Plants having enhanced nitrogen use efficiency and methods of producing same
09695436 ยท 2017-07-04
Assignee
Inventors
- Chandra McAllister (Edmonton, CA)
- Perrin Beatty (Edmonton, CA)
- Jayne D'Entremont (Barriere, CA)
- Allen Good (Edmonton, CA)
Cpc classification
C12N15/8261
CHEMISTRY; METALLURGY
Y02A40/146
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
Methods are provided for enhancing yield and nitrogen use efficiency in plants, and to methods of increasing biomass and seed yield in plants grown under nitrogen limiting conditions using variants of enzymes involved in nitrogen assimilation or metabolism from non-plant organisms.
Claims
1. A transgenic plant, plant cell, or plant part comprising a polynucleotide encoding an alanine amino transferase (AlaT) protein from a non-plant organism gene operably linked to a PBpr1 promoter which comprises the nucleotide sequence of SEQ ID No. 5, wherein the polynucleotide comprises a nucleotide sequence encoding a protein having at least 80% amino acid identity to the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16.
2. The transgenic plant, plant cell, or plant part according to claim 1, wherein the plant is corn, wheat, maize, rice, barley, canola, soybean, cotton, alfalfa, safflower, tomato or potato.
3. A seed obtained from the transgenic plant according to claim 1, comprising said polynucleotide operably linked to said PBpr1 promoter.
4. The transgenic plant, plant cell, or plant part according to claim 1, wherein the plant cell is a rice plant cell, a barley plant cell, a wheat plant cell or a maize plant cell.
5. The transgenic plant, plant cell, or plant part according to claim 1, wherein the plant cell is a rice plant cell.
6. The transgenic plant, plant cell, or plant part according to claim 1, wherein the gene encoding the AlaAT protein comprises a nucleotide sequence having 95% identity to SEQ ID No. 3 or 4.
7. The transgenic plant, plant cell, or plant part according to claim 6, wherein the plant cell is a rice plant cell, a barley plant cell, a wheat plant cell or a maize plant cell.
8. A genetic construct comprising a polynucleotide encoding an alanine amino transferase (AlaT) protein from a non-plant organism operably linked to a PBpr1 promoter which comprises the nucleotide sequence of SEQ ID No. 5, wherein the polynucleotide comprises a nucleotide sequence encoding a protein having at least 80% amino acid identity to the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16.
9. The genetic construct according to claim 8, wherein the polynucleotide comprises a nucleotide sequence having 95% identity to SEQ ID No. 3 or 4.
10. A method of generating a plant having increased biomass, increased nitrogen use efficiency or increased seed yield comprising: transforming a plant cell with the genetic construct according to claim 9, and growing the transformed plant cell to produce a plant that expresses the protein, thereby generating a plant having increased biomass, increased nitrogen use efficiency or increased seed yield, wherein the increased biomass, increased nitrogen use efficiency or increased seed yield is relative to the biomass, nitrogen use efficiency or seed yield of a wild-type plant grown under identical conditions.
11. A method of generating a plant having increased nitrogen use efficiency, increased biomass or increased seed yield comprising: transforming a plant cell with the genetic construct according to claim 8, and growing the transformed plant cell to produce a plant that expresses the protein, thereby generating a plant having increased nitrogen use efficiency, increased biomass or increased seed yield, wherein the increased nitrogen use efficiency, increased biomass or increased seed yield is relative to the nitrogen use efficiency, biomass or seed yield respectively of a wild-type plant grown under identical conditions.
12. The method according to claim 11, wherein the plant is corn, wheat, maize, rice, barley, canola, soybean, cotton, alfalfa, safflower, sugarcane, tomato or potato.
13. A method for producing a plant having increased nitrogen use efficiency, increased biomass, increased seed yield, earlier development time, or a combination thereof, comprising: growing a plant from a plant, plant part or seed, comprising the genetic construct according to claim 8, thereby producing a plant having increased nitrogen uptake, increased biomass, increased seed yield, earlier development time, or a combination thereof, wherein the increased nitrogen uptake, increased biomass and increased seed yield is relative to the nitrogen uptake, biomass and seed yield of a wild-type plant grown under identical conditions.
14. The method of claim 13, further comprising providing a fertilizer to the plant or the habitat of the plant.
15. The method of claim 14, wherein the fertilizer is a nitrogen containing fertilizer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
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DETAILED DESCRIPTION
(20) The present invention relates to monocot plants having enhanced yield and/or nitrogen utilization efficiency (NUE), to methods of producing such plants, to methods for enhancing yield and NUE in monocot plants, and to methods of increasing biomass and seed yield in monocot plants grown under nitrogen limiting conditions. This invention also relates to variants of AlaAT and their operative linkage with different promoters, for example, without limitation, the cauliflower mosaic virus promoter CaMV 35S and tissue specific promoters (such as OsANT1 and PBpr1; Shrawat et al., 2008; Lock, 2011). The present invention also relates to the use of AlaAT encoding polynucleotides from non-plant organisms in plants, and the use of different variants of AlaAT encoding polynucleotides from organisms such as Archaea and mammals in plants under control of a plant-expressible promoter.
