgenic plants of the present invention express at least one double stranded RNA molecule at a level sufficient to reduce the proliferation of nematodes as compared to the proliferation of nematodes observed in a nontransgenic plant. Promoters. An estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, stradiolactivated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the 46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have little detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating trans gene expression in plants (Zuo, et al., Plant J. 2000 October; 24(2):265-73, herein incorporated by reference in its entirety).
(180) E. Vectors.
(181) In general, these vectors comprise a nucleic acid sequence encoding a RNAi silencing construct of the present invention (as described above) operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant. In one embodiment, the present invention contemplates a composition comprising a nucleic acid sequence encoding a tgd translation regulator of the present invention that is operatively linked to an appropriate promoter and inserted into a suitable vector for a particular transformation technique. Many methods may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are widely described in the art (See for example, Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., all of which are herein incorporated by reference in their entirety). Numerous transformation vectors are available for plant cell transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra (1982) Gene 19: 259; Bevan et al. (1983) Nature 304:184, all of which are herein incorporated by reference in their entirety), the bar gene which confers resistance to the herbicide phosphinothricin (White et al. (1990) Nucl Acids Res. 18:1062; Spencer et al. (1990) Theor. Appl. Genet. 79:625, all of which are herein incorporated by reference in their entirety), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann (1984) Mol. Cell. Biol. 4:2929, herein incorporated by reference in its entirety), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al. (1983) EMBO J., 2:1099, herein incorporated by reference in its entirety).
(182) In one embodiment, the present invention contemplates a composition comprising a nucleic acid sequence encoding a tgd RNAi construct of the present invention that is operatively linked to an appropriate promoter and inserted into a suitable vector for a particular transformation technique. Recombinant DNA, such as that described above, can be introduced into a plant cell in a number of ways. The choice of any specific method might depend on the type of plant targeted for transformation. In some embodiments, a vector is maintained episomally (i.e., for example, transient transformation). In other embodiments, a vector is integrated into the genome (i.e., for example, stable transformation). In one embodiment, any gene whose use in an RNAi expression vector results in the accumulation of oil in vegetative tissues such as leaf and stem is contemplated.
(183) In yet other embodiments, the nucleic acids of the present invention are utilized to construct vectors derived from plant (+) RNA viruses (i.e., for example, brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof). Generally, the inserted polynucleotide of the present invention can be expressed from these vectors as a fusion protein (for example, coat protein fusion protein) or from its own subgenomic promoter or other promoter. Methods for the construction and use of such viruses are described. U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785, all of which are incorporated herein by reference in their entirety.
(184) In some embodiments of the present invention the nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (WO 93/07278, herein incorporated by reference in its entirety).
(185) Numerous transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra (1982) Gene 19: 259; Bevan et al. (1983) Nature 304:184; all of which are herein incorporated by reference), the bar gene which confers resistance to the herbicide phosphinothricin (White et al. (1990) Nucl Acids Res. 18:1062; Spencer et al. (1990) Theor. Appl. Genet. 79:625; all of which are herein incorporated by reference), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann (1984) Mol. Cell. Biol. 4:2929; herein incorporated by reference), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al. (1983) EMBO J., 2:1099; herein incorporated by reference).
(186) In some embodiments, the vector is adapted for use in an Agrobacterium mediated transfection process (See e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are incorporated herein by reference). Construction of recombinant plasmids encoding a tgd RNAi construct in addition to recombinant Ti and Ri plasmids in general follows methods typically used with common bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with endogenous plasmids and sometimes constructed from foreign sequences. These may include but are not limited to structural genes for antibiotic resistance as selection genes.
(187) Exemplary systems of using recombinant plasmid vectors that are compatible with the present invention include, but are not limited to the cointegrate and binary systems. In the cointegrate system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic plasmid that contains both the cis-acting and trans-acting elements required for plant cell transformation as, for example, in the pMLJ1 shuttle vector and the non-oncogenic plasmid pGV3850. The second system is called the binary system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic plasmid PAL4404. These and other vectors useful for these systems are commercially available.
(188) In one embodiment, plant transformation was achieved by the floral dip method (Clough and Bent, 1998). Resistant seedlings were selected on MS medium containing 40 mg/mL of kanamycin.
(189) Recombinant DNA, such as that described above, can be introduced into a plant cell in a number of ways. The choice of any specific method might depend on the type of plant targeted for transformation. In some embodiments, a vector is maintained episomally (i.e., for example, transient transformation). In preferred embodiments, a vector is integrated into the genome (i.e., for example, stable transformation).
(190) F. Other Parts of Silencing Vector and Overexpression Vector Constructs.
(191) The expression cassettes for both RNAi and overexpression vector constructs may further comprise any sequences required for expression of the silencing sequences and heterologous sequence constructs. Such additional sequences include, but are not limited to, transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles, cell compartments, tissues, etc. A heterologous gene encoding a tgd silencing construct or overexpressed gene of the of the present invention, including variants or mutations of tgd RNAi constructs or overexpression vectors for WRI1 and F-Box, includes any suitable sequence of the inventions as described herein. Preferably, the tgd RNAi construct is provided within an expression vector such that transformation with the vector results in expression of the ds RNA for reducing translation of at least one type of TGD protein. In further embodiments, the heterologous gene is provided within an expression vector such that transformation with the vector results in expression of the heterologous protein, i.e. WRIK1 and/or F-Box polypeptide. Examples of suitable vectors are described herein.
(192) For example, adapters or linkers can be used to join (ligate) the DNA sequence fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.
(193) A variety of transcriptional terminators are available for use in expression of sequences using the promoters of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (See e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet., 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627; all of which are herein incorporated by reference). Another form of gene regulation for altering oil content is using and expressing a dominant negative mutation in plants, for example, see, European Patent Application EP0945514. Oil production was also modified by the use of a silencing construct that instead of interfering with endogenous transcription or translation revealed a dominant negative form of regulation.
(194) In addition, in some embodiments, constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Calais et al. (1987) Genes Develop. 1:1183; herein incorporated by reference). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
(195) In some embodiments of the present invention, the construct for expression of the nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Calderone et al. (1984) Cell 39:499; Lassoer et al. (1991) Plant Molecular Biology 17:229; all of which are herein incorporated by reference), a plant translational consensus sequence (Joshi (1987) Nucleic Acids Research 15:6643; herein incorporated by reference), an intron (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81; herein incorporated by reference), and the like, operably linked to the nucleic acid sequence encoding a TGD.
(196) G. Transformation Techniques.
(197) In some embodiments of the present invention the nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (WO 93/07278; herein incorporated by reference).
(198) Once a nucleic acid sequence encoding a tgd RNAi construct of the present invention is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.
(199) In some embodiments, the vector is introduced through ballistic particle acceleration (i.e. gene gun) using devices (e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.). (See e.g., U.S. Pat. No. 4,945,050; and McCabe et al. (1988) Biotechnology 6:923). See also, Weissinger et al. (1988) Annual Rev. Genet. 22:421; Sanford et al. (1987) Particulate Science and Technology, 5:27 (onion); Svab et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8526 (tobacco chloroplast); Christou et al. (1988) Plant Physiol., 87:671 (soybean); McCabe et al. (1988) Bio/Technology 6:923 (soybean); Klein et al. (1988) Proc. Natl. Acad. Sci. USA, 85:4305 (maize); Klein et al. (1988) Bio/Technology, 6:559 (maize); Klein et al. (1988) Plant Physiol., 91:4404 (maize); Fromm et al. (1990) Bio/Technology, 8:833; and Gordon-Kamm et al. (1990) Plant Cell, 2:603 (maize); Koziel et al. (1993) Biotechnology, 11:194 (maize); Hill et al. (1995) Euphytica, 85:119 and Koziel et al. (1996) Annals of the New York Academy of Sciences 792:164; Shimamoto et al. (1989) Nature 338: 274 (rice); Christou et al. (1991) Biotechnology, 9:957 (rice); Datta et al. (1990) Bio/Technology 8:736 (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (1993) Biotechnology, 11: 1553 (wheat); Weeks et al. (1993) Plant Physiol., 102:1077 (wheat); Wan et al. (1994) Plant Physiol. 104: 37 (barley); Jahne et al. (1994) Theor. Appl. Genet. 89:525 (barley); Knudsen and Muller (1991) Planta, 185:330 (barley); Umbeck et al. (1987) Bio/Technology 5: 263 (cotton); Casas et al. (1993) Proc. Natl. Acad. Sci. USA 90:11212 (sorghum); Somers et al. (1992) Bio/Technology 10:1589 (oat); Torbert et al. (1995) Plant Cell Reports, 14:635 (oat); Weeks et al. (1993) Plant Physiol., 102:1077 (wheat); Chang et al., WO 94/13822 (wheat) and Nehra et al. (1994) The Plant Journal, 5:285 (wheat); all of which are herein incorporated by reference.
(200) In other embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; PCT application WO 95/16783; all of which are herein incorporated by reference). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g., using biolistics or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al. (1990) PNAS, 87:8526; Staub and Maliga, (1992) Plant Cell, 4:39; all of which are herein incorporated by reference). The presence of cloning sites between these markers allowed creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga (1993) EMBO J., 12:601; all of which are herein incorporated by reference). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3-adenyltransferase (Svab and Maliga (1993) PNAS, 90:913; herein incorporated by reference). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of the RNAs encoded by the DNA molecule.
(201) In other embodiments, vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway (1985) Mol. Gen. Genet, 202:179; herein incorporated by reference). In still other embodiments, the vector is transferred into the plant cell by using polyethylene glycol (Krens et al. (1982) Nature, 296:72; Crossway et al. (1986) BioTechniques, 4:320; all of which are herein incorporated by reference); fusion of protoplasts with other entities, either mini cells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al. (1982) Proc. Natl. Acad. Sci., USA, 79:1859; herein incorporated by reference); protoplast transformation (EP 0292435; herein incorporated by reference); direct gene transfer (Paszkowski et al. (1984) EMBO J., 3:2717; Hayashimoto et al. (1990) Plant Physiol. 93:857; all of which are herein incorporated by reference).
(202) In still further embodiments, the vector may also be introduced into the plant cells by electroporation (Fromm, et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602; all of which are herein incorporated by reference). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus which are grown into whole plants.
(203) H. Agrobacterium-Mediated Plant Transformation.
(204) In some embodiments, the vectors comprising a nucleic acid sequence encoding a tgd RNAi construct or overexpression construct of the present invention are used in Agrobacterium-mediated transformation (Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd ed. Kluwer Academic Publ., Dordrecht 1995 in Sect., Ringbuc Zentrale Signatur: BT11-P; Glick, Bernard R. and Thompson, John E. Methods in Plant Molecular Biology and Biotechnology, S. 360, CRC Press, Boca Raton 1993; herein incorporated by reference). For example, Agrobacterium mediated transformation can be performed using the GV3 (pMP90) (Koncz and Schell 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Arabidopsis thaliana can be grown and transformed according to standard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1194-1199; Bent et al. 1994, Science 265:1856-1860). Additionally, rapeseed can be transformed with the constructs of the present inventions via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Use of antibiotic for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Additionally, Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al. (1994, Plant Cell Report 13:282-285). Alternatively, plants may be transformed in vivo, such as by transformation of a whole plant by Agrobacteria infiltration of adult plants, as in a floral dip method (Bechtold N, Ellis J, Pelletier G (1993) Cr. Acad. Sci. IIIVie 316:1194-1199; herein incorporated by reference). Transformation of soybean can be performed using for example a technique described in EP 0424 047, U.S. Pat. No. 5,322,783 (Pioneer Hi-Bred International) or in EP 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770 (University Toledo). Soybean seeds are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours. The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air-dried to moisture content less than 20% (fresh weight) in a sealed Petri dish until further use. The method of plant transformation is also applicable to Brassica and other crops. In particular, seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes, at room temperature with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. The seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period, the seeds lose approximately 85% of their water content. The seeds are then stored at room temperature in a sealed Petri dish until further use. Agrobacterium tumefactions culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and re-suspended in MS (Murashige and Skoog, 1962) medium supplemented with 100 M acetosyringone. Bacteria cultures are incubated in this pre-induction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 44% moisture content were imbibed for 2 hours at room temperature with the pre-induced Agrobacterium suspension culture. (The imbibition of dry embryos with a culture of Agrobacterium was also applicable to maize embryo axes). The embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at room temperature. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to kill the Agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25 C., under 440 mol m.sup.2sec.sup.1 and 12 hours photoperiod. Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favor the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25 C., under 440mol m-2 sec-1 and 12 hours photoperiod for about 80 days. Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization wherein DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labeled probe by PCR, and used as recommended by the manufacturer.
(205) I. Host Plants.
(206) Host cells, tissues and plants that are transformed with a heterologous recombinant silencing gene encoding a tgd gene or tgd gene fragment of the present invention include, but are not limited to, those plant organisms that naturally express oils comprising triacylglycerols (TAGs) and those plant organisms in which it is commercially feasible to grow for harvesting large amounts of the TAG products. The methods of the present invention are not limited to any particular plant, including monocotyledons (monocots) and dicotyledons (dicots). Such plant organisms include but are not limited to, oleaginous plants. Examples of oleaginous plants include oil seed-producing plants, such as soybean, rutabaga, rapeseed (Brassica napus L. and B. campestris (canola)), sunflower, cotton, corn, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut, rice, etc. Many commercial cultivars of these plants, i.e. elite cultivars, may be transformed with heterologous recombinant silencing genes of the present inventions. In a preferred embodiment, a host plant is a mustard family (Brassicaceae; Cruciferae) plant, such as Arabidopsis thaliana, Brassica napus L. and B. campestris plants, rutabaga plants, rape plants, field cabbage plants, for harvesting oils, such as rape oil, canola oil, mustard oil, oil of Colza, and the like. In a preferred embodiment, a host organism comprises a heritable heterologous recombinant silencing gene passed on to progeny plants. In another aspect, the invention provides transgenic plant cells comprising any of the genetic constructs described herein. In certain useful embodiments, the transgenic plant cell is Arabidopsis thaliana. In some embodiments, the plant cells is a cell from an oil-producing agricultural crop such as soybean, palm, rapeseed (canola), safflower, grape seed or sunflower. In other embodiments, the transgenic plant cell is an agricultural crop plant, such as corn or rice.
(207) In one embodiment, the inventors contemplate inserting vectors comprising heterologous transgenic tgd RNAi silencing genes into a plant host cell and plant host tissue. A heterologous recombinant silencing gene comprising a tgd gene or tgd gene fragment of the present invention, including variants or mutations of a tgd gene or tgd gene fragment, includes any suitable tgd sequence that results in accumulation of oil in plant cells, plant leaf tissue and plant stem tissue in addition to the entire plant. Whole plants will be grown from transformed host cells or tissues, for example, from protoplast cells, such as hypocotyls protoplasts, cotyledons, shoots, embryo, plantlet meristem tissue, shoots, in vitro microspore culture, and the like. In one embodiment, the inventors contemplate inserting a silencing vector of the present invention comprising a heterologous transgenic tgd RNAi silencing gene into any plant cell or plant tissue capable of being grown into a first generation whole plant, for example, mesophyllprotoplasts, hypocotyl explants, cotyledon explants, an embryo, callus, embryogenic callus, a plantlet, and the like.
(208) Preferably, the recombinant-silencing gene comprising a tgd gene is provided within an expression vector comprising a promoter in operable combination with the tgd silencing gene such that transformation with the vector results in expression of the silencing gene. In some embodiments, expression is contingent upon induction of the prompter. In preferred embodiments, the silencing gene constructs are integrated into the plant genome. Suitable vectors are described herein.
(209) In yet other embodiments of the present invention, a transgenic organism (i.e., for example, a transgenic plant cell, plant seed, plant tissue or plant seedling) comprises a gene encoding a heterologous recombinant silencing gene of the present invention operably linked to an inducible promoter, is grown either in either the presence or absence of the inducing agent and/or inducing environmental condition (i.e., for example, where the inducing agent is a chemical), or is germinated or grown into a seedling or mature plant and then exposed to an inducing agent. In still other embodiments of the present invention, a transgenic organism comprises a gene encoding a heterologous recombinant silencing gene comprising a tgd gene of the present invention is operably linked to a promoter which is either species, cell, and/or tissue specific or developmentally specific, and is grown to the point at which the organism is developed or the developmental stage at which the developmentally-specific promoter is activated. Such promoters include, but are not limited to, tissue specific promoters.
(210) In some embodiments, when gene expression is silenced, i.e. reduced, one result is increased oil content of a plant tissue. It is not intended to limit the type of plant tissue. Indeed, a variety of plant tissues are contemplated for use for producing oil of the present inventions including but not limited to ground tissue, parenchyma tissue, leaf tissue, petiole tissue, stem tissue, stalk tissue, shoot tissue, epidermal tissue, dermal tissue, phloem tissue, vascular tissue, pith tissue, photosynthetic tissue, lamina tissue, chlorenchyma tissue, mesophyll tissue, stoma tissue, etc. In some embodiments, the invention provides an RNAi expression vector for silencing a gene in a cell. In particular for silencing genes for altering oil biosynthesis. In some embodiments, the silenced gene increases oil content of a plant cell. It is not intended to limit the type of plant cell used for increasing oil content. Indeed, a variety of plant cells are contemplated for producing oil of the present inventions including but not limited to a meristern cell, a mesophyll cell, a spongy mesophyll cell, a protoplast cell, a dermal cell, an epidermal cell, a parenchyma cell, collenchyma, a sclerenchyma cell, a companion cell, a guard cell, a subsidiary cell, an epidermal hair cell (a trichome), etc. In one embodiment, a plant cell for use in the present inventions comprises a plastid. In one embodiment, a plant cell for use in the present inventions comprises a chromoplast. In one embodiment, a plant cell for use in the present inventions comprises a leucoplast, such as a leucoplast exposed to light. In a preferred embodiment, a plant cell for use in the present inventions comprises chlorophyll. In another preferred embodiment, a plant cell for use in the present inventions comprises a chloroplast. In a further embodiment, the silencing genes result in an increase of production of fatty acids. In a further embodiment, increase of production of fatty acids refers to an increase (accumulation) of a fatty acid including but not limited to a fatty acid for use in a biofuel. Indeed, a fatty acid accumulated by the compositions and methods of the present inventions include but are not limited to monogalactosyldiacylglycerol (MGDG), monogalactosyl-diacylglycerol (MGDG) (sn-2) trigalactosyldiacylglycerol (TGDG), triacylglycerol (TAG) and triacylglycerol (TAG) 18:0, diacylglycerol (DAG), alpha-naphthol straining fatty acid, trigalactosyldiacylglycerol (triGDG), digalactosyl diglycerides (DGDG), digalactosyl diglycerides (DGDG) (sn-2), SQDG tetragalactosyldiacylglycerol (TetraGDG), specifically 16:0, 16:1, 18:0 18:1. In one embodiment, a transgenic organism (i.e., for example, a transgenic Brassicaceae plant cell, plant seed, plant tissue or plant seedling) is grown under conditions sufficient to effect increased production of TAGs in vegetative tissues.
(211) J. Growing and Breeding Plants of the Present Inventions.
(212) The plant life cycle for oil production with this invention is 4 to 6 weeks in a laboratory environment. It takes 4 weeks for the plants to form canopies, at which time alteration of a TGD gene occurs. The promoter system controlling the TGD gene function is inducible by using estrogen. Disruption of the genes prior to plant growth and development is harmful to the health of the plant, and causes the plant to turn yellow. Hence, the plant must be harvested after the oil accumulates in the leaf and stem structures. Harvesting is contemplated 1 to 2 weeks after disruption of the TGD gene.
(213) 1. Growing Transformed Cells into Plants.
(214) After selecting for transformed plant material that can express the heterologous gene encoding a tgd RNAi construct of the present invention, whole plants are regenerated. Plant regeneration from cultured protoplasts is described in Evans et al. (1983) Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co. New York); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. III (1986); herein incorporated by reference. It is known that many plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, and monocots (e.g., the plants described above). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.
(215) Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.
(216) Transgenic lines are established from transgenic plants by tissue culture propagation. The presence of nucleic acid sequences encoding a tgd RNAi construct of the present invention (including mutants or variants thereof) may be transferred to related varieties by traditional plant breeding techniques. In some embodiments of the present invention, a plant oil comprising TAG is produced in vivo, by providing an organism transformed with a heterologous gene encoding a tgd RNAi construct of the present invention and growing the transgenic organism under conditions sufficient to effect production of TAGs.
(217) In other embodiments of the present invention, a plant oil comprising TAG is produced in vivo by transforming an organism with a heterologous gene encoding a tgd RNAi construct of the present invention and growing the transgenic organism under conditions sufficient to effect production of TAGs.
(218) 2. Breeding Transgenic Plants.
(219) After each first generation transgenic plant is constructed, progeny from each of the first generation transgenic plants are bred, for example as in cross-breeding by sexual fertilization to produce second generation transgenic plants comprising various combinations of both a silencing construct, and in some embodiments an overexpression vector, and commercially desired traits. In a further embodiment, transgenic plants with one silencing gene are cross-bred to transgenic plants comprising other silencing genes. For example, various combinations of progeny from the first generation transgenic plants are crossbred to other corn plants in order to produce second generation transgenic showing a reduction in oil content. Progeny of the second generation (F.sub.2) transgenic plants are cross-bred by man using sexual fertilization to other F.sub.2 generation plants or with first generation transgenic plants to produce third generation transgenic plants that contain one or more silenced genes, or combinations thereof. For example, in one embodiment, cross-breeding a second generation transgenic plant with a reduction in tgd expression with another second generation transgenic plant with a reduction in tgd expression produces a third generation transgenic plant with reduced tgd expression that is economically desirable for (accumulating) oil in vegetative tissues. In other embodiments, transgenic plants with various combinations of reduced oil enzymes can be made by cross-breeding progeny from a particular transgenic plants. For example, Zhang et al, Theor. Appl. Genet. 92: 752-761, (1996), Zhong et al, Plant Physiol. 110:1097-1107, (1996), and Zhong et al, Planta, 187:483-489, (1992), herein incorporated by reference, provide methods for making transgenic plants by sexual fertilization.
(220) Plants provided by the present inventions are contemplated for use in breeding to other plants, for producing commercial plant lines have increased oil in vegetative tissues along with other desired agronomic traits, such as insect resistance, pathogen resistance, drought resistance, cold resistance, fertility, etc.
(221) Alternatively, plant material is transformed as above with a plasmid containing a heterologous RNAi expression cassette encoding a tgd-silencing construct. The transgenic plant is grown from the transformed plant material. Next, plant material from this transgenic plant is transformed with a second plasmid containing a heterologous expression cassette encoding a different tgd silencing construct and a second selectable marker for stacking tgd-silencing constructs. The double transgenic plant is then grown from the transformed plant material. Such that any desired combination of silenced genes in transgenic plants are contemplated. In a preferred embodiment, the above heterologous RNAi sequence expression cassettes further include therein nucleotide sequences that encode one or more selectable markers that enable selection and identification of transgenic plants that express the modified oil of the present invention. In some embodiments, the selectable markers confer additional benefits to the transgenic plant such as herbicide resistance, insect resistance, and/or resistance to environmental stress.
(222) Alternatively, the above transformations are performed by co-transforming the plant material with a first plasmid containing a gene expression cassette encoding a selectable marker and a second plasmid containing a heterologous RNAi sequence expression cassette encoding a silencing construct. The advantage of using a separate plasmid is that after transformation, the selectable marker can be removed from the transgenic plant by segregation, which enables the selection method for recovering the transgenic plant to be used for recovering transgenic plants in subsequent transformations with the first transgenic plant. Examples of preferred markers that provide resistance to herbicides include, but are not limited to, the bar gene from Streptomyces hygroscopicus encoding phosphinothricin acetylase (PAT), which confers resistance to the herbicide glufonsinate; mutant genes which encode resistance to imidazalinone or sulfonylurea such as genes encoding mutant form of the ALS and AHAS enzyme as described by Lee at al. EMBO J. 7: 1241 (1988) and Miki et al., Theor. Appl. Genet. 80: 449 (1990), respectively, and in U.S. Pat. No. 5,773,702, all of which are herein incorporated by reference genes which confer resistance to glycophosphate such as mutant forms of EP SP synthase and aroA; resistance to L-phosphinothricin such as the glutamine synthetase genes; resistance to glufosinate such as the phosphinothricin acetyl transferase (PAT and bar) gene; and resistance to phenoxy propionic acids and cyclohexones such as the ACCAse inhibitor-encoding genes (Marshall, et al. Theor. Appl. Genet. 83: 435 (1992), herein incorporated by reference). The above list of genes which can import resistance to an herbicide is not inclusive and other genes not enumerated herein but which have the same effect as those above are within the scope of the present invention. Examples of preferred genes which confer resistance to pests or disease include, but are not limited to, genes encoding a Bacillus thuringiensis protein such as the delta-endotoxin, which is disclosed in U.S. Pat. No. 6,100,456, herein incorporated by reference; genes encoding lectins, (Van Damme et al., Plant Mol. Biol. 24: 825 (1994), herein incorporated by reference); genes encoding vitamin-binding proteins such as avidin and avidin homologs which can be used as larvicides against insect pests; genes encoding protease or amylase inhibitors, such as the rice cysteine proteinase inhibitor (Abe, et al., J. Biol. Chem. 262: 16793 (1987), herein incorporated by reference) and the tobacco proteinase inhibitor I (Hubb, et al., Plant Mol. Biol. 21:985 (1993)); genes encoding insect-specific hormones or pheromones such as ecdysteroid and juvenile hormone, and variants thereof, mimetics based thereon, or an antagonists or agonists thereof; genes encoding insect-specific peptides or neuropeptides which, upon expression, disrupts the physiology of the pest; genes encoding insect-specific venom such as that produced by a wasp, snake, etc.; genes encoding enzymes responsible for the accumulation of monoterpenes, sesquiterpenes, asteroid, hydroxamine acid, phenylpropanoid derivative or other non-protein molecule with insecticidal activity; genes encoding enzymes involved in the modification of a biologically active molecule (see U.S. Pat. No. 5,539,095 to Sticklen et al., which discloses a chitinase that functions as an anti-fungal); genes encoding peptides which stimulate signal transduction; genes encoding hydrophobic moment peptides such as derivatives of Tachyplesin which inhibit fungal pathogens; genes encoding a membrane permease, a channel former or channel blocker (for example, cecropin-beta lytic peptide analog renders transgenic tobacco resistant to Pseudomonas solanacerum) (Jaynes, et al. Plant Sci. 89:43 (1993), herein incorporated by reference); genes encoding a viral invasive protein or complex toxin derived therefrom (viral accumulation of viral coat proteins in transformed cells of some transgenic plants impart resistance to infection by the virus the coat protein was derived as shown by Beachy, et al. Ann. Rev. Phytopathol. 28:451 (1990); genes encoding an insect-specific antibody or antitoxin or a virus-specific antibody (Tavladoraki, et al. Nature 366: 469 (1993), herein incorporated by reference); and genes encoding a developmental-arrestive protein produced by a plant, pathogen or parasite which prevents disease. The above list of genes which can import resistance to disease or pests is not inclusive and other genes not enumerated herein but which have the same effect as those above are within the scope of the present invention. Examples of genes which confer resistance to environmental stress include, but are not limited to, mtld and HVA1, which are genes that confer resistance to environmental stress factors; rd29A and rd19B, which are genes of Arabidopsis thaliana that encode hydrophilic proteins which are induced in response to dehydration, low temperature, salt stress, or exposure to abscisic acid and enable the plant to tolerate the stress (Yamaguchi-Shinozaki, et al., Plant Cell 6: 251-264 (1994)).
(223) 3. Evaluation of Seed Viability/Fertility.
(224) An exemplary method for determining viability, i.e. germination levels of seeds from plants of the present inventions is contemplated as a Warm Germination Test. Seeds may be placed in a row on a piece of wet filter paper, covered by another piece of wet filter paper, rolled up, then wrapped with a piece of waxed paper and placed in a large beaker with 1 inch of water at the bottom. The beaker is incubated in a growth chamber at 25 C. Seed germination is evaluated at day 5 of incubation.
(225) 4. Evaluation of Agronomic Traits by Field Trials.
(226) In one embodiment, the transgenic plants are evaluated for agronomic performance associated with a silenced tgd gene. Agronomic field trials may be conducted as two row plots planted and thinned to a density of 30,000 plants per acre. Field trials are contemplated for being conducted as two-row plots seeded at nine seeds per foot in 12-foot rows with a 3-foot border on 30-inch-row spacing. Seed may be sampled from every plot and submitted for proximate analysis using near-infrared transmittance. Oil and protein data may be reported on a dry matter basis. Outlier analysis based on deleted studentized residuals using Statistical Analysis Software (SAS) may be performed on data prior to statistical analysis. Statistical analyses may be run using mixed model procedures in SAS. The analysis may be performed using a split-plot model to compare positive to negative isolines within the transgenic event. The analysis may be run across locations, with locations, replications within locations, and their interactions with the fixed effects considered random effects.
(227) K. Harvesting/Isolating Oils from Plants of The Present Inventions.
(228) In some embodiments of the present invention, the methods for accumulating oils in vegetative tissues, i.e. including TAGs, further comprise isolating the oils and TAGs produced. Several methods have been reported, and include harvesting the transgenic plants and extracting TAGs (see, for example, Christie, (1982) Lipid Analysis. 2nd Edition (Pergamon Press, Oxford); and Kates, (1986) Techniques of Lipidology (Elsevier, Amsterdam)). Extraction procedures preferably include solvent extraction, and typically include disrupting cells, as by chopping, mincing, grinding, and/or sonicating, prior to solvent extraction. In one embodiment, lipids are extracted from the tissue according to the method of Bligh and Dyer (1959) (Can J Biochem Physiol 37: 911-917). In yet other embodiments of the present invention, the TAGs are further purified, as for example by thin layer liquid chromatography (TLC), gas-liquid chromatography, counter current chromatography, high performance liquid chromatography, and the like.