(21) The following description is of a preferred embodiment.
(22) The headings provided are not meant to be limiting of the various embodiments of the invention. Terms such as comprises, comprising, comprise, includes, including and include are not meant to be limiting. In addition, the use of the singular includes the plural, and or means and/or unless otherwise stated. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
(23) The language tissue-specific expression of a target nucleotide sequence is known in the art and includes the expression of a target nucleotide sequences in only selected tissues; although the target nucleotide sequence may be present in multiple tissues, it is expressed in only a subset of those tissues. Such selective expression may be due to the influence of one or more regulatory genetic elements, for example but not limited to promoter elements, repressor elements, enhancer elements, or other regulatory factors that may interact with DNA, RNA.
(24) The language target gene or target nucleotide sequence is art-recognized, and includes any nucleotide sequence which is desirably expressed in one or more selected plant tissues. Examples of target genes or nucleotide sequences which may advantageously be utilized in conjunction with the methods of the invention include genes or nucleotide sequences involved in nitrogen assimilation and/or utilization, genes or nucleotide sequences involved in stress resistance or disease and pest resistance, and genes or nucleotide sequences involved in nutrient uptake and utilization. Such genes or nucleotide sequences are well known to one of skill in the art.
(25) The language plant is art-recognized, and includes any monocotyledenous or dicotyledenous plant. Examples of plants for use in the invention include but are not limited to canola, barley, maize, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato, Arabidopsis thaliana and certain tree genera, including conifers and Populus species.
(26) The language non-plant includes any other organisms that are not plants, such as, but not limited to, microorganisms (for example, without limitation, bacteria, yeast, fungi, algae, Archaea, virus), mammals and non-mammals (for example, without limitation, arthropods, birds, reptiles, amphibians and fish).
(27) The language operative linkage is art-recognized, and includes the placement of a target nucleotide sequence relative to a nucleic acid regulatory sequence such that the expression of the target nucleotide sequence is controlled by the regulatory sequence. This regulatory sequence can have a positive effect (increase) on the expression of the target gene or nucleotide sequence (e.g., the regulatory sequence is a promoter or an enhancer element), or the regulatory sequence can reduce the expression of the target gene or nucleotide sequence (e.g., the regulatory sequence is a repressor element). The regulatory sequence may be physically located 5 or 3 of the target gene or nucleotide sequence, may be within the coding sequence of the target gene or nucleotide sequence, or may be contained on an intron within the target gene or nucleotide sequence.
(28) The language nulls is art-recognized and includes a plant that has undergone tissue culturing but does not carry a transgene or selectable marker.
(29) Production of plants which express one or more target genes or nucleotide sequences under a plant-expressible promoter are described herein. The invention further provides seeds containing one or more target genes or nucleotide sequences under the control of a plant-expressible promoter, such as a tissue-specific promoter element that specifically directs the tissue-specific expression of the gene or nucleotide sequence. By the methods of the invention, it is possible to produce plants having one or more desired traits or properties in selected tissues; e.g., to alter specifically the genetic and/or physiological properties of the fruit or the roots of the plant. The invention further provides methods of producing plants having root and leaf-specific expression of one or more desired nucleotide sequences, using the OsANT1 and PBpr1 promoter elements (Shrawat et al., 2008; Lock, 2011).
(30) The methods of the invention for the production of plants involve the operative linkage of one or target genes or nucleotide sequences to a genetic regulatory element, such as, but not limited to, a promoter. As is known in the art, promoters are nucleic acid sequences that allow for regulation of transcription of a gene or nucleotide sequence. Promoters can allow for constitutive expression, such as the well known cauliflower mosaic virus promoter CaMV 35S, inducible expression, such as the stress inducible promoter rd29A (Pino et al., 2007), tissue-specific expression, such as the root-specific OsANT1 promoter (Good et al., US 2009/0288224), and developmentally specific expression, such as the senescence induced IPT promoters (Ma 2008). Promoters can also be weak or strong, suggesting that whenever or wherever they are induced, they will allow for expression of the attached gene or nucleotide sequence at varying levels.
(31) The methods of the invention for the production of plants having tissue-specific expression of one or more target genes or nucleotide sequences are accomplished through the use of a genetic regulatory element which directs the tissue-specific expression of the target gene(s). This regulatory element may be either negative or positive in activity: a plant tissue-specific promoter or enhancer element permits the expression of the target gene or nucleotide sequence(s) in one or more specific tissues, whereas a plant tissue-specific repressor suppresses the expression of the target genes or nucleotide sequences in one or more specific tissues, while expression in the other tissue(s) continues unabated. For the purposes of the present invention, it will be understood that promoter sequences constitute the preferred genetic regulatory elements of the invention.
(32) It will be understood by one skilled in the art that modifications may be made to the promoters used in the methods and constructs of the invention to improve or modulate the activity of the promoter. Multiple copies of a selected promoter may be operatively linked to a single target gene or nucleotide sequence to thereby alter the expression level of the linked gene or nucleotide sequence, or a selected promoter may be operatively linked to one or more target genes or nucleotide sequences such that the expression of each target gene or nucleotide sequence is coordinately regulated. A promoter may be of any size appropriate to permit the tissue-specific functioning of the promoter. A promoter may be modified (e.g., by mutagenesis, deletion, insertion, or truncation) to alter the degree to which the operatively linked gene or nucleotide sequence is expressed in the selected tissue, or to alter the specificity of tissue expression directed by the promoter. Further, the placement of the promoter relative to the operatively linked target gene or nucleotide sequence may be modulated (e.g., moved further away or closer together) to attain a desired level of promoter-directed expression.
(33) A target gene or nucleotide sequence of the invention may be any gene or nucleotide sequence which is desirably expressed in a plant, including the expression in a tissue-specific manner in a plant. General classes of target genes or nucleotide sequences that may be employed in the methods and constructs of the invention include one or more genes or nucleotide sequences encoding plant structural proteins, genes or nucleotide sequences encoding proteins involved in the transport and/or uptake of nutrients, genes or nucleotide sequences encoding enzymes and proteins involved in nutrient utilization, genes or nucleotide sequences encoding proteins involved in stimulation or continuation of plant growth. Further, the target gene or nucleotide sequence may be a nucleotide sequence which, when transcribed, is antisense to a native sequence, the transcription and translation of which is desired to be suppressed.
(34) For example, which is not to be considered limiting in any manner, the genes or nucleotide sequences of interest are those encoding enzymes in the assimilation and/or metabolism of nitrogen. The genes or nucleotide sequences of interest may include genes or nucleotide sequences which encode proteins involved in assimilating ammonia into amino acids or use the formed amino acids in biosynthetic reactions, that is, nitrogen utilization proteins. Example of nitrogen utilization proteins include, but are not limited to, nitrate ammonium and amino acid transporters, glutamine synthetase (GS), asparagine synthetase (AS), glutamate synthase (also known as glutamate 2:oxogluturate amino transferase and GOGAT), asparaginase (ANS), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT) and alanine aminotransferase (AlaAT) and those genes or nucleotide sequences which may be involved in Nutrient Use Efficiency (NUE) as described by Beatty et al. (2009), and in U.S. Pat. No. 7,589,257 (which is incorporated herein by reference).
(35) The target gene or nucleotide sequence may be a gene or nucleotide sequence naturally expressed in a selected plant (endogenous), or it may be not naturally expressed in the selected plant (non-endogenous). The gene or nucleotide sequence may originate from a plant or from a non-plant, including, without limitation, viral, bacterial, Archaea or animal sources. Preferably, the gene or nucleotide sequence is heterologous to the promoter to which it is linked, in that it is not linked to an unmodified, inducible promoter to which the gene or nucleotide sequence is naturally linked. Thus, as used herein, the term heterologous with respect to a nucleic acid or DNA refers to a nucleic acid or DNA which is operably linked to a nucleic acid to which it is not naturally linked in nature. For example, a promoter is said to be heterologous with respect to a coding region, when that promoter is not naturally linked to that coding region.
(36) As used herein, the term endogenous with respect to a nucleic acid molecule or a polypeptide in an organism refers to a nucleic acid molecule or a polypeptide having a nucleotide sequence or respectively an amino acid sequence, which is naturally occurring within cells of that organism. Nucleic acid molecules or polypeptides do not become endogenous to a particular organism because that organism or cells of that organism have been modified using, for example, recombinant DNA techniques to artificially contain such nucleic acid molecule or polypeptide. Therefore, as used herein, a non-endogenous nucleic acid molecule or polypeptide is a nucleic acid molecule or polypeptide that is not naturally occurring with cells of an organism, and may have been artificially introduced.
(37) It is therefore contemplated herein that the target gene or nucleotide sequence be a non-plant, non-endogenous AlaAT for expression in a plant, such as SEQ ID NOs: 1, 3 or 4 and Mus musculus AlaAT2, which is known in the art (see for example McAllister et.al, 2013; which is incorporated herein by reference). The target gene or nucleotide sequence can be any non-plant nucleotide sequence having an nucleic acid sequence similarity of approximately 70% to the nucleic acid sequence of barley (Hordeum vulgare) AlaAT (SEQ ID NO:1), mouse (Mus musculus) AlaAT (SEQ IDNO:3), Mus musculus AlaAT2, (McAllister et.al, 2013), or Pyrococcus furiosus AlaAT (SEQ ID NO:4), or any amount between 70% and 100% amino acid sequence, for example, but not limited, to 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleic acid sequence similarity to the nucleic acid sequence of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT, Mus musculus AlaAT2, (McAllister et.al, 2013), or Pyrococcus furiosus AlaAT, or any amount therebetween.