(229) In one exemplary embodiment, leaf lipid extracts of the wild type and the tgd1-1 and tgd2-1 mutants were compared by TLC. In the tgd2-1 sample a lipid staining positive for sugar and co-chromatographing with authentic trigalactolipid of tgd1-1 is present. See,
(230) In summary, for exemplary plants such as Arabidopsis thaliana plants, tgd-1, 2, and 3 mutant genes were shown to contribute significantly to TAG accumulation in leaves of TAGs through ER mechanisms using a variety of mutant tgd gene genotypes with a range of phenotypes. In general, tgd mutants were impaired in the ER-pathway while showing an increased 16-carbon-to-18-carbon fatty acid ratio in their galactolipids. (Awai (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 10817-10822). This was particularly visible for digalactosyldiacylglycerol, which is to a large extent derived from the ER-pathway (Heinz, (1983) Plant Physiol. 72:273-279). Accordingly, for example, the 16-carbon-to-18-carbon ratio for the digalactolipid increased from 0.29 to 0.76 in the tgd3 mutant (Table 3). This phenomenon was contemplated as due to the substrate specificities of the different acyltransferases in the plastid and the ER leading to 18-carbon fatty acids at the sn-1 position and 16-carbon fatty acids at the sn-2 position of the diacylglycerol backbone for plastid derived lipids. Those lipids derived from the ER-pathway carry 18-carbon fatty acids in both positions (Heinz, (1983) Plant Physiol. 72:273-279). Positional analysis using Rhizopus lipase confirmed an increase in 16-carbon fatty acids in the sn-2 position of monogalactosyldiacylglycerol and digalactosyldiacylglycerol of the tgd3 mutant.
(231) Processes for extracting plant oils from seeds for biofuel feedstock are well known and practiced. The inventors contemplate using an oil extraction process for leaves and stems similar to the process used for extracting oil from peppermint leaves. In an exemplary embodiment, vegetative oils and seed oil may be isolated. Starting with 50 g of ground plant tissues or seeds suspended in buffer, centrifugation (20,000 g, 30 min) yielded a floating fat pad, liquid fraction containing soluble and membrane-associated proteins, and a pellet.
(232) VI. Nucleic Acid and Protein Detection.
(233) In some embodiments, the inventors contemplate monitoring the expression of a gene silencing construct of the present inventions in a plant cell, plant tissue or plant seed. In some embodiments, the expression of the gene-silencing construct is by integration of a marker gene in the construct. In some embodiments, the residual expression of the TGD protein in monitored.
(234) Leaves of developing T1 (F1) plants may be analyzed in a PCR-based assay designed to quantitate the amount of a specific DNA target to determine if the plants were homozygous, heterozygous, or null for the trans gene. Events where the transgene and the selectable marker are linked (closely co-localized on the same chromosome) can be identified by Southern analysis.
(235) A. Detection of DNA and RNA.
(236) In some embodiments, the inventors contemplate monitoring the level of gene silencing by determining the amount of expressed TGD genes. DNA and mRNA expression may be measured by any suitable method, including but not limited to, those disclosed below. In some embodiments, RNA is detection by Northern blot analysis. Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe.
(237) In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or cDNA using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. In some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978 (each of which is herein incorporated by reference) is utilized. Exemplary, semi-quantitative RT-PCR analysis of TGD3 mRNA levels in wild-type (Col-2) and tgd3 plants,
(238) B. Detection of TGD Protein.
(239) In some embodiments, the inventors contemplate monitoring the level of gene silencing by determining the amount of expressed TGD proteins. Thus, in some embodiments, gene expression may be detected by measuring the expression of a protein or polypeptide. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by immunohistochemistry. In other embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is described below. In one example, a polyclonal antibody against TGD2 was made and used for detecting TGD2, Awai, et al., 2006. Similar methods are contemplated for making antibodies for detecting TGD1, TGD3, and TGD4.
(240) Antibody binding may be detected by many different techniques including, but not limited to, (e.g., immunoelectron microscopy, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
(241) In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled.
(242) VII. Summary.
(243) While attempting to overcome limitations from using mutant tgd genes in plants for increasing accumulated oil in vegetative tissues, the inventors developed at least two types of RNAi tgd gene fragment constructs. One under control of a constitutive promoter and one under control of an inducible promoter along with methods comprising this RNAi construct for controlling the initiation of oil accumulation in leaves, stems and shoots. Thus in some embodiments, compositions and methods are used to produce whole plants, such as seedlings, immature, mature and senescing plants, whose vegetative tissues have accumulated oils, including accumulated TAGs. In some embodiments, plants having accumulated oils are plants having reduced TGD3 protein. In other embodiments, plants having accumulated oils in vegetative tissues are plants have reduced TGD4 protein. In yet other embodiments, plants having accumulated oils in vegetative tissues have reduced TGD2. In yet further embodiments, plants having accumulated oils in vegetative tissue are plants having reduced TGD1. In even further embodiments, having accumulated oils in vegetative tissues have more than one TGD protein that is reduced. The engineered plants of the present inventions are contemplated for use in a variety of ways, including but not limited to increasing the total amount of oil harvested from an individual plant since harvesting oil from vegetative tissues in addition to seeds. Additional advantages of making and using engineered plants would be the added flexibility of growing these plants for a shorter amount of time since oil produced in vegetative tissues were harvested before the plant matured and produced seeds. Thus growing and harvesting oil in young plants would reduce the overall cost of growing the plants. Further, the inventors contemplate growing several crops per growing season instead of one crop due to the early production of vegetative oils before seeds are ready for harvesting. Further, engineered plants having reduced production of TGDs would produce higher levels of TAGs in younger plants. The inventors further contemplate the following additional benefits of using the compositions and methods of the present inventions: enables more efficient feedstock crops per plant, increases the capacity of feedstock crops to store oil, hence, more oil can be produced, stored, and harvested per crop acreage, thus reducing the cost of biodiesel feedstock, while increasing efficiencies gained from higher oil yielding crops reduce oils costs and translate into lower biodiesel costs, technology is stackable and compatible with other transgenic crop technologies, and technology can be coupled with other transgenic crop technologies that aim to provide more advantageous feedstock. The oil obtained from vegetative tissues and seeds of engineered plants is contemplated for several uses including but not limited to biodiesel production, biofuels, and bioplastics production in manufacturing plants.
EXPERIMENTAL
(244) The following examples serve to illustrate certain embodiments and aspects of the present invention and are not to be construed as liming the scope thereof. In the experimental disclosures which follow, the following abbreviations apply: N (normal); M (molar); mM (millimolar); M (micromolar); mol (moles); mmol (millimoles); mol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); g (micrograms); ng (nanograms); pg (picograms); L and l (liters); ml (milliliters); l (microliters); cm (centimeters); mm (millimeters); m (micrometers); nm (nanometers); U (units); min (minute); s and sec (second); k (kilometer); deg (degree); C. (degrees Centigrade/Celsius), and polymerase chain reaction (PCR).
Example I
(245) This example describes exemplary materials and methods used during the development of the present inventions.
(246) Briefly, exemplary lipid assays were used for analyzing lipid content in lipids extracted from Col-2 wild type, seeds and plants of the present inventions. In some examples, tgd mutant seedlings were grown on Murashige and Skoog (MS) medium with 1% sucrose. In particular, fatty acid methylesters were prepared as described by Drmann (Plant Cell 7:1801-1810 (1995), herein incorporated by reference in its entirety) and quantified by gas chromatography according to Rossak, et al. (Arch. Biochem. Biophys. 340:219-230 (1997), herein incorporated by reference in its entirety). Polar lipids were analyzed as described by Drmann (Plant Cell 7:1801-1810 (1995), herein incorporated by reference in its entirety) on activated ammonium sulfate-impregnated silica gel TLC plates (Si250PA; Mallinckrodt, Baker, N.J.) developed with chloroform/methanol/acetic acid/water (85/25/10/4, v/v). Neutral lipids were separated on untreated TLC plates developed with petroleum ether/ether/acetic acid (80/20/1, v/v). Lipids were visualized by brief exposure to iodine vapor or staining with -naphthol to detect glycolipids (Benning, et al., Arch. Biochem. Biophys. 317:103-111 (1995), herein incorporated by reference in its entirety). The fatty acid compositions at the sn-2 position of individual lipids were determined using Rhizopus lipase (Sigma, St. Louis, Mo.) digestion according to (Siebertz, et al., Naturforsch. 32:193-205 (1977), herein incorporated by reference in its entirety), with modifications as described in (Miguel, et al., Plant Physiol. 117, 923-930 (1998), herein incorporated by reference in its entirety). Fatty acid methyl esters were formed from lyso-lipids, and the fatty acid methylesters were quantified by gas chromatography.
(247) Exemplary mass spectrometry methods were used to characterize lipid extracts using liquid chromatography/mass spectrometry (LC/MS) on a Waters LCT Premier time-of-flight mass spectrometer equipped with Shimadzu LC-20AD pumps and SIL-5000 autosampler. Extracts were analyzed using V-mode operation and electrospray ionization in both positive and negative ion modes similar to described protocols (Houjou, et al., Rapid Commun. Mass Spectrom. 19:654-666 (2005), herein incorporated by reference in its entirety) except that, in place of MS/MS spectra, mass spectra were acquired in alternating acquisition functions at low (15 V) and high (75 V) potentials on aperture one to generate spectra with, and without in-source collision-induced dissociation (CID). The latter conditions allow for production of characteristic fragment ions that support structure assignments. HPLC separations were performed using a Restek Allure C18 column (1150 mm) using a ternary gradient based on: (a) 10 mM aqueous ammonium acetate, (b) methanol, and (c) 2-propanol.
Example II