(38) Sequence identity or sequence similarity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (for example, using, but not limited to, the following parameters: GAP penalty 5, # of top diagonals 5, fixed GAP penalty 10, k-tuple 2, floating gap 10, and window size 5). However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482), Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (e.g. GAP, BESTFIT, FASTA, and BLAST), or by manual alignment and visual inspection.
(39) Also included are nucleotide sequences that hybridize under stringent hybridization conditions to SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:4, or Mus musculus AlaAT2, (McAllister et.al, 2013). The present invention also includes a nucleotide sequence that hybridizes under stringent hybridization conditions to a compliment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:4, or Mus musculus AlaAT2, (McAllister et.al, 2013). These nucleotide sequences that hybridize to SEQ ID 1, 3, 4, or Mus musculus AlaAT2, (McAllister et.al, 2013), a complement of SEQ ID 1, 3, 4, or Mus musculus AlaAT2, (McAllister et.al, 2013), encode a protein that exhibits AlaAT activity.
(40) Hybridization under stringent hybridization conditions is known in the art (see for example Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 and supplements; Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A Laboratory Manual, 3.sup.rd edition 2001; each of which is incorporated herein by reference). An example of one such stringent hybridization conditions may be about 16-20 hours hybridization in 4SSC at 65 C., followed by washing in 0.1SSC at 65 C. for an hour, or 2 washes in 0.1SSC at 65 C. each for 20 or 30 minutes. Alternatively, an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4SSC at 42 C., followed by washing in 0.1SSC at 65 C. for an hour, or 2 washes in 0.1SSC at 65 C. each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO.sub.4 buffer pH 7.2; 10 mM EDTA) at 65 C., with 2 washes either at 50 C. in 0.1SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65 C. in 2SSC, 0.1% SDS for 20 or 30 minutes each.
(41) In addition, the target gene or nucleotide sequence can be any non-plant nucleotide sequence having an amino acid sequence similarity of approximately 80% to the amino acid sequence of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT or Pyrococcus furiosus AlaAT, or any amount between 80% and 100% amino acid sequence, for example, but not limited, to 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence similarity to the amino acid sequence of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT or Pyrococcus furiosus AlaAT, or any amount therebetween (see
(42) TABLE-US-00001 mRNA NCBI Protein NCBI Species Gene Reference Reference H. sapiens GPT NM_005309.2 NP_005300.1 M. mulatta GPT XM_001093616.2 XP_001093616.1 C. lupus GPT XM_847258.2 XP_852351.2 B. taurus GPT NM_001083740.1 NP_001077209.1 M. musculus Gpt NM_182805.2 NP_877957.1 R. norvegicus Gpt NM_031039.1 NP_112301.1 D. rerio gpt21 NM_001142774.1 NP_001136246.1 D. rerio LOC100148522 XM_001919861.2 XP_001919896.1 S. cerevisiae ALT1 NM_001181976.1 NP_013190.1 K. lactis KLLA0F19162g XM_455940.1 XP_455940.1 S. pombe SPBC582.08 NM_001021084.1 NP_595176.1 318829 MGG_06503 XM_369988.2 XP_369988.2 N. crassa NCU03973 XM_952519.2 XP_957612.1 A. thaliana ALAAT2 NM_105892.4 NP_565040.2 A. thaliana AlaAT1 NM_101591.5 NP_173173.3 O. sativa Os10g0390500 NM_001071039.1 NP_001064504.1 Genbank NCBI Species Protein Accession No. Reference Thermococcus Alanine AEK72704.1 sp. aminotransferase Thermococcus Aspartate EEB72915.1 YP_002581272.1 sp. aminotransferase Thermococcus Aspartate ADT83250.1 YP_004070473.1 barophilus aminotransferase Thermococcus Alanine ZP_11216073.1 zilligii aminotransferase Thermococcus aminotransferase AFL94660.1 YP_006424454.1 sp. 2 Thermococcus Alanine ACS33579.1 YP_002959443.1 gammatolerans aminotransferase Thermococcus Alanine ACJ15716.1 YP_002306613.1 onnurineus aminotransferase Thermococcus Alanine ACS90484.1 YP_002994833.1 sibiricus aminotransferase Thermococcus alanine BAD85283.1 YP_183507.1 kodakarensis aminotransferase Thermococcus Alanine AAK98527.1 ZP_09730551.1 litoralis aminotransferase Pyrococcus Alanine AEH24862.1 YP_004624134.1 yayanosii aminotransferase Pyrococcus sp. alanine AEC52815.1 YP_004424819.1 aminotransferase Pyrococcus sp. alanine AFK22834.1 YP_006354909.1 aminotransferase Pyrococcus alanine NP_143210.1 horikoshii aminotransferase Pyrococcus alanine NP_126507.1 abyssi aminotransferase Pyrococcus Chains A, B, 1XI9_A furiosus C and D alanine aminotransferase
(43) The target gene or nucleotide sequence may be, but not necessarily, modified as required. For example, the gene or nucleotide sequence may be modified to be transcribable and translatable in a plant system; for example, the gene or nucleotide sequence can be modified such that it contains all of the necessary poly-adenylation sequences, start sites and termination sites which allow the coding sequence to be transcribed into messenger ribonucleic acid (mRNA) and the mRNA to be translated into a functional protein in the selected plant system. Further, the target gene or nucleotide sequence may be modified such that its codon usage is more similar to that of native genes or nucleotide sequences or nucleotide sequences of the selected plant. Such target gene or nucleotide sequence modifications and the methods by which they may be made are well known in the art.
(44) The methods and genetic constructs disclosed herein may be used to produce a plant or a plant part of any species capable of utilizing the promoter such that the transgenic plant, or non-natural plant, has tissue-specific expression of one or more desired genes or nucleotide sequences. Both monocotyledenous and dicotyledenous plants are amenable to such alteration. The invention is intended to be particularly applicable to, for example, crop plants (especially those of the genus Oryza), ornamental plants, and trees (particularly conifers and the genus Populus). Particularly suitable plants for the practice of the present invention include, but are not limited to, canola, barley, sugar cane, corn, canola, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato, aspen, cottonwood, Arabidopsis thaliana, conifers and poplar, or parts of any of these plants, for example, roots, root tips, leaves, stems, flowers, apical buds, meristematic tissues, and the like.
(45) The transgenic plants (non-natural plants), plant parts, and seeds produced according to the present invention may be further useful in breeding programs for the production of plant species having more than one desired trait. For example two transgenic plants of the invention each having expression of a desired transgene in differing plant tissues may be crossed to result in progeny transgenic plants having tissue-specific expression of both transgenes; or two transgenic plants of the invention each having expression of a different desired transgene in the same plant tissue may be crossed to result in progeny transgenic plants having tissue-specific expression of both transgenes. In this fashion it is possible to produce transgenic plants having a combination of desirable traits in selected tissue(s) of the plant.
(46) Furthermore, it will be understood by one skilled in the art that different species of plants may be more or less amenable to genetic manipulation in general, and that, therefore, it may be advantageous to first transform a related species of the desired plant by the methods and with the constructs of the invention and to subsequently introduce the tissue-specific expression of the target gene or nucleotide sequence into the desired plant species by cross-breeding techniques. Such techniques and appropriately related plant species are well known to one skilled in the art.
(47) Plant cells or protoplasts that have been transformed with the gene constructs of the present invention can be regenerated into differentiated plants using standard nutrient media supplemented with shoot-inducing or root-inducing hormone, using methods known to those skilled in the art (see, for example, Shahin, E. A. U.S. Pat. No. 4,634,674 and references therein, incorporated herein by reference in their entirety). Seeds may additionally be harvested from such transgenic plants using methods well known in the art and further used to re-grow the transgenic plants and hybrids of the invention.
Uses of the Invention
(48) The methods and constructs of the invention allow the production of plants and seeds having expression of one or more desired genes or nucleotide sequences in one or more selected tissues of the plant. Thus, the methods and constructs of the invention permit the production of plants having one or more desired traits limited to selected plant tissues, thereby enabling the targeting of a trait to the tissue to which it is best suited, or avoiding the expression of a desirable gene or nucleotide sequence in a tissue where its effects are unwanted. There are a wide variety of specific applications of the invention, including, but not limited to, the production of plants having increased yield, stress tolerance, having improved nutrient uptake and/or utilization, having improved nutrient content and/or yields of desired compounds. Specific applications of the invention are further described below.
(49) One application of the invention is in the production of plants better able to thrive on nutrient-poor soils. It is well known in the art that certain plant species, particularly crop plants, deplete the soil of nutrients necessary to sustain growth, such as nitrogen, phosphate, and potassium. In order to replenish the lacking nutrients, it is necessary either to fertilize the soil (an expensive and environmentally damaging procedure) or to cultivate plants known to deposit the depleted nutrient into the soil (e.g., clover or soybean in the case of nitrogen depletion), which may be crops that are less profitable or nutritive and therefore less desirable to grow. Frequent fertilizing to maintain optimal nutrients in the soil is costly in terms of agriculture outputs such as labour and fuel therefore crop plants are usually in a state of either nutrient abundance or depletion. Another application of the invention is in the production of plants better able to capture and utilize nitrogen when it is present in either an adequate or abundant supply in the soil before nitrogen loss due to leaching, volatilization or microbial degradation occurs. The methods of the invention permit the targeted expression of genes or nucleotide sequences involved in nutrient uptake (e.g., transport molecules) to those tissues in which the uptake occurs (e.g., the root or root hairs) to thereby improve the ability of the plant to absorb the nutrient from the environment. The invention may also be used to produce plants which express heterologous nutrient utilization nucleotide sequences, or optimized (for example, optimized for plant expression) native nutrient utilization nucleotide sequences, in selected tissues (e.g., the root or leaves) that permit more efficient use of the nutrient, such that less of the nutrient is required for the normal growth and functioning of the plant. Further, it is possible, using the methods of the invention, to express genes or nucleotide sequences involved in the use and uptake of nutrients not normally used by the plant in those plant tissues which are directly exposed to the different nutrient (e.g., root and leaf). In this fashion, plants which are able to grow and thrive on different nutrient sources (e.g., different nitrogen sources) may be produced.