(248) This example describes exemplary compositions and methods for making RNAi constructs of the present inventions.
(249) Various tgd RNAi silencing constructs were made using a 5 fragment of a cDNA coding region of TGD. Specifically, the inventors designed RNAi sequences for silencing a tgd gene, i.e. a sense tgd fragment (for examples, see, Table 6A) and antisense tgd fragment (for examples, see, Table 6B) attached with a spacer sequence, for example, a fragment of a tgd3 intron, see, Table 6C. Examples of TGD RNAi silencing constructs examples, see, Table 6C.
(250) TABLE-US-00021 TABLE6A Exemplarytgdsensegenesequencesforuse intgdgenesilencingaspartofRNAiconstructsequences. Exemplarysequenceforuseingenesilencing Targetgene vectors:Sensesequence,i.e. SEQ forsilencing: whenpromoterislocated5:5-3 IDNO: TGD4 ATGAACAGAATGAGATGGGTCGGAGAGGGAGACATCTGGG 12 AT3G06960 ACCTCGATATGTCAACTCCGGTGACGCTCGAGGGCACCGC ACGAGCTGTTCCTGACGATCCTCTTCCTCTAGGTCTCTCT AGAGGCACTCGTCTATCTCGCCCTAAGCAAGTTGAGTTCT TCCACCGCTTCATGGCCTCACCTCTCATCCCTTCCTTCTC CCCTATCCGTCCCAACACCGGAGATGGAGGCGGTGGTGGA TTCTCTCTTCAAAGAGTCCTCACTCTTCCTTTCTCCAACA ACTGGCTTGTGTCTCTTCTGGGCCAATTCGATGTTCAGAG ATTCGTAACGGAGATAGATAAGACTAAAGCTTTTGGTCGA GGGTCTTCGTCTACAGTAGCTTCTCGTTTAAACACAATTG GCAAGCATTTGAAGGATAAATCTTTGTACGCATTGGGTTT TTGTTCTGAGTTTTTGTTATCACCAGATGATACTTTGCTT CTTAGCTATGATGCTTACAA TGD4PDE320 ATGAACAGAATGAGATGGGTCGGAGAGGGAGACATCTGGG 13 (PIGMENT ACCTCGATATGTCAACTCCGGTGACGCTCGAGGGCACCGC DEFECTIVE ACGAGCTGTTCCTGACGATCCTCTTCCTCTAGGTCTCTCT 320) AGAGGCACTCGTCTATCTCGCCCTAAGCAAGTTGAGTTCT TCCACCGCTTCATGGCCTCACCTCTCATCCCTTCCTTCTC CCCTATCCGTCCCAACACCGGAGATGGAGGCGGTGGTGGA TTCTCTCTTCAAAGAGTCCTCACTCTTCCTTTCTCCAACA ACTGGCTTGTGTCTCTTCTGGGCCAATTCGATGTTCAGAG ATTCGTAACGGAGATAGATAAGACTAAAGC TGD3 AACGAAAAATGGCAATGTGACTCACTCAATCGGTGACTCG 14 ATNAP11 CTATAGTCTGTGAAGAAAGGCCAATTTCGCCATAAAGTTC ATNAP11 ACACCTTTGATCTCCTTTGTTTCTGGGTTTCTCCTAAATC (Arabidopsis ATCCAAATTGGTATCGAATTTGCCCTTCTCCGATTCAATT thaliananon- TCTTCACGATCTCAA intrinsicabc protein11) TGD2 ATGATTGGGAATCCAGTAATTCAAGTTCCATCATCACTAA 15 AT3G20320 TGCCATCATCCTCCATGATTGCTTGTCCTCGAGTTTCACC CAATGGGGTTCCTTATCTTCCACCAAAACCTAGAACTAGG CATTTAGTGGTCAGAGCTGCATCCAATTCCGATGCTGCTC ATGGTCAACCATCGTCTGATGGGGGGAAGAATCCTCTCAC CGTTGTTTTGGATGTGCCCAGGAATATATGGAGACAGACT TTAAAACCTTTGAGTGATTTTGGGTTTGGTAAGAGAAGTA TTTGGGAAGGTGGTGTTGGTTTGTTTATTGTCTCTGGAGC TACTCTTCTTGCTCTTAGCTGGGCTTGGTTGCGAGGTTTT CAAATGCGGTCGAAGTTTAGGAAATATCAGACTGTGTTTG AGCTTAGTCATGCTTCTGGTATTTGCACGGGAACACCGGT TAGGATCCGTGGGGTTACTGTTGGTACGATTATCCGTGTT AATCCTTCCTTGAAGAATAT TGD2 CCTAGAACTAGGCATTTAGTGGTCAGAGCTGCATCCAATT 16 CCGATGCTGCTCATGGTCAACCATCGTCTGATGGGGGGAA GAATCCTCTCACCGTTGTTTTGGATGTGCCCAGGAATATA TGGAGACAGACTTTAAAACCTTTGAGTGATTTTGGGTTTG GTAAGAGAAGTATTTGGGAAGGTGGTGTTGGTTTGTTTAT TGTCTCTGGAGCTACTCTTCTTGCTCTTAGCTGGGCTTGG TTGCGAGGTTTTCAAATGCGGTCGAAGTTTAGGAAATATC AGACTGTGTTTGAGCTTAGTCATGCTTCTGGTATTTGCAC GGGAACACCGGTTAGGATCCGTGGGGTTACTGTTGGTACG ATTATCCGTGTTAATCCTTCCTTGAAGAATATTGAAGCTG TTGCTGAGATAGAAGATGA TGD1 ATGATGCAGACTTGTTGTATCCATCAATCGTTTTGTTTCC 17 CTCATAGAGTCTTTCCACGGTTTGATGCTTCGATTGGTAT TAAGCCCCCAAAGCTTTGTCAAGTTGGTTTCATTGGAAAG ACTCAATCTTATGGGATTTCAAGTCCGATACGGCAAAGAA GATTATATGTGAATTTGAATGCTAATGATGGTCACCCATC CATGTCTATGTTGGAAGAAGAAACCTCTACTGAAAACAAC GCACCCAGTCAAGAAGCCGAGCTTCCGTTCAGCAAATGGT CACCTTCTAAGTACATATGGAGAGGTTTATCAGTTCCTAT TATAGCAGGACAAGTCGTTCTCCGGATTTTAAAGGGTAAG ATTCACTGGAGAAACACTCTTCAACAGCTGGAGAGAACCG GACCGAAATCTCTAGGAGTTTGTCTTCTGACTTCTACATT TGTTGGTATGGCTTTCACAATCCAGTTCGTTAGAGAATTC ACTAGACTAGGTCTAAACAG TGD1 TGTGTGTTGTTGTTGTTGGCACTGTGCCACTTTCTCTCTC 18 GATGAACCCTCTCAAGCAAGCTTCTTCGATCTTCCGAGCT TAGTTTCGTTTCTAAATTAGAGATTTCACCTAGATTGGTC CGTACATATCTTATACTGGGATTCGAATTTGGCTGCCTCA GAGTCAGAGATTGATTAATTGATCAGATTCAGCTGTTGAA ATCGTGCTTATTGCTACAAATTGAGAGGCACTAAATCAGT GAGGTCGTAAAGAAGAAGGCAACCACAATGATGCAGACTT GTTGTATCCATCAATCGTTTTGTTTCCCTCATAGAGTCTT TCCACGGTTTGATGCTTCGATTGGTATTAAGCCCCCAAAG CTTTGTCAAGTTGGTTTCATTGGAAAGACTCAATCTTATG GGATTTCAAGTCCGATACGGCAAAGAAGATTATATGTGAA TTTGAATGCTAATGATGGTCACCCATCCATGTCTATGTTG GAAGAAGAAACCTCTACTGAAAACAACGCACCCAGTCAAG AAGCCGAGCTTCCGTTCAGCAAATGGTCACCTTCTAAGTA CATATGGAGAGGTTTATCAGTTCCTATTATAGCAGGACAA GTCGTTCTCCGGATTTTAAAGGGTAAGATTCACTGGAGAA ACACTCTTCAACAGCTGGAGAGAACCGGACCGAAATCTCT AGGAGTTTGTCTTCTGACTTCTACATTTGTTGGTATGGCT TTCACAATCCAGTTCGTTAGAGAATTCACTAGACTAGGTC TAAACAGATCCATTGGAGGTGTCTTGGCTTTAGCCTTCTC TAGAGAGCTAAGTCCAGTCATCACATCGATTGTTGTTGCT GGACGAATGGGAAGTGCATTTGCAGCTGAACTAGGGACAA TGCAAGTCTCAGAGCAAACTGATACACTCCGTGTTTTAGG AGCTGACCCAATTGATTATCTAATCACTCCAAGAGTCATC GCCTCGTGTTTGGCTCTACCGTTTCTGACACTCATGTGTT TCACTGTTGGTATGGCTTCAAGCGCTCTGCTCTCTGATGC AGTTTACGGGATCAGCATTAACATAATCATGGACTCGGCT CACCGAGCACTTAGACCATGGGACATTGTGAGTGCCATGA TTAAATCTCAAGTCTTTGGAGCTATAATATCGGTAATTAG TTGTTCTTGGGGAGTAACCACTACTGGAGGTGCTAAAGGT GTTGGAGAATCTACAACTTCTGCTGTCGTCATGTCTCTTG TCGGAATCTTCATCGCGGACTTTGTGCTTTCTTCCTTCTT CTTTCAAGGTGCTGGAGATTCTTTGAAGAACTGTGTTTGA CATATTATTTTCTGTCTTCTTTTGTTGTGGTTTAGATGGG TTTATGTAAATCAGTTGTCTTAAATTGAGAAAGTAACATC ATTTTAGAAAGAACAGAAAGATTGCTATATTTCTATTCCA ATAATGATACACATTGAATAAT
(251) TABLE-US-00022 TABLE6B Exemplarytgdantisensegenesequencesforuseintgdgenesilencing aspartofRNAiconstructsequences. Targetgene Exemplarysequenceforuseingenesilencingvectors: SEQ forsilencing: antisensesequence,i.e.whenpromoterislocated5:5-3 IDNO: TGD4: TTGTAAGCATCATAGCTAAGAAGCAAAGTATCATCTGGTGATAACAAAAACTCAGAACA 51 AAAACCCAATGCGTACAAAGATTTATCCTTCAAATGCTTGCCAATTGTGTTTAAACGAG AAGCTACTGTAGACGAAGACCCTCGACCAAAAGCTTTAGTCTTATCTATCTCCGTTACG AATCTCTGAACATCGAATTGGCCCAGAAGAGACACAAGCCAGTTGTTGGAGAAAGGAAG AGTGAGGACTCTTTGAAGAGAGAATCCACCACCGCCTCCATCTCCGGTGTTGGGACGGA TAGGGGAGAAGGAAGGGATGAGAGGTGAGGCCATGAAGCGGTGGAAGAACTCAACTTGC TTAGGGCGAGATAGACGAGTGCCTCTAGAGAGACCTAGAGGAAGAGGATCGTCAGGAAC AGCTCGTGCGGTGCCCTCGAGCGTCACCGGAGTTGACATATCGAGGTCCCAGATGTCTC CCTCTCCGACCCATCTCATTCTGTTCAT TGD3: ATGCTTTCGTTATCATGCTCTTCTTCTTCTTCTTCGTTGCTTCCTCCGAGTTTACACTA 52 CCACGGTTCTTCTTCTGTTCAGTCCATCGTTGTACCAAGAAGGAGTCTTATCTCGTTTC GTCGGAAAGTCTCTTGCTGTTGCATAGCTCCACCTCAGAACTTGGACAACGAT TGD2: ATATTCTTCAAGGAAGGATTAACACGGATAATCGTACCAACAGTAACCCCACGGATCCT 53 AACCGGTGTTCCCGTGCAAATACCAGAAGCATGACTAAGCTCAAACACAGTCTGATATT TCCTAAACTTCGACCGCATTTGAAAACCTCGCAACCAAGCCCAGCTAAGAGCAAGAAGA GTAGCTCCAGAGACAATAAACAAACCAACACCACCTTCCCAAATACTTCTCTTACCAAA CCCAAAATCACTCAAAGGTTTTAAAGTCTGTCTCCATATATTCCTGGGCACATCCAAAA CAACGGTGAGAGGATTCTTCCCCCCATCAGACGATGGTTGACCATGAGCAGCATCGGAA TTGGATGCAGCTCTGACCACTAAATGCCTAGTTCTAGGTTTTGGTGGAAGATAAGGAAC CCCATTGGGTGAAACTCGAGGACAAGCAATCATGGAGGATGATGGCATTAGTGATGATG GAACTTGAATTACTGGATTCCCAATCAT TGD1: CTGTTTAGACCTAGTCTAGTGAATTCTCTAACGAACTGGATTGTGAAAGCCATACCAAC 54 AAATGTAGAAGTCAGAAGACAAACTCCTAGAGATTTCGGTCCGGTTCTCTCCAGCTGTT GAAGAGTGTTTCTCCAGTGAATCTTACCCTTTAAAATCCGGAGAACGACTTGTCCTGCT ATAATAGGAACTGATAAACCTCTCCATATGTACTTAGAAGGTGACCATTTGCTGAACGG AAGCTCGGCTTCTTGACTGGGTGCGTTGTTTTCAGTAGAGGTTTCTTCTTCCAACATAG ACATGGATGGGTGACCATCATTAGCATTCAAATTCACATATAATCTTCTTTGCCGTATC GGACTTGAAATCCCATAAGATTGAGTCTTTCCAATGAAACCAACTTGACAAAGCTTTGG GGGCTTAATACCAATCGAAGCATCAAACCGTGGAAAGACTCTATGAGGGAAACAAAACG ATTGATGGATACAACAAGTCTGCATCAT
(252) TABLE-US-00023 TABLE6C Exemplarysilencingsequencescomprisinginoperablecombination, senseandantisensefragments,whereaspacerelementsequenceis locatedinbetweenthesenseandantisensesequences. Exemplarysequenceforuseingenevectors:Inoperable combination:sensesequence-spacer-antisense,i.e.when Targetgene promoterislocated5:5-3 (Senseunderlined-spacer SEQ forsilencing: sequenceinBOLD-antisenseinlowercase) IDNO: TGD4: Atgaacagaatgagatgggtcggagagggagacatctgggacctcgatatgtcaac 19 AT3G06960 tccggtgacgctcgagggcaccgcacgagctgttcctgacgatcctcttcctctag gtctctctagaggcactcgtctatctcgccctaagcaagttgagttcttccaccgc ttcatggcctcacctctcatcccttccttctcccctatccgtcccaacaccggaga tggaggcggtggtggattctctcttcaaagagtcctcactcttcctttctccaaca actggcttgtgtctcttctgggccaattcgatgttcagagattcgtaacggagata gataagactaaagcttttggtcgagggtcttcgtctacagtagcttctcgtttaaa cacaattggcaagcatttgaaggataaatctttgtacgcattgggtttttgttctg agtttttgttatcaccagatgatactttgcttcttagctatgatgcttacaaAACC CAGAAGAAAGAATCttgtaagcatcatagctaagaagcaaagtatcatctggtgat aacaaaaactcagaacaaaaacccaatgcgtacaaagatttatccttcaaatgctt gccaattgtgtttaaacgagaagctactgtagacgaagaccctcgaccaaaagctt tagtcttatctatctccgttacgaatctctgaacatcgaattggcccagaagagac acaagccagttgttggagaaaggaagagtgaggactctttgaagagagaatccacc accgcctccatctccggtgttgggacggataggggagaaggaagggatgagaggtg aggccatgaagcggtggaagaactcaacttgcttagggcgagatagacgagtgcct ctagagagacctagaggaagaggatcgtcaggaacagctcgtgcggtgccctcgag cgtcaccggagttgacatatcgaggtcccagatgtctccctctccgacccatctca ttctgttcat TGD3 AACGAAAAATGGCAATGTGACTCACTCAATCGGTGACTCGCTATAGTCTGTGAAGA 20 Underlined AAGGCCAATTTCGCCATAAAGTTCACACCTTTGATCTCCTTTGTTTCTGGGTTTCT regionis CCTAAATCATCCAAATTGGTATCGAATTTGCCCTTCTCCGATTCAATTTCTTCACG 5-3 exon1 ATCTCAAAACCCAGAAGAAAGAATCatgctttcgttatcatgctcttcttcttctt cttcgttgcttcctccgagtttacactaccacggttcttcttctgttcagtccatc gttgtaccaagaaggagtcttatctcgtttcgtcggaaagtctcttgctgttgcat agctccacctcagaacttggacaacgat TGD2: atgattgggaatccagtaattcaagttccatcatcactaatgccatcatcctccat 21 AT3G20320 gattgcttgtcctcgagtttcacccaatggggttccttatcttccaccaaaaccta gaactaggcatttagtggtcagagctgcatccaattccgatgctgctcatggtcaa ccatcgtctgatggggggaagaatcctctcaccgttgttttggatgtgcccaggaa tatatggagacagactttaaaacctttgagtgattttgggtttggtaagagaagta tttgggaaggtggtgttggtttgtttattgtctctggagctactcttcttgctctt agctgggcttggttgcgaggttttcaaatgcggtcgaagtttaggaaatatcagac tgtgtttgagcttagtcatgcttctggtatttgcacgggaacaccggttaggatcc gtggggttactgttggtacgattatccgtgttaatccttccttgaagaatatAACC CAGAAGAAAGAATCatattcttcaaggaaggattaacacggataatcgtaccaaca gtaaccccacggatcctaaccggtgttcccgtgcaaataccagaagcatgactaag ctcaaacacagtctgatatttcctaaacttcgaccgcatttgaaaacctcgcaacc aagcccagctaagagcaagaagagtagctccagagacaataaacaaaccaacacca ccttcccaaatacttctcttaccaaacccaaaatcactcaaaggttttaaagtctg tctccatatattcctgggcacatccaaaacaacggtgagaggattcttccccccat cagacgatggttgaccatgagcagcatcggaattggatgcagctctgaccactaaa tgcctagttctaggttttggtggaagataaggaaccccattgggtgaaactcgagg acaagcaatcatggaggatgatggcattagtgatgatggaacttgaattactggat tcccaatcat TGD1: atgatgcagacttgttgtatccatcaatcgttttgtttccctcatagagtctttcc 22 AT1G19800 acggtttgatgcttcgattggtattaagcccccaaagctttgtcaagttggtttca ttggaaagactcaatcttatgggatttcaagtccgatacggcaaagaagattatat gtgaatttgaatgctaatgatggtcacccatccatgtctatgttggaagaagaaac ctctactgaaaacaacgcacccagtcaagaagccgagcttccgttcagcaaatggt caccttctaagtacatatggagaggtttatcagttcctattatagcaggacaagtc gttctccggattttaaagggtaagattcactggagaaacactcttcaacagctgga gagaaccggaccgaaatctctaggagtttgtcttctgacttctacatttgttggta tggctttcacaatccagttcgttagagaattcactagactaggtctaaacagAACC CAGAAGAAAGAATCctgtttagacctagtctagtgaattctctaacgaactggatt gtgaaagccataccaacaaatgtagaagtcagaagacaaactcctagagatttcgg tccggttctctccagctgttgaagagtgtttctccagtgaatcttaccctttaaaa tccggagaacgacttgtcctgctataataggaactgataaacctctccatatgtac ttagaaggtgaccatttgctgaacggaagctcggcttcttgactgggtgcgttgtt ttcagtagaggtttcttcttccaacatagacatggatgggtgaccatcattagcat tcaaattcacatataatcttctttgccgtatcggacttgaaatcccataagattga gtctttccaatgaaaccaacttgacaaagctttgggggcttaataccaatcgaagc atcaaaccgtggaaagactctatgagggaaacaaaacgattgatggatacaacaag tctgcatcat
(253) In one tgd RNAi construct for reducing TGD3, SEQ ID NO:20 was used to make two types of constructs. One construct had a constitutive promoter (S35) and the other construct had an inducible gene silencing promoter (for general examples of inducible gene silencing see, Guo et al. (The Plant J. 34(3): 383-92 (2003)), incorporated herein by reference in its entirety). In this example, the spacer region DNA was an intron of tgd3.