(50) Development of Novel Cereal Crops:
(51) As described below, Oryza sativa (rice) plants may be transformed with novel gene contracts such that the plants ectopically express a nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism, for example, but not limited to, alanine dehydrogenase, glutamine synthetase, asparagine synthetase, glutamate synthase, asparaginase, glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, and those nucleotide sequences which may be involved in Nutrient Use Efficiency described by Beatty et al. (2009), and U.S. Pat. No. 7,589,257. The present invention contemplates transformation of Oryza sativa with a novel gene construct, as described above, operatively linked to a tissue-specific promoter. The transformed plant is grown under laboratory conditions to determine the beneficial effects of tissue-specific expression on plant growth and yield under controlled growth conditions.
(52) As indicated in Example 4 below, transgenic plants expressing a gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism under the control of a tissue-specific promoter (pPBpr1) exhibited higher biomass and seed weight than those plants expressing a gene or nucleotide sequence expressed under a constitutive promoter (maize ubiquitin 1 promoter, ubi-1) in the presence of an adequate supply of nitrogen.
(53) Transgenic Oryza plants ectopically expressing a gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism under the control of a tissue-specific promoter (pPBpr1) are also capable of optimising the utilization of available nitrogen under a range of environmental conditions thereby resulting in an increase in plant biomass, seed weight or a combination thereof.
(54) Therefore, the present invention provides a method for increasing seed yield of a plant comprising; transforming the plant with a target gene or nucleotide sequence in operative linkage with a promoter element, to produce a transformed plant, the target gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism; and growing the transformed plant.
(55) The above description is not intended to limit the claimed invention in any manner, furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.
(56) The present invention will be further illustrated in the following examples. In order to assess whether enzyme isoforms of AlaAT have different kinetics and if different isoforms favor an NUE phenotype, AlaAT from mouse and Pyrococcus were tested.
EXAMPLES
(57) Materials and Methods
(58) Expression Vector Construction and Arabidopsis thaliana Transformation
(59) The alanine aminotransferase enzymes assayed were chosen based on differences in their amino acid sequence as described by McAllister et al. 2012 (see also
(60) TABLE-US-00002 TABLE 1 List of promoters, genes and sequences used to build the specific expression vectors for transformation into Arabidopsis thaliana. Ter- Vector Vector Accession mi- Back- Name Promoter Gene # nator bone Arabi- 2XCaMV35S HvAlaAT Z26322 nos binary dopsis vector pMDC32 Arabi- 2XCaMV35S Mm1AlaAT NP_877957 nos binary dopsis vector pMDC32 Arabi- 2XCaMV35S Mm2AlaAT NP_776291 nos binary dopsis vector pMDC32 Arabi- 2XCaMV35S PfAlaAT NP_579226 nos binary dopsis vector pMDC32 Arabi- OsAnt1 HvAlaAT Z26322 nos binary dopsis vector pMDC32
(61) Binary vectors were transformed into Agrobacterium tumefaciens strain GU3101 and selected using kanamycin. Agrobacterium cultures (OD.sub.600 0.3-0.7) were then used to transform Arabidopsis thaliana plants, both COL (ecotype Columbia background) and alaat1;2 knockout backgrounds, using floral dip (alaat1;2 knockout background is explained in detail in Miyashita, 2008). Transformed plants were selected by sowing seeds on hygromycin (Hyg) and using a protocol modified from Harrison et al. (2006), with plants left covered at 4 C. for 4 days and at room temperature for 3 days; PCR was carried out on all primary transgenics with primers specific to the AlaAT insert: for HvAlaAT, 5-GAGGTTCTTGCCCTTTGTGA-3 (SEQ ID No: 19) and 5-TTCAGCTCGTTGCAAGTAA-3 (SEQ ID No: 20); for MmAlaAT1, 5-CCAGAGGATGCCAAGAGAAG-3 (SEQ ID No: 21) and 5-GCTCCGTGAGTTTAGCCTTG-3 (SEQ ID No: 22); for MmAlaAT2, 5-GCAGGCTTGTGGTGGAAA-3 (SEQ ID No: 23) and 5-GCACTTTCTTAAAGGAGTGGAATC-3 (SEQ ID No: 24); for PfAlaAT, 5-GCGCTCTACGACAAAAAGACACTTGA-3 (SEQ ID No: 25) and 5-CGTTAGTCCTGCTATAGCTGCGAATT-3 (SEQ ID No: 26). T3 seed was sown on Hyg media to select for homozygous lines; PCR was used to verify the presences of the specific AlaAT insertions. Three independent Arabidopsis insertion lines for each AlaAT construct, in both COL and alaat1;2 knockout backgrounds, were selected for and used for future analyses.