(254) TABLE-US-00024 TABLE7 Exemplaryprimersusedforproviding sequencesforuseinRNAiconstructs. Gene ForwardPrimer-sense ReversePrimer-antisense TGD4 5-CATGGATCCATGAACAG 5-ACAGTCGACCTAGTGCTCAAA AATGAGATGGGT-3 GAAACGAAGC-3 SEQIDNO:23 (XU,2008) SEQIDNO:24 TGD3 5-ACGGTACCATGCTTTCG 5-CTGGTACCCTAGTATCTGATT TTATCATGCTC-3 GGTCCAT-3 SEQIDNO:25 SEQIDNO:26 Binbin,etal.JBC,2007 TGD2 5-GTCGACATGATTGGGAA 5-GTCGACTCATAGTAGCCTGCT TCCAGTAATTCAAG-3 TAGGG-3 SEQIDNO:27 SEQIDNO:28 Awaietal,ProcNatl AcadSciUSA.2006 TGD1 5-AATACTAGTGGCGCGCC 5-CCAGGATCCATTTAAATTCAA ATGATGCAGACTTGTT-3 ACACAGTTCTT-3 SEQIDNO:29 SEQIDNO:30
(255) TABLE-US-00025 TABLE8 ExemplarypromotersforusewithRNAiconstructsandvectors. Promoter Type: Examples: SEQIDNO: Exemplaryuse Inducible -estradiolpromoteri.e.regulatory SEQIDNO:32 Incombination regionofthehumanestrogen tctgctggagacatgagagctgc withexemplary receptorcarboxyterminalregion caacctttggccaagcccgctcat RNAisequences residues282-295),asoneexample, gatcaaacgctctaagaagaacag NCBI:NM_000125encoding,forone cctggccttgtccctgacggccga example, ccagatggtcagtgccttgttgga SEQIDNO:31: tgctgagccccccatactctattc SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVS cgagtatgatcctaccagaccctt ALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRE cagtgaagcttcgatgatgggctt LVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMI actgaccaacctggcagacaggga GLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIF gctggttcacatgatcaactgggc DMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF gaagagggtgccaggctttgtgga LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTL tttgaccctccatgatcaggtcca QQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNV ccttctagaatgtgcctggctaga VPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLA gatcctgatgattggtctcgtctg TAGSTSSHSLQKYYITGEAEGFPATV gcgctccatggagcacccagggaa gctactgtttgctcctaacttgct cttggacaggaaccagggaaaatg tgtagagggcatggtggagatctt cgacatgctgctggctacatcatc tcggttccgcatgatgaatctgca gggagaggagtttgtgtgcctcaa atctattattttgcttaattctgg agtgtacacatttctgtccagcac cctgaagtctctggaagagaagga ccatatccaccgagtcctggacaa gatcacagacactttgatccacct gatggccaaggcaggcctgaccct gcagcagcagcaccagcggctggc ccagctcctcctcatcctctccca catcaggcacatgagtaacaaagg catggagcatctgtacagcatgaa gtgcaagaacgtggtgcccctcta tgacctgctgctggagatgctgga cgcccaccgcctacatgcgcccac tagccgtggaggggcatccgtgga ggagacggaccaaagccacttggc cactgcgggctctacttcatcgca ttccttgcaaaagtattacatcac gggggaggcagagggtttccctgc cacggtctga Constitutive CaMV35Spromoter SEQIDNO:33 Incombination tgagacttttcaacaaaggg withexemplary taatatccggaaacctcctc genesilencing ggattccattgcccagctat sequencefor ctgtcactttattgtgaaga RNAisequences tagtggaaaaggaaggtggc tcctacaaatgccatcattg cgataaaggaaaggccatcg ttgaagatgcctctgccgac agtggtcccaaagatggacc cccaccccacgaggagcatc gtggaaaaagaagacgttcc aaccacgtcttcaaagcaag tggattgatgtgatatctcc actgacgtaagggatgacgc acaatcccactatccttcgc aagacccttcctctatataa ggaagttcatttcatttgga gagga Constitutive 35Sminimalpromoter cccactatccttcgc Operably aagacccttcctctatataa combinedwith ggaagttcatttcatttgga genesturned gagga onby inducible promoter
(256) TABLE-US-00026 TABLE9 Exemplaryspacersequences forseparatingsenseandantisenseDNAinRNAivectors. spacersequences: Exemplaryuse: glucuronidaseintron SEQIDNO:35 RNAivectorsusingfull tatacgccatttgaagccgatgtcacgccgt lengthTGD1 atgttattgccgggaaaagtgtacgtatcac cgtttgtgtgaacaacgaactgaactggcag actatcccgccgggaatggtgattaccgacg aaaacggcaagaaaaagcagtcttacttcca tgatttctttaactacgccgggatccatcgc agcgtaatgctctacaccacgccgaacacct gggtggacgatatcaccgtggtgacgcatgt cgcgcaagactgtaaccacgcgtctgttgac tggcaggtggtggccaatggtgatgtcagcg ttgaactgcgtgatgcggatcaacaggtggt tgcaactggacaaggcaccagcgggactttg caagtggtgaatccgcacctctggcaaccgg gtgaaggttatctctatgaactgtgcgtc acagccaaaagccagacagagtgtgatatct acccgctgcgcgtcggcatccggtcagtggc agtgaagggcgaacagttcctgatca accac TGD3intron AACCCAGAAGAAAGAATC incombinationwith SEQIDNO:36 exemplarygenesilencing sequenceforTGD3
(257) TABLE-US-00027 TABLE 10 Exemplary vectors. inducible vector pER8, Zuo, (2000) An estrogen receptor- based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273. silencing vector pGSA1285 (ABRC, Ohio State University, Columbus, OH; CD3-454)
(258) 1. Silencing Tgd3 Using a Constitutive Promoter.
(259) The construct TGD3RNAi was introduced into Arabidopsis wild-type Col-2 background by Agrobacterium-mediated transformation (Xu et al. Plant Cell 17: 3094-3110 (2005), herein incorporated by reference in its entirety). T2 seeds from independent transformation lines were germinated on the regular MS medium containing Hygromycin B. After two weeks, resistant plants were transferred into a liquid MS solution. Oils (plant samples) were harvested from these plants after 6 days in liquid MS solution without an inducing compound. Harvested oils were analyzed by TLC. Constitutive overexpression of TGD3RNAi produced plants having increased levels of TGDG and TAG, see, lane labeled tgd-3,
(260) 2. Silencing Tgd3 Using a Chemical Inducible TGD3RNAi System.
(261) The construct TGD3RNAi-pER8, see,
(262)
(263) As shown in
(264) However, in the control wild type Col-2 plant treated the same way, a faint band migrated at the same position as TGDG was also observed, suggesting that the presence of extra oligogalactolipids in the controls might be due to a stress condition e.g. hypoxia (seedlings immersed under liquid solution for 6 days). Other ways of applying the -estradiol inducer such as spraying the surface of leaves or direct germination of seeds on inducer-containing MS medium did not lead to these positive results.
Example III
(265) This example describes a contemplated method for making and using plants having lowered tgd expression (i.e. expressing an RNAi construct of the present inventions) combined with overexpression of heterologous WRINKLED1 protein genes. RNAi vectors and methods of engineering plants comprising these gene silencing constructs for exemplary tgd 3 or tgd4 are described herein. Constructs for overexpressing heterologous genes, i.e. WRI1 and F-box, and plants engineered for expressing these constructs are provided as described herein, and in Cernac and Benning, The Plant J, 40:575-585 (2004) and WO 2007 038345 respectively, herein incorporated by reference in their entirety. In some embodiments, WRI1 and/or F-box genes are expressed constitutively within the whole plant, for example, the constitutive CaMV 35S promoter is contemplated for use.
(266) A. Examples of Making Transgenic Plants Having Reduced TGD Protein.
(267) Plants having reduced TGD protein are described herein, including in Example II. Such plants expressing tgd RNAi vector constructs are contemplated for use as host cells, tissues or plants for use in transfection with vectors as described herein for overexpressing WRI1 transcription factor proteins. In one embodiment, instead of using wild type seedlings, as described below, seedlings having reduced TGD proteins are used for transfection.
(268) B. Examples of Making Transgenic Plants Overexpressing Heterologous WRINKLED1.
(269) A complete cDNA for WRI1 DNA was obtained by RT-PCR employing a kit from Qiagen (Valencia, Calif., USA) using wild type (Col-2) silique RNA and the PCR primers 5-GGTACCAAATCTAAACTTTCTCAGAG-3 (SEQ ID NO:60) and 5-GGTACCGGCAAAGACATTGATTATTC-3 (SEQ ID NO:61). The RT-PCR product was cloned using TA-overhangs into the pCR2.1TOPO vector from Invitrogen (Carlsbad, Calif., USA). Sequencing was performed at the MSU Genomics Technology Support Facility. The cDNA sequence for WRI1 was deposited at GenBank (AY254038). The cDNA was excised with KpnI and inserted into the binary vector pBinAR-Hyg (Dormann and Benning, 1998) or into a pCAMBIA1300 derivative (CAMBIA, Can berra, Australia), which contained the EcoRI/HindIII expression cassette from pBIN121 (Clontech, Palo Alto, Calif., USA).
(270) One example of a WRI1 gene for use in a construct for overexpressing mRNA for WRI1 in a plant is an Arabidopsis thaliana WRINKLED1 (WRI1) with Accession number AY254038 (in FAST A format), provided below as SEQ ID NO:37.
(271) TABLE-US-00028 1 AAACCACTCTGCTTCCTCTTCCTCTGAGAAATCAAATCAC 41 TCACACTCCAAAAAAAAATCTAAACTTTCTCAGAGTTTAA 81 TGAAGAAGCGCTTAACCACTTCCACTTGTTCTTCTTCTCC 121 ATCTTCCTCTGTTTCTTCTTCTACTACTACTTCCTCTCCT 161 ATTCAGTCGGAGGCTCCAAGGCCTAAACGAGCCAAAAGGG 201 CTAAGAAATCTTCTCCTTCTGGTGATAAATCTCATAACCC 241 GACAAGCCCTGCTTCTACCCGACGCAGCTCTATCTACAGA 281 GGAGTCACTAGACATAGATGGACTGGGAGATTCGAGGCTC 321 ATCTTTGGGACAAAAGCTCTTGGAATTCGATTCAGAACAA 361 GAAAGGCAAACAAGTTTATCTGGGAGCATATGACAGTGAA 401 GAAGCAGCAGCACATACGTACGATCTGGCTGCTCTCAAGT 441 ACTGGGGACCCGACACCATCTTGAATTTTCCGGCAGAGAC 481 GTACACAAAGGAATTGGAAGAAATGCAGAGAGTGACAAAG 521 GAAGAATATTTGGCTTCTCTCCGCCGCCAGAGCAGTGGTT 561 TCTCCAGAGGCGTCTCTAAATATCGCGGCGTCGCTAGGCA 601 TCACCACAACGGAAGATGGGAGGCTCGGATCGGAAGAGTG 641 TTTGGGAACAAGTACTTGTACCTCGGCACCTATAATACGC 661 AGGAGGAAGCTGCTGCAGCATATGACATGGCTGCGATTGA 721 GTATCGAGGCGCAAACGCGGTTACTAATTTCGACATTAGT 761 AATTACATTGACCGGTTAAAGAAGAAAGGTGTTTTCCCGT 801 TCCCTGTGAACCAAGCTAACCATCAAGAGGGTATTCTTGT 841 TGAAGCCAAACAAGAAGTTGAAACGAGAGAAGCGAAGGAA 881 GAGCCTAGAGAAGAAGTGAAACAACAGTACGTGGAAGAAC 921 CACCGCAAGAAGAAGAAGAGAAGGAAGAAGAGAAAGCAGA 961 GCAACAAGAAGCAGAGATTGTAGGATATTCAGAAGAAGCA 1001 GCAGTGGTCAATTGCTGCATAGACTCTTCAACCATAATGG 1041 AAATGGATCGTTGTGGGGACAACAATGAGCTGGCTTGGAA 1081 CTTCTGTATGATGGATACAGGGTTTTCTCCGTTTTTGACT 1121 GATCAGAATCTCGCGAATGAGAATCCCATAGAGTATCCGG 1161 AGCTATTCAATGAGTTAGCATTTGAGGACAACATCGACTT 1201 CATGTTCGATGATGGGAAGCACGAGTGCTTGAACTTGGAA 1241 AATCTGGATTGTTGCGTGGTGGGAAGAGAGAGCCCACCCT 1281 CTTCTTCTTCACCATTGTCTTGCTTATCTACTGACTCTGC 1321 TTCATCAACAACAACAACAACAACCTCGGTTTCTTGTAAC 1361 TATTTGGTCTGAGAGAGAGAGCTTTGCCTTCTAGTTTGAA 1401 TTTCTATTTCTTCCGCTTCTTCTTCTTTTTTTTCTTTTGT 1441 TGGGTTCTGCTTAGGGTTTGTATTTCAGTTTCAGGGCTTG 1481 TTCGTTGGTTCTGAATAATCAATGTCTTTGCCCCTTTTCT 1521 AATGGGTACCTGAAGGGCGA
(272) Proper orientation and integrity were confirmed by restriction analysis and sequencing. For generation of transgenic plants, seedlings for wild type plants were grown and transferred to 3.5 inch pots that had been set up for dipping by mounding with potting soil and were covered with a plastic window screen with 20 evenly spaced holes of 0.5 cm diameter. Growth conditions were as described herein. When the primary inflorescence had bolted and the first three or four flowers had opened (approximately 4 weeks), the plants were transformed using the floral dip method (Clough and Bent, 1998, herein incorporated by reference in its entirety). Three to four pots were used per construct. The plants were dipped again in the same manner 2 or 3 weeks later. Competent cells of Agrobacterium tumefaciens strain GV3101, C58C1, pMP90 (Koncz and Schell, 1986, herein incorporated by reference in its entirety) were prepared and transformed according to Shen and Forde (1989). Transformation efficiencies ranged from 1 to 2% for pBinAR-Hyg or pCAMBIA constructs and 0.5-0.8% for pBIC20. Wild type plants transformed with the WRI1 cDNA were grown in separate flats in the same walk-in chamber and plants were individually tied to stakes. Leaf tissue taken for RNA analysis was harvested from 28-day-old rosettes. The stems used were taken from 35-day-old plants. Seedlings were 8 days old from the time the plates were placed into the incubator. Roots and shoots were excised from 10-day-old plantlets. Tissues were snap frozen in liquid nitrogen. Flowers consisted of the inflorescence meristem through the first three opened flowers. Whole silique tissue was harvested from approximately 40-day-old plants. Green siliques of each stage were taken. The age of the seed for the time course was determined by staging flowers as previously described (Focks and Benning, 1998, herein incorporated by reference in its entirety). Seeds were dissected out of the silique using fine tweezers. Seeds from 10 to 20 siliques were harvested onto a glass slide on ice and then gathered and placed into a 1.5 ml polypropylene tube in dry ice. This was repeated until the tagged siliques for each particular time point were harvested (10-14 h for one person). Tissue was stored at 80 C. until extraction. RNA extraction was performed according to the methods of Hosein (2001). Northern analysis (5 g total RNA) was performed as previously described (Drmann and Benning, 1998, herein incorporated by reference in its entirety).
(273) The blots were analyzed using a phosphor imager (Molecular Dynamics, Amersham, Piscataway, N.J., USA). Background integrals of areas equal in size to those of the bands located between the wells and the bands on the blots were subtracted. Using the image quantification software provided by the manufacturer (image quant 5.2 and fragment analysis 1.1), the ratio of each background-corrected band intensity to the 16S rRNA band intensity for the respective time point was calculated and the lowest ratio was arbitrarily set to 1, to which other signals in each series were normalized. In experiments using RT-PCR, RNA was extracted from 11-day-old seedlings of each genotype using a Qiagen RN easy kit, as per the manufacturer's instructions. Reverse transcription was performed on 1 g of total RNA using a Qiagen Omniscript kit. The primers used to distinguish WRI1 were designed to span the region of the first intron (5-CCGACGCAGCTCTATCTACA-3 SEQ ID NO:38 and 5-AGCCTCCCATCTTCCGTTGT-3 SEQ ID NO:39). Actin (At2g3762) was used for control purposes (5-TGCGACAATGGAACTGGAATGG-3 SEQ ID NO:40 and 5-AACAATCGATGGACCTGACTCG-3 SEQ ID NO:41). The cycle conditions were 3.5 min at 94 C., 0.5 min at 94 C., 0.75 min at 54 C., 1 min at 72 C., 30 cycles followed by 5 min at 72 C. In the wild type, the predicted PCR product is 358 bp, in the event of genomic DNA contamination; there is a product of 1080 bp. The same reaction conditions were used for the actin mRNA quantification, except that 25 cycles were used. For both, 1 unit of Invitrogen Taq polymerase was used with a final MgCl.sub.2 concentration of 1.5 mM, dNTP concentration of 0.125 mM, and 10 pmol of each primer. Lipid extractions for the purpose of thin-layer chromatography were carried out as previously described (Focks and Benning, 1998) with the exception that the solvent system was 80:20:1, petroleum ether:ethyl ether:acetic acid. Seed oil quantitation by total fatty acid methylester analysis was carried out as previously described (Focks and Benning, 1998; Rossak et al., 1997). For time course experiments on seeds and seedlings, the sum of 20:0, 20:1, 22:0, and 22:1 LFA methylesters was reported.
Example IV
(274) The following are examples of making transgenic plants overexpressing heterologous F-Box for lipid modification and/or increased lipid production in vegetative tissues using f-box genes.
(275) This example describes a contemplated method for making and using plants having lowered tgd expression (i.e. expressing an RNAi construct of the present inventions) combined with overexpression of heterologous F-Box genes.
(276) RNAi vectors and methods of engineering plants comprising these gene-silencing constructs for tgd 3 or tgd4 are described above. Constructs for overexpressing heterologous genes, i.e. F-box, and plants engineered for expressing these constructs are provided as described herein, and in WO 2007 038345, herein incorporated by reference in its entirety.
(277) In some embodiments, plants of the present invention for increasing oil production in vegetative tissues were based, in part, upon incorporating the unexpected finding that overexpression of F-box genes in plants results in the increased production of plant oil. In particular, the overexpression of the Arabidopsis thaliana F-box protein gene (GenBank Accession Nos. NM_111499 (cDNA, SEQ ID NO:57) and NP 566277 (protein, SEQ ID NO:42) using the seed specific promoter phaseolin, produced seeds with a higher oil content phenotype (see,
(278) The sequence of the Arabidopsis thaliana F-box/Kelch-Repeat protein with GenBank Accession No. NP 566277 (protein, SEQ ID NO:42) is shown below.
(279) TABLE-US-00029 1 MKAIQLLWEAIMEATKRERRREDDDGEKASPESLVLPPEI 41 ITEILLRLPAKSIGRFRCVSKLFCTLSSDPGFAKIHLDLI 81 LRNESVRSLHRKLIVSSHNLYSLDFNSIGDGIRDLAAVEH 121 NYPLKDDPSIFSEMIRNYVGDHLYDDRRVMLKLNAKSYRR 161 NWVEIVGSSNGLVCISPGEGAVFLYNPTTGDSKRLPENFR 201 PKSVEYERDNFQTYGFGFDGLTDDYKLVKLVATSEDILDA 241 SVYSLKADSWRRICNLNYEHNDGSYTSGVHFNGAIHWVFT 281 ESRHNQRVVVAFDIQTEEFREMPVPDEAEDCSHRFSNFVV 321 GSLNGRLCVVNSCYDVHDDIWVMSEYGEAKSWSRIRINLL 361 YRSMKPLCSTKNDEEVLLELDGDLVLYNFETNASSNLGIC 401 GVKLSDGFEANTYVESLISPNSYGIES
(280) Accordingly, in one aspect, the present invention provides a genetic construct for the overexpression of an F-box protein in plants and plants overexpressing F-Box protein for use with tgd silencing constructs and plants expressing tgd silencing constructs of the present inventions. In general, such genetic constructs of the invention include embodiments described in WO/2007/038345, herein incorporated in its entirety in combination (i.e. stacked) with tgd RNAi of the present inventions. In particular, it is contemplated that aspects of the present inventions comprise overexpression of a heterologous F-box protein in plants in addition to reducing expression of a TGD gene resulting in lowered expressing of TGD proteins in the same plants resulting in a corresponding increase in TAG in vegetative tissues and seeds in plants having commercial value. In further embodiments, WRI1 and F-box genes are expressed within a plant having reduced TGD protein production.
Example V
(281) This example describes exemplary generation of single transgenic lines for use in breeding double and multiple transgenic-engineered plants of the present inventions.
(282) Double and multiple transgenic plant lines are contemplated to be established from individual transgenic plants. In one embodiment, transformed plants containing tgd RNAi constructs were grown to maturity and used in breeding methods of the present inventions. Thus, in one embodiment, a breeding method is contemplated comprising propagating transformed plants for use in developing plants and lines as source plants comprising RNAi constructs of the present inventions. In a further embodiment, these original or source plants are contemplated for breeding, such as in crossing, i.e. artificially or naturally, with other engineered plants in order to produce plants having additional modified gene expression of heterologous genes, i.e. stacked or pyramid genes. Thus, the presence of nucleic acid sequences encoding a heterologous wri1 gene and/or f-box gene of the present invention (including mutants or variants thereof) may be combined with traits of tgd RNAi transgenic plants by traditional plant breeding techniques. Such traditional techniques include but are not limited to artificial cross pollination, natural pollination, self-pollination, etc., and breeding methods including but not limited to back-crossing, in breeding, out crossing, hybridization, etc., wherein such crossing results in multiple generations for developing
(283) Accordingly, methods of making these engineered plants with increased TAG amounts comprises breeding these f-box or WRINKLED1 overexpressing plants with engineered plants having reduced expression of a TGD gene as the result of transformation of the host plant with a TGD RNAi gene silencing vector construct of the present interventions. In a preferred embodiment, these methods of breeding further comprise using breeding stock with desired agronomic traits, such as large amounts of TAG production, large seed sizes, a particular oil composition, strong growth characteristics, growth characteristics for a particular hardiness zone, high levels of fertility, etc., and/or desirable economic traits, such as roundup ready varieties or other plant lines having glyphosate-resistance.
(284) Thus, in another embodiment, an overexpression construct of the present inventions is provided as a double gene vector for overexpressing both an AGPase RNAi silencing vector combined with an expression construct for an oil regulating transcript factor such as a WRINKLED1 gene in a host plant expressing a tgd RNAi construct having reduced TGD protein for increasing lipid production without significantly impacting host plant viability and fertility. Thus methods of the present inventions further comprise transforming plants, in particular oil seed plants, with a double gene overexpression vector that when expressed increases the amount of mRNA for encoding an oil transcription factor, such as encoding a WRI1 protein along with RNAi for reducing TGD proteins in a plant.
Example VI
(285) This example describes material and methods comprising a double gene vector for overexpressing WRI1 in combination with reducing AGPRNAi.
(286) Overexpression of WRI1 in combination with reduced AGPase (i.e. expressing a silencing construct AGPRNAi) caused a growth phenotype. Independent homozygous T3 transgenic Arabidopsis lines expressing a WRINKLED (WRI1) cDNA or suppressing the APS1 gene by RNAi under the control of the 35S cauliflower mosaic virus (CaMV) promoter or the Patatin B33 promoter in various combinations, summarized in
(287) The WRINKLED cDNA was obtained by PCR primers 5CCAAggatcc-AAATCTAAACTTTCTCAGAGT3 (SEQ ID NO:58) and 5CCTTacgcgtGGCAAA-GACATTGATTATT3 (SEQ ID NO:59). However, several WRINKLED cDNA sequences are available. For example, the WRINKLED cDNA can have the sequence provided with NCBI accession number NM_202701.2 (GI:145362489) and shown below as SEQ ID NO:44.
(288) TABLE-US-00030 1 CAGGGTTTATTTAACTTGCCCTTTCTCGTTTCCTCCTTTT 41 TTTCTTAAACCACTCTGCTTCCTCTTCCTCTGAGAAATCA 81 AATCACTCACACTCCAAAAAAAAATCTAAACTTTCTCAGA 121 GTTTAATGAAGAAGCGCTTAACCACTTCCACTTGTTCTTC 161 TTCTCCATCTTCCTCTGTTTCTTCTTCTACTACTACTTCC 201 TCTCCTATTCAGTCGGAGGCTCCAAGGCCTAAACGAGCCA 241 AAAGGGCTAAGAAATCTTCTCCTTCTGGTGATAAATCTCA 281 TAACCCGACAAGCCCTGCTTCTACCCGACGCAGCTCTATC 321 TACAGAGGAGTCACTAGACATAGATGGACTGGGAGATTCG 361 AGGCTCATCTTTGGGACAAAAGCTCTTGGAATTCGATTCA 401 GAACAAGAAAGGCAAACAAGGTTTCGAGCATATGACAGTG 441 AAGAAGCAGCAGCACATACGTACGATCTGGCTGCTCTCAA 481 GTACTGGGGACCCGACACCATCTTGAATTTTCCGGCAGAG 521 ACGTACACAAAGGAATTGGAAGAAATGCAGAGAAGGAAGA 561 AGGAAGAATATTTGGCTTCTCTCCGCCGCCAGAGCAGTGG 601 TTTCTCCAGAGGCGTCTCTAAATATCGCGGCGTCGCTAGG 641 CATCACCACAACGGAAGATGGGAGGCTCGGATCGGAAGAG 681 TGTTTGGGAACAAGTACTTGTACCTCGGCACCTATAATAC 721 GCAGGAGGAAGCTGCTGCAGCATATGACATGGCTGCGATT 761 GAGTATCGAGGCGCAAACGCGGTTACTAATTTCGACATTA 801 GTAATTACATTGACCGGTTAAAGAAGAAAGGTGTTTTCCC 841 GTTCCCTGTGAACCAAGCTAACCATCAAGAGGGTATTCTT 881 GTTGAAGCCAAACAAGAAGTTGAAACGAGAGAAGCGAAGG 921 AAGAGCCTAGAGAAGAAGTGAAACAACAGTAGAAGGAAGA 961 ACCACCGCAAGAAGAAGAAGAGAAGGAAGAAGAGAAAGCA 1001 GAGCAACAAGAAGCAGAGATTGTAGGATATTCAGAAGAAG 1041 CAGCAGTGGTCAATTGCTGCATAGACTCTTCAACCATAAT 1081 GGAAATGGATCGTTGTGGGGACAACAATGAGCTGGCTTGG 1121 AACTTCTGTATGATGGATACAGGGTTTTCTCCGTTTTTGA 1161 CTGATCAGAATCTCGCGAATGAGAATCCCATAGAGTATCC 1201 GGAGCTATTCAATGAGTTAGCATTTGAGGACAACAACAAC 1241 TTCATGTTCGATGATGGGAAGCACGAGTGCTTGAACTTGG 1281 AAAATCTGGATTGTTGCGTGGTGGGAAGAGAGAGCCCACC 1321 CTCTTCTTCTTCACCATTGTCTTGCTTATCTACTGACTCT 1361 GCTTCATCAACAACAACAACAACAACCTCGGTTTCTTGTA 1401 ACTATTTGGTCTGAGAGAGAGAGCTTTGCCTTCTAGTTTG 1441 AATTTCTATTTCTTCCGCTTCTTCTTCTTTTTTTTCTTTT 1481 GTTGGGTTCTGCTTAGGGTTTGTATTTCAGTTTCAGGGCT 1521 TGTTCGTTGGTTCTGAATAATCAATGTCTTTGCCCCTTTT 1561 CTAATGCTCCAAGTTCAGAT
(289) The AGPRNAi constructs were designed for this experiment and contemplated experiments with B. napus plants and therefore targeted a region of the sequence conserved across Arabidopsis and Brassica. A sequence from B. napus AGPase (accession AJ271162, SEQ ID NO:46) with 82% DNA sequence identity to the most similar Arabidopsis gene APS1 (At5g48300, SEQ ID NO:45) was used to design the RNAi construct. APS1 encodes one of the major catalytic isoforms of the small subunit of AGPase, which was found in the organs of the plant (Crevillen et al., 2005).
(290) The following shows exemplary parts of an RNAi AGPase construct for use in operable combination with a promoter sequence. These parts are followed by an exemplary assembled construct for use in operable combination with a promoter.
(291) The Arabidopsis thaliana ADPG pyrophosphorylase small subunit (APS1) with accession number At5g48300 is shown below and provided herein as SEQ ID NO:45.
(292) TABLE-US-00031 1 ATGGCGTCTGTATCTGCAATTGGAGTTCTCAAGGTACCTC 41 CTGCTTCGACTTCCAATTCCACCGGAAAAGCCACGGAGGC 81 GGTTCCCACGAGGACTCTTTCTTTCTCCTCCTCTGTTACT 121 TCATCCGACGACAAGATTTCACTCAAATCCACCGTCTCCC 161 GTCTTTGTAAATCTGTTGTTCGCAGGAATCCGATCATCGT 201 CTCTCCCAAAGCCGTCTCAGATTCTCAAAACTCTCAAACT 241 TGTCTCGATCCTGATGCTAGCAGCAGTGTTTTGGGGATAA 281 TCTTAGGAGGTGGAGCTGGAACTCGTCTTTATCCACTTAC 321 GAAGAAGAGAGCGAAACCAGCTGTGCCTCTTGGTGCTAAC 361 TATAGGCTTATTGATATTCCTGTGAGCAACTGTTTGAATA 401 GCAACATATCCAAGATCTATGTTCTTACTCAGTTCAATTC 441 CGCGTCTTTGAATCGTCATCTTTCACGAGCTTATGCTAGT 481 AACATGGGAGGTTATAAGAATGAAGGATTCGTTGAAGTTC 521 TCGCTGCTCAACAGAGTCCTGAAAACCCCAACTGGTTCCA 561 GGGGACAGCTGATGCCGTCAGGCAATACTTGTGGTTGTTC 601 GAGGAGCATAATGTCTTGGAGTATCTCATTCTTGCTGGGG 641 ATCATTTGTATAGAATGGACTATGAGAAGTTTATTCAAGC 681 ACATAGGGAGACTGATGCTGATATCACAGTAGCTGCATTA 721 CCAATGGACGAGCAACGAGCCACTGCTTTTGGGCTGATGA 761 AGATTGATGAGGAAGGACGTATTATTGAATTTGCTGAAAA 801 ACCAAAAGGGGAGCACCTAAAGGCCATGAAGGTTGACACA 841 ACAATTCTAGGTCTTGATGATCAGAGAGCCAAGGAGATGC 881 CTTTCATTGCTAGTATGGGTATTTATGTTGTAAGCAGAGA 921 TGTAATGCTAGACTTACTACGGAATCAGTTTCCTGGAGCT 961 AATGACTTTGGAAGTGAAGTCATTCCCGGTGCCACTTCCC 1001 TTGGACTGAGGGTGCAAGCTTACCTATATGATGGTTACTG 1041 GGAAGACATTGGTACTATAGAGGCATTTTATAACGCTAAT 1081 CTTGGAATCACCAAGAAACCAGTTCCTGATTTTAGTTTCT 1121 ATGACCGTTCTGCTCCGATCTACACACAGCCGCGTTATTT 1161 ACCACCGTCTAAGATGCTTGATGCTGATGTTACTGACAGT 1201 GTCATCGGAGAGGGCTGTGTTATCAAGAACTGCAAAATTC 1241 ATCACTCTGTGGTTGGACTCCGTTCCTGCATATCAGAAGG 1281 TGCTATTATTGAAGATTCGTTATTAATGGGAGCTGATTAT 1321 TACGAGACTGCTACGGAAAAGAGCCTCTTAAGCGCGAAAG 1361 GAAGTGTACCCATAGGTATTGGGAAAAACTCGCACATCAA 1401 AAGGGCCATCATCGACAAAAACGCACGTATCGGTGACAAT 1441 GTCAAGATCATAAACAGCGACAACGTGCAAGAGGCAGCGA 1481 GAGAGACTGATGGATATTTCATAAAGAGCGGAATTGTAAC 1521 GGTTATCAAAGACGCCTTAATCCCAACCGGCACTGTCATC 1561 TGAAGTACACATAATGCTCCTTTGTTTATTTCTT
(293) Fragments can be used of a Brassica napus mRNA of cDNA encoding an ADP-glucose pyrophosphorylase small subunit (AGPase), for example, accession number AJ271162 (gi: 7688094), provided herein as SEQ ID NO:46.