(62) Plate Assays
(63) Sterilized seeds were stratified in 0.15% (w/v) agar for approximately 48 hrs; sterilized seeds were sown onto square 10010015 mm petri plates containing modified MS media (0.5% (w/v) sucrose and 0.8% (w/v) agar) with 0 mM KNO.sub.3.sup., 2 mM KNO.sub.3.sup., 0.25 mM KNO.sub.3.sup., 2.5 mM alanine or 2.5 mM glutamate as the sole nitrogen source. Seeds from both control lines, COL and alaat1;2, as well as three independent insertion lines each of OsANT1:HvAlaAT, CaMV35S:PfAlaAT, CaMV35S:HvAlaAT, CaMV35S:MmAlaAT1 and CaMV35S:MmAlaAT2, in both a COL and an alaat1;2 knockout background, were used for analysis on 0 mM, 0.25 mM and 2 mM KNO.sub.3.sup. plates.
(64) Transgenic plants containing OsANT1:HvAlaAT, CaMV35S:PfAlaAT and CaMV35S:MmAlaAT1, in both COL and alaat1;2 knockout backgrounds, were used for analysis on 2.5 mM glutamate and alanine. Plants were sown horizontally across square petri plates approximately 2 cm from the top of each plate; 6 plants were sown per plate. Control plants and transgenics were sown on the same plates in an alternating fashion. Three independent insertion lines for each AlaAT were assayed in quadruplicate along with control plants. Plants were grown vertically at 21 C., 60% humidity and a lighting cycle of 16 light/8 dark. Chambers were blocked for variations in lighting conditions, resulting in four blocks, with final lighting blocks containing a maximum difference of 20% across a single block with an average light intensity of 170 E m.sup.2 sec.sup.1. Plates were moved within lighting blocks daily and monitored for changes in growth and development. Changes in vertical tap root length were measure (cm) between 0-5 after sowing (DAS), 5-8 DAS and 8-12 DAS for plants grown on 2 mM and 0.25 mM KNO.sub.3.sup.. The vertical growth of tap roots of plants grown on 2.5 mM glutamate and alanine was measured (cm) between 0-5, 5-8, 8-12, 12-15, 18 and 21 DAS for changes in root length. Changes in tap root lengths over these time periods were then used to analyze the rate of tap root growth between transgenics and controls. At 18 DAS or 21 DAS (earlier if plants were senescing or had grown too large for plates) tap root lengths were measured (cm) on all MS plates (top of hypocotyl to root tip).
(65) MS plates were also prepared varying nitrogen and carbon amounts. Modified MS (0.8% (w/v) agar) containing 1 mM KNO.sub.3.sup. and 0% (w/v) sucrose was used to grow transgenic plants containing OsANT1:HvAlaAT, CaMV35S:PfAlaAT and CaMV35S:MmAlaAT1, in both COL and alaat1;2 knockout backgrounds, as well as control lines, in two different lighting conditions: 170 E m.sup.2 sec.sup.1 (high) and 100 E m.sup.2 sec.sup.1 (low) (see
(66) Phenotypic similarity between individual plant lines expressing the same AlaAT construct in all plating conditions was assessed via one-way ANOVA (=0.05, P value<0.05). Final tap root lengths of control plants and transgenic lines were compared using two-way ANOVA (P value<0.05), analyzing the genotype of plants and the lighting block plants were grown in.
(67) Analysis of AlaAT Primary Structure
(68) The enzymes with AlaAT activity that were obtained for expression studies and kinetic analysis and the amino acid sequences were compared using ClustalW software (full primary sequence comparison is provided in McAllister et al. 2012 (see
(69) Construction of Binary Vectors and Agrobacterium Mediated Transformation
(70) The sequences of the genes using in the cloning are described in
(71) The constructs were transformed separately into Agrobacterium tumefaciens strain EHA105 by the freeze thaw method (Weigel and Glazebrook, 2002). The transformation of Arabidopsis thaliana is as described above.
(72) Rice callus (Oryza sativa c.v. Nipponbare (NB)) was transformed with constructs, using an Agrobacterium transformation system developed in our laboratory (Shrawat and Good, 2011). The final AlaAT constructs for transformation into Oryza sativa c.v. Nipponbare (NB), the initial AlaAT source, the promoter used to drive AlaAT expression in vivo, as well as the binary vectors utilized for transformation are summarized in Table 2.
(73) TABLE-US-00003 TABLE 2 List of promoters, genes and sequences used to build the specific expression vectors for transformation into Oryza sativa. Vector Accession Termi- Vector Name Promoter Gene # nator Backbone pGPMn PBpr1 Mm1AlaAT NP_877957 nos pGPro2 pGPPn PBpr1 PfAlaAT NP_579226 nos pGPro2 pGUMn UBi1 Mm1AlaAT NP_877957 nos pGPro2 pGUPn UBi2 PfAlaAT NP_579226 nos pGPro2
EXAMPLE 1
(74) Novel AlaAT gene constructs (CaMV35S::MmAlaAT1;CaMV35S::PfAlaAT; and OsAnt1::HvAlaAT1) were introduced into either the alaat1;2 double knock out (DNK) (
(75) The novel AlaAT gene constructs reduced the time to flowering in the transgenic Arabidopsis thaliana.