(294) TABLE-US-00032 1 AAGAGTGAAGCTCCTCCTCTCGAACAACAACAATGGCGAC 41 AATGGCTGCAATCGGATCCTTAAAAGTCCCTTCCTCTTCC 81 TCGAACCACACCCGTAGATTATCATCTTCTTCTCAACGGA 121 AGACTCTCTCGTTCTCGTCGTCTTCTCTTACCGGAGAGAA 161 ACTCAACCCGACGCAGGAGATCATCATCTCCAATCTCCCA 201 CGTGGCAACGAGAGAAGAACGCCATCGATCGTCTCTCCAA 241 AAGCAGTTTCCGATTCGCAAAACTCGCAAACTTGCCTTGA 281 TCCTGACGCTAGCAGAAGTGTGTTGGGGATAATTCTGGGA 321 GGTGGTGCTGGAACGAGATTGTATCCGCTAACGAAGAAGA 361 GAGCGAAGCCAGCTGTTCCTCTCGGCGCTAACTACCGTCT 401 CATTGATATCCCAGTGAGCAATTGCTTGAACAGTAACATC 441 TCCAAGATCTATGTCCTTACTCAGTTCAACTCAGCTTCTC 481 TCAACCGCCATCTCTCCCGAGCTTACGCCAGCAACATGGG 521 TGGTTACAAGAACGAAGGCTTCGTCGAGGTTCTTGCTGCT 561 CAACAGAGTCCCGAGAATCCCAATTGGTTTCAGGGGACTG 601 CTGATGCAGTGAGGCAGTACTTGTGGCTGTTTGAAGAGCA 641 CAATGTGTTGGAGTTTTTGGTTCTTGCAGGGGATCATTTG 681 TACCGTATGGACTACGAGAAGTTCATTCAAGCGCATCGTG 721 AGACCGACGCTGATATCACTGTTGCTGCTCTTCCTATGGA 761 TGAGAAACGTGCCACGGCTTTTGGACTTATGAAGATTGAT 801 GACGAAGGAAGGATCATTGAGTTTGCTGAGAAGCCTAAAG 841 GAGAGCAGTTAAAGGCTATGAAGGTTGATACAACAATCTT 881 GGGACTTGATGACGAAAGGGCCAAAGAGATGCCCTTTATT 921 GCTAGTATGGGGATATATGTTGTTAGCAAGAATGTGATGT 961 TGGACTTGCTCCGAGACCAGTTCCCTGGAGCTAATGACTT 1001 CCGGAGAGAAGTTATCCCTGGTGCTACTGATCTTGGACTC 1041 AGAGTGCAAGCTTATCTGTATGATGGATACTGGGAAGATA 1081 TTGGTACCATTGAAGCCTTTTACAATGCTAATCTTGGGAT 1121 CACCAAGAAACCGAGACCAGATTTCAGCTTCTATGACCGT 1161 TCAGCACCAATCTACACACAGCCTCGGTACTTACCTCCAT 1201 CCAAGATGCTTGATGCCGATGTTACCGATAGTGTGATCGG 1241 TGAAGGTTGTGTCATCAAGAATTGCAAAATCCACCATTCC 1281 GTCATTGGTCTTCGGTCTTGCATATCGGAGGGTGCAATCA 1321 TAGAAGACACCTTGTTGATGGGTGCTGACTACTACGAGAC 1361 TGATGCGGATAGGACACTCCTGGCTGCAAAAGGCAGCATC 1401 CCGATTGGCATTGGCCGAGACTCTCACATTAAAAGAGCTA 1441 TCATTGACAAGAATGCTCGTATTGGTGACAACGTCAAGAT 1481 CATCAACACGGACAATGTGCAAGAAGCTGCCAGAGAGACG 1521 GATGGATACTTCATCAAGAGCGGCATTGTTACAGTGATCA 1561 AGGATGCTCTGATTCCTAGTGGAACTGTTATCTAAGAGAC 1601 ACCACCACCCCGTTTGACAATCTTCTTAATATCTCATGGT 1641 TTTGACCTCGAGTTAGCTTCCACTGATGCTATCTCTAATG 1681 TTATCCGAGGTCAGGGCCTATGCATCCTCCTGCCTTATCC 1721 CTAAATAATATTCTCTACTTTGTATTCTAGTCTTTGGCGA 1761 T
(295) For an RNAi construct (e.g., an RNAi cassette) one example of an AGPase sense sequence for RNAi is the following sequence identified herein as SEQ ID NO:47:
(296) TABLE-US-00033 1 5-GGACTACGAGAAGTTCATTCAAGCGCATCGTGAGACCGAC 41 GCTGATATCACTGTTGCTGCTCTTCCTATGGATGAGAAAC 81 GTGCCACGGCTTTTGGACTTATGAAGATTGATGACGAAGG 121 AAGGATCATTGAGTTTGCTGAGAAGCCTAAAGGAGAGCAG 161 TTAAAGGCTATGAAGGTTGATACAACAATCTTGGGACTTG 201 ATGACGAAAGGGCCAAAGAGATGCCCTTTATTGCTAGTAT 241 GGGGATATATGTTGTTAGCAAGAATGTGATGTTGGACTTG 281 CTCCGAGACCAGTTCCCTGGAGCTAATGACTTCGGGAGTG 321 AAGTTATCCCTGGTGCTACTGATCTTGGACTCAGAGTGCA 361 AGCTTATCTGTATGATGGATACTGGGAAGATATTGGTACC 401 ATTGAAGCCTTTTACAATGCTAATCTTGGGATCACCAAGA 441 AACCAGTACCAGATTTCAGCTTCTATGACCGTTCAGCACC 481 AATCTACACACAGCCTCGGT-3
(297) A spacer DNA sequence with SEQ ID NO:48.
(298) TABLE-US-00034 1 5-AGCTCCTGCTCTGTGAATTTCTCCGCTCAC GATAGATCTG 41 CTTATACTCCTTACATTCAACCTTAGATCTGGTCTCGATT 81 TCTGTTTCTCTGTTTTTTTCTTTTGGTCGAGAATCTGATG 121 TTTGTTTATGTTCTGTCACCATTAATAATAATGAACTCTC 161 TCATTCATACAATGATTAGTTTCTCTCGTCTACAAAACGA 201 TATGTTGCATTTTCACTTTTCTTCTTTTTTTCTAAGATGA 241 TTTGCTTTGACCAATTTGTTTAGATCTTTATTTTATTTTA 281 TTTTCTGGTGGGTTGGTGGAAAGGAAAACAAAAAAAAAAC 321 AGCATAAATTGTTATTTGTTAATGTATTCATTTTTTGGCT 361 ATTTGTTCTGGGTAAAAATCTGCTTCTACTATTGAATCTT 401 TCCTGGATTTTTTACTCCTATTGGGTTTTTAAGGAAAACA 441 TACATAATAAAAGGAAAACAAAAGTTTTATAGATTCTCTT 481 AAACCCCTTACGATAAAAGTTGGAATCAAAATAATTCAGG 521 ATCAGATGCTCTTTGATTGATTCAGATGCGATTACAGTTG 561 CATGGCAAATTTTCTAGATCCGTCGTCACATTTTATTTTC 601 TGTTTAAATATCTAAATCTGATATATGATGTCGACAAATT 641 CTGGTGGCTTATACATCACTTCAACTGTTTTCTTTTGGCT 681 TTGTTTGTCAACTTGGTTTTCAATACGATTTGTGATTTCG 721 ATCGCTGAATTTTTAATACAAGCAAACTGATGTTAACCAC 761 AAGCAAGAGATGTGACCTGCCTTATTAACATCGTATTACT 801 TACTACTAGTCGTATTCTCAACGCAATCGTTTTTGTATTT 841 CTCACATTATGCCGCTTCTCTACTCTTTATTCCTTTTGGT 881 CCACGCATTTTCTATTTGTGGCAATCCCTTTCACAACCTG 921 ATTTCCCACTTTGGATCATTTGTCTGAAGACTCTCTTGAA 961 TCGTTACCACTTGTTTCTTGTGCATGCTCTGTTTTTTAGA 1001 ATTAATGATAAAACTATTCCATAGTCTTGAGTTTTCAGCT 1041 TGTTGATTCTTTTGCTTTTGGTTTTCTGCAGGTCAATTC-3
(299) The AGPase antisense sequence for the RNAi was as follows (with SEQ ID NO:49).
(300) TABLE-US-00035 1 5-ACCGAGGCTGTGTGTAGATTGGTGCTGAACGGTCATAGAA 41 GCTGAAATCTGGTACTGGTTTCTTGGTGATCCCAAGATTA 81 GCATTGTAAAAGGCTTCAATGGTACCAATATCTTCCCAGT 121 ATCCATCATACAGATAAGCTTGCACTCTGAGTCCAAGATC 161 AGTAGCACCAGGGATAACTTCACTCCCGAAGTCATTAGCT 201 CCAGGGAACTGGTCTCGGAGCAAGTCCAACATCACATTCT 241 TGCTAACAACATATATCCCCATACTAGCAATAAAGGGCAT 281 CTCTTTGGCCCTTTCGTCATCAAGTCCCAAGATTGTTGTA 321 TCAACCTTCATAGCCTTTAACTGCTCTCCTTTAGGCTTCT 361 CAGCAAACTCAATGATCCTTCCTTCGTCATCAATCTTCAT 401 AAGTCCAAAAGCCGTGGCACGTTTCTCATCCATAGGAAGA 441 GCAGCAACAGTGATATCAGCGTCGGTCTCACGATGCGCTT 481 GAATGAACTTCTCGTAGTCC-3
(301) One full sequence for an RNAi construct is as follows (SEQ ID NO:50).
(302) TABLE-US-00036 5-GGACTACGAGAAGTTCATTCAAGCGCATCG TGAGACCGAC 41 GCTGATATCACTGTTGCTGCTCTTCCTATGGATGAGAAAC 81 GTGCCACGGCTTTTGGACTTATGAAGATTGATGACGAAGG 121 AAGGATCATTGAGTTTGCTGAGAAGCCTAAAGGAGAGCAG 161 TTAAAGGCTATGAAGGTTGATACAACAATCTTGGGACTTG 201 ATGACGAAAGGGCCAAAGAGATGCCCTTTATTGCTAGTAT 241 GGGGATATATGTTGTTAGCAAGAATGTGATGTTGGACTTG 281 CTCCGAGACCAGTTCCCTGGAGCTAATGACTTCGGGAGTG 321 AAGTTATCCCTGGTGCTACTGATCTTGGACTCAGAGTGCA 361 AGCTTATCTGTATGATGGATACTGGGAAGATATTGGTACC 401 ATTGAAGCCTTTTACAATGCTAATCTTGGGATCACCAAGA 441 AACCAGTACCAGATTTCAGCTTCTATGACCGTTCAGCACC 481 AATCTACACACAGCCTCGGTAGCTCCTGCTCTGTGAATTT 521 CTCCGCTCACGATAGATCTGCTTATACTCCTTACATTCAA 561 CCTTAGATCTGGTCTCGATTTCTGTTTCTCTGTTTTTTTC 601 TTTTGGTCGAGAATCTGATGTTTGTTTATGTTCTGTCACC 641 ATTAATAATAATGAACTCTCTCATTCATACAATGATTAGT 681 TTCTCTCGTCTACAAAACGATATGTTGCATTTTCACTTTT 721 CTTCTTTTTTTCTAAGATGATTTGCTTTGACCAATTTGTT 761 TAGATCTTTATTTTATTTTATTTTCTGGTGGGTTGGTGGA 801 AAGGAAAACAAAAAAAAAACAGCATAAATTGTTATTTGTT 841 AATGTATTCATTTTTTGGCTATTTGTTCTGGGTAAAAATC 881 TGCTTCTACTATTGAATCTTTCCTGGATTTTTTACTCCTA 921 TTGGGTTTTTAAGGAAAACATACATAATAAAAGGAAAACA 961 AAAGTTTTATAGATTCTCTTAAACCCCTTACGATAAAAGT 1001 TGGAATCAAAATAATTCAGGATCAGATGCTCTTTGATTGA 1041 TTCAGATGCGATTACAGTTGCATGGCAAATTTTCTAGATC 1081 CGTCGTCACATTTTATTTTCTGTTTAAATATCTAAATCTG 1121 ATATATGATGTCGACAAATTCTGGTGGCTTATACATCACT 1161 TCAACTGTTTTCTTTTGGCTTTGTTTGTCAACTTGGTTTT 1201 CAATACGATTTGTGATTTCGATCGCTGAATTTTTAATACA 1241 AGCAAACTGATGTTAACCACAAGCAAGAGATGTGACCTGC 1281 CTTATTAACATCGTATTACTTACTACTAGTCGTATTCTCA 1321 ACGCAATCGTTTTTGTATTTCTCACATTATGCCGCTTCTC 1361 TACTCTTTATTCCTTTTGGTCCACGCATTTTCTATTTGTG 1401 GCAATCCCTTTCACAACCTGATTTCCCACTTTGGATCATT 1441 TGTCTGAAGACTCTCTTGAATCGTTACCACTTGTTTCTTG 1481 TGCATGCTCTGTTTTTTAGAATTAATGATAAAACTATTCC 1521 ATAGTCTTGAGTTTTCAGCTTGTTGATTCTTTTGCTTTTG 1561 GTTTTCTGCAGGTCAATTCACCGAGGCTGTGTGTAGATTG 1601 GTGCTGAACGGTCATAGAAGCTGAAATCTGGTACTGGTTT 1641 CTTGGTGATCCCAAGATTAGCATTGTAAAAGGCTTCAATG 1681 GTACCAATATCTTCCCAGTATCCATCATACAGATAAGCTT 1721 GCACTCTGAGTCCAAGATCAGTAGCACCAGGGATAACTTC 1761 ACTCCCGAAGTCATTAGCTCCAGGGAACTGGTCTCGGAGC 1801 AAGTCCAACATCACATTCTTGCTAACAACATATATCCCCA 1841 TACTAGCAATAAAGGGCATCTCTTTGGCCCTTTCGTCATC 1881 AAGTCCCAAGATTGTTGTATCAACCTTCATAGCCTTTAAC 1721 TGCTCTCCTTTAGGCTTCTCAGCAAACTCAATGATCCTTC 1761 CTTCGTCATCAATCTTCATAAGTCCAAAAGCCGTGGCACG 1801 TTTCTCATCCATAGGAAGAGCAGCAACAGTGATATCAGCG 1841 TCGGTCTCACGATGCGCTTGAATGAACTTCTCGTAGTCC-3
(303) Three representative lines were selected from each group for further analysis. On MS medium supplemented with 1% sucrose, the transgenic lines with single-gene constructs germinated and established into complete seedlings within 10-15 days. However, delayed development and expansion of cotyledons and leaves were observed in two of the AGPRNAi-WRI1 double gene transgenic lines 14 and 28, with these seedlings developing a glossy pale green appearance (
(304) On soil, the AGPRNAi-WRI1 lines established well and underwent normal growth and development compared to wild-type plants (
(305) As APS1 (At5g48300) was the most similar Arabidopsis gene to the B. napus AGPase-encoding sequence chosen for the RNAi constructs, the relative expression of APS1 mRNA in the transgenic lines was analyzed. In 15-day-old seedlings of AGPRNAi-WRI1 lines, in which the AGPRNAi cassette was expressed under the control of the Patatin B33 promoter, the abundance of APS1 mRNA was decreased between 1.4-fold and 1.9-fold (
(306) Presence of AGPRNAi led to the inhibition of starch biosynthesis and accumulation of sugars. To determine the effects of the transgenes on carbohydrate metabolism, starch, sucrose, glucose and fructose levels of 15-day-old seedlings from the two types of AGPRNAi lines were analyzed. Compared to wild-type plants, the level of starch was reduced by about half in the 35S-AGPRNAi and AGPRNAi-WRI1 lines (
(307) These results confirmed that the construct was effective in targeting starch biosynthesis and reducing carbon partitioning into starch. 35S-AGPRNAi and AGPRNAi-WRI1 lines accumulated considerably higher levels of sucrose, with a three to four-fold increase compared to wild-type plants (
(308) Correspondingly line 17 possessed comparatively lower WRI1 mRNA levels (90-fold) than the other two lines, in which the expression was almost 105-120-fold that of wild type (
(309) The first leaf pair and main root of the high oil accumulator line 28 and of a wild type was analyzed by confocal microscopy. As revealed by Nile red staining, the numbers and size of oil droplets were very distinct and high in line 28 leaf and root samples. The droplets were distributed close to the chloroplast in the leaf. In contrast, very few oil droplets were observed in the wild-type leaf and root samples.
(310) Expression of WRI1 target genes in high-oil lines is increased. To probe the efficacy of the WRI1 expression construct was examined. The relative mRNA abundance of known WRI1 target genes involved in glycolysis or fatty acid biosynthesis in 35S-WRI1 line 7, AGPRNAi-WRI1 line 28 and wild-type seedlings grown for 15 days on 1% sucrose. When normalized to wild-type values, the transcript levels for sucrose synthase 2 (SUS2) At5g49190 (14-fold), biotin carboxyl carrier protein isoform 2 (BCCP2) At5g15530 (3.7-fold) and acyl carrier protein 1 (ACP1) At3g05020 (5.6-fold) were considerably higher in AGPRNAi-WRI1 line 28 compared to 35S-WRI1 line 7 (3.2-, 2.2- and 3.6-fold, respectively;
(311) TABLE-US-00037 TABLE 11 Triacylglycerol (TAG) contribution to carbon and energy density of dry biomass compared to carbohydrates. Carbohydrate* TAG Carbo- TAG Starch TAG Carbon (molC/gDW) (molC/gDW hydrate* % % % % WT 4446.32 42.73 99.04 0.96 97.99 2.01 28 8619.19 248.52 97.2 2.8 79.83 20.17 Energy Carbohydrate*, TAG Carbo- TAG Starch TAG density (kJ/gDW) (molC/gDW) hydrate* % % % % WT 2.07 0.03 98.7 1.3 97.4 2.6 28 4.02 0.15 96.3 3.8 75.0 25.0 *Includes starch, sucrose, glucose and fructose. Based on 2803 kJ/mol for glucose CRC Handbook of Chemistry and Physics (2008), herein incorporated by reference. Based on 35 114 kJ/mol for triolein (Freedman and Bagby, 1989, herein incorporated by reference). Energy stored in TAG as percentage. WT, wild type; 28: AGPRNAi-WRINKLED1.
(312) TABLE-US-00038 TABLE12 Primersusedinmethodsdescribedherein. Actin2 At3g18780 F TGTGACAATGGTACCGGTATGG R GCCCTGGGAGCATCATCTC (SEQIDNO:62) ACP1 At3g05020 F TGTCTGGCAACAACAAGGATTAGT R GCGGAGGTTGAAGGATAGATTAGTC (SEQIDNO:63) 30AR At1g24360 F AAAGCCGTCGCGAAGCTA R GGAGCCAATTGTCGGATTTG (SEQIDNO:64) APS1 At5g48300 F ATGGCGTCTGTATCTGCAATTGGAG R GGATTTGAGTGAAATCTTGTCGTCG (SEQIDNO:65) BCCP2 At5g15530 F AACAGGCGGGTCGGATCT R GCGGCTGCCATCTTTGAG (SEQIDNO:66) PDHE1a At1g01090 F GAGGCCAAGGTGGATCCAT R CAAAGCCACCAAGCATGTTG (SEQIDNO:67) P1-PKb1 At5g52920 F CCAACGGTGGATCTGTGTCTAC R TCACTGCAAAACTCGCTGGTT (SEQIDNO:68) SUS2 AT5G49190 F CAGTCTGCAGAGGAAGCCATAGT R AGGTCTGGGACGTATAGCCAAA (SEQIDNO:69) TAG1 At2g19450 F TGGATTCTGCTGGCGTTACTAC R AGCCTATCAAGATCGACGAACTCT (SEQIDNO:70) WRI1 At3g54320 F AAACGAGCCAAAAGGGCTAAG R GGGCTTGTCGGGTTATGAGA (SEQIDNO:71) F- = Forward R = Reverse
Exemplary Materials and Methods.
(313) Plasmid construction. A double gene construct was made for duel function: an RNAi cassette (construct) for reducing AGPase translation (i.e. silencing AGPase) while overexpressing WRI1, see Cernac et al., 2004, herein incorporated by reference in its entirety. The WRI1 cDNA insert from Arabidopsis thaliana (Cernac and Benning, 2004) was obtained by PCR primers 5CCAAggatcc-AAATCTAAACTTTCTCAGAGT3 (SEQ ID NO:58) and 5CCTTacgcgtGGCAAA-GACATTGATTATT3 (SEQ ID NO:59). A fragment of 1463 bp containing the complete open reading frame was digested with BamHI and MluI. This fragment was then placed between a CaMV 35S (35S)/Patatin B33 (B33) promoter (Koster-Topfer et al., 1989, herein incorporated by reference), and a nos terminator to form an intermediate vector p35S-WRI1-nos and sequencing was performed. A C-terminal His-Tag was introduced by site-directed mutagenesis with two inverse primers WRI1HisR 5 gtgatgatgGACCAAATAGTTACAAGAAAC3 (SEQ ID NO:72) and WRI1HisF 5 catcaccatTGAGAGAGAGAGCTTT3 (SEQ ID NO:73). This vector was then digested with SfiI and inserted into a pLH6000 vector (DNA cloning service, Hamburg, Germany) to form 35S-WRI1 and B33-WRI1 (
(314) Plant material and transformation. Arabidopsis wild-type (Col2) seeds or transgenic seeds were surface sterilized and grown on Murashige and Skoog (MS) agar plates containing 1% sucrose in a growth chamber set to 16 h light/8 h dark (70-80 lm/m 2/s) at 22 C. after 2 days of stratification at 4 C. Fifteen-day-old wild-type and transgenic plants were transferred onto soil and grown in a growth chamber at 16 h light/8 h dark (70-80 lm/m 2/s) and 22 C. The binary vectors were introduced into Agrobacterium strain GV3101 by electroporation, and the T-DNAs were mobilized into Arabidopsis by flower-dip transformation (Clough and Bent, 1998). Transgenic plants (T1) were selected on MS agar plates containing 1% sucrose and 20 mg/L hygromycin or 100 mg/L kanamycin. In general, wild-type and homozygous transgenic seedlings were grown on the same shelf of the growth chamber when used for metabolite analysis and other assays.
(315) Quantitative real-time PCR (qRT-PCR). Total RNA was extracted from 15-day-old plants using an RNeasy Plant Mini Kit (Qiagen, Valencia, Calif.), and samples were stored at 80 C. Approximately 0.5 g of total RNA was reverse transcribed by SuperScript III (Invitrogen, Carlsbad, Calif.), according to the manufacturers protocol. The cDNA (1.0 L) was used as a template in a 23-L PCR consisting of forward and reverse gene-specific primers (Table 12) and Fast SYBR Green master mix (Applied Biosystems, Forest City, Calif.) on an Applied Biosystems PRISM 7000 Sequence Detection System. The copy numbers of the cDNAs were quantified using a standard curve method, and the copy numbers of each sample was standardized to ACTIN2. The relative expression (as fold change) was scaled such that the expression level of the wild type was equal to one in the graphs.
(316) Metabolite analysis. Fifteen-day-old wild-type and transgenic Arabidopsis whole seedlings were freeze-dried. Neutral lipids were extracted from dried samples using chloroform:methanol with 100 M internal standard tri15:0 TAG and separated on silica plate using a mixture of solvents consisting of petroleum ether:ethyl ether:acetic acid (80:20:1, by volume). After thin-layer chromatography (TLC), TAG bands were visualized with iodine vapor. For quantitative analysis, TAG bands were isolated from the TLC plate, dissolved in toluene with 1 M internal standard tri13:0 TAG and assayed using ESI-MS as described previously (Durrett et al., 2010, herein incorporated by reference in its entirety). Starch and sugars were analyzed by using an NAD(P)H-linked assay (Lowry & Passonneau, A flexible system of enzymatic analysis (Academic Press NY 1972), herein incorporated by reference in its entirety). About 5- to 10-mg dried whole seedling samples were ground into fine powder in a Retsch Mill at a frequency of 30 (maximum speed) for 30 s, soluble sugars were extracted by the addition of 1 mL 3.5% perchloric acid. Tubes were incubated on ice for 5 min before being centrifuged at 20,000 g for 10 min at 4 C. Approximately 750 L of supernatant was recovered and processed as described below for glucose, fructose and sucrose assays. The pellet was washed twice with 70% alcohol, resuspended in 500 L 0.2 M KOH and incubated at 95 C. for 30 min. Samples were allowed to cool for 2-5 min before adding 90 L of 1 M acetic acid to bring the pH to approximately 5. Starch in the pellet was broken down to glucose by the addition of an enzyme cocktail (50 L) consisting of 6.6 U amyloglucosidase (E-AMGDF; Megazyme, Wicklow, Ireland) and 50 U of alpha-amylase (E-ANAAM; Megazyme) and incubated for 2 days on a shaker at room temperature. Samples were then centrifuged for 30 min at 20,000 g, and the supernatant was transferred to fresh microfuge tubes. Five to ten microliters of the starch sample were assayed for glucose as described below. The 750-L supernatant recovered as described above was neutralized to pH 7 by the addition of 150 L of buffer consisting of 2 M KOH, 150 mM Hepes and 10 mM KCl. Samples were frozen in liquid nitrogen to help precipitate salts. Samples were then thawed and centrifuged at 20,000 g, and the supernatant was transferred to a fresh microfuge tube. Both starch and soluble sugars were assayed on a plate reader (Spectra Max M2, Molecular devices, Sunnyvale, Calif.) using an NADP(H)-linked assay at 340 nm. Samples were read in triplicate, using standard 96-well clear bottom plates. Each well that was used was 15 mM MgCl.sub.2, 3 mM EDTA, 500 nmol NADP, 500 nmol ATP and 0.4 U glucose-6-phosphate dehydrogenase (G-8529; Sigma, St Louis, Mo.). Five microliters of sample was added to each well, and the reaction was started by adding 0.5 U of Hexokinase (H-4502; Sigma). Fructose and sucrose were determined by the sequential addition of 4 U Phosphoglucose isomerase (P-9544; Sigma) and 25 U Invertase (I-4504; Sigma). Change in optical density ( OD) were determined by taking endpoint assays before and after the addition of each enzyme. Because the Spectra Max M2 plate reader can also determine the path length of aqueous samples, absorbance units were normalized to a 1-cm path length and absolute glucose amounts were determined using an extinction coefficient of 6220 L/mol/cm for Nicotinamide 2-phosphoadenine dinucleotide (NADPH) at 340 nm (Lowry and Passonneau, 1972, herein incorporated by reference).
(317) Microscopy. For oil droplet visualization, the first pair of leaves and main root samples from 15-day-old wild-type and transgenic seedlings were used. Freshly harvested leaf and root samples mounted in Nile red (2.5 g/mL methanol) in 75% glycerol on a slide were examined using an Olympus FluoView 1000 Laser Scanning Confocal Microscope. Oil droplets were observed at 570-630 nm emission following 559-nm excitation by a solid-state laser. Chloroplasts were observed at 655-755-nm emission using the same laser excitation. Images were captured with the Olympus FluoView 1000 ASW software (Olympus, Center Valley, Pa.).
Example VII
(318) This example illustrates that plants generated using the methods described herein accumulate higher levels of triacylglycerols (TAGs) in their vegetative tissue.
(319) Arabidopsis lines overexpressing WRINKLED1 (WRI1) alone, plants overexpressing WRINKLED with down-regulation of AGPase, and plants that overexpress WRINKLED with down regulation of AGPase in tgd1-1 mutant background were generated as described herein.
(320) After growth for fifteen days, vegetative tissues were collected from seedlings and dried. Triacylglycerol (TAG) levels in these vegetative tissues of the 15-day-old-seedling were measured by electrospray ionization mass spectrometry (ESI-MS) quantification.
(321)
(322) However, plants having combined WRINKLED overexpression, down regulation of AGPase, and reduced tgd function had about 10.3 nmoles triacylglycerols per mg dry weight of tissue. As illustrated wild type plants have substantially no detectable levels of triacylglycerols in their tissues.
(323) These data show that the methods and genetic constructs described herein provide plants with significantly more oil content than is present in wild type plants, or even in plants with increased WRINKLED expression and reduced AGPase expression.
(324) All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, plant biology, plant disease, agriculture, biofuels, biochemistry, chemistry, and plant pathogens or related fields are intended to be within the scope of the following claims. All references cited herein are incorporated in their entirety.