(76) Furthermore, the introduction of a novel AlaAT gene into either the normal genetic background or the DNK background, under two different conditions (as described above) increases the rate of root growth of the transgenic plant, and this rate changes as the plants get older (see
(77) The effect of the novel AlaAT gene constructs (CaMV35S::MmAlaAT1; CaMV35S::PfAlaAT; and OsAnt1::HvAlaAT1) introduced into the normal genetic background (COL background) on root growth in Arabidopsis plants was determined at 18 DAS. As noted above, these plants were grown on MS with 2.5 mM alanine or 2.5 mM glutamate as the only nitrogen source. The CaMV35S:PfAlaAT and CaMV35S:MmAlaAT grew significantly larger than the COL control; and both of them grew better than the OsANT1 promoter driving HvAlaAT. COL and AlaAT over-expressers (OE) were grown on the same plate and blocks are replicates that were moved accordingly.
EXAMPLE 2
(78) In
(79) In
(80)
EXAMPLE 3
(81) In
(82) In
(83) In
(84) In
(85) The experiments carried out for
EXAMPLE 4: TRANSGENIC RICE
(86) Heterozygous T.sub.0 Seed Yield and Biomass Preliminary Screens
(87) Transgenic rice (Oryza sativa) containing the specific genetic constructs above and outlined in
(88) It was examined whether the amount of available nitrogen would have an effect on plant biomass and seed weight on plants expressing PBpr1::AlaAT grown under laboratory conditions when compared to similar plants that ectopically expressed the target nucleotide sequence under a constitutive promoter (maize ubiquitin 1).
(89) Seed weights and biomass of the transgenic plants grown under laboratory conditions were examined at the dates provided below in Table 3. At the T.sub.0 stage, several of the transgenic plants expressing AlaAT in a tissue-specific manner had higher above ground biomass and seed weight compared to transgenic plants expressing AlaAT in a constitute manner (Table 3).
(90) TABLE-US-00004 TABLE 3 Biomass and seed weight for transgenic Oryza sativa plants expressing AlaAT in a tissue-specific or constitutive manner. bio- seed prod mass wt plant # seeding date flowering date tillers (g) (g) pGPMn1/1-1 12 Jun. 2012 28 Aug. 2012 10 31.5 10.88 pGPMn1/1-2 12 Jun. 2012 17 Aug. 2012 9 25.73 10.06 pGPMn1/1-3 12 Jun. 2012 19 Aug. 2012 10 27.6 5.67 pGPMn1/1-4 12 Jun. 2012 19 Aug. 2012 8 30.26 11.52 pGPMn1/1-5 12 Jun. 2012 27 Aug. 2012 14 31.62 10.11 pGPMn1/1-6 12 Jun. 2012 18 Aug. 2012 13 34.64 8.26 pGPMn1/1-7 12 Jun. 2012 17 Aug. 2012 10 33.98 11.20 pGPMn1/1-8 12 Jun. 2012 23 Aug. 2012 9 31.78 12.16 pGPMn1/1-9 12 Jun. 2012 19 Aug. 2012 12 33.41 10.14 pGPMn1/1-10 12 Jun. 2012 17 Aug. 2012 10 29.43 9.66 pGPMn1/1-11 12 Jun. 2012 18 Aug. 2012 12 33.6 8.95 pGPMn1/1-12 12 Jun. 2012 23 Aug. 2012 10 29.28 9.30 pGPMn1/1-13 12 Jun. 2012 31 Aug. 2012 13 30.86 11.79 pGPMn1/1-14 12 Jun. 2012 21 Sep. 2012 10 28.51 9.98 pGPMn1/1-15 12 Jun. 2012 21 Sep. 2012 15 33.38 12.45 pGPMn1/1-16 25 Jun. 2012 2 Sep. 2012 13 28.78 5.91 pGPMn1/1-17 25 Jun. 2012 4 Sep. 2012 13 26.14 5.95 pGPMn1/1-20 25 Jun. 2012 9 Sep. 2012 16 36.15 14.88 pGPMn1/1-21 25 Jun. 2012 9 Sep. 2012 13 29.01 5.20 pGPPn2/1-2 12 Jun. 2012 20 Aug. 2012 10 32.07 12.99 pGPPn2/1-3 12 Jun. 2012 4 Sep. 2012 7 26.35 7.06 pGPPn2/1-4 12 Jun. 2012 28 Aug. 2012 8 24.56 8.29 pGPPn2/1-5 12 Jun. 2012 28 Aug. 2012 7 24.61 8.06 pGPPn2/1-6 12 Jun. 2012 20 Aug. 2012 9 27.35 9.69 pGPPn2/1-7 25 Jun. 2012 30 Sep. 2012 9 26.3 3.70 pGPPn2/1-8 25 Jun. 2012 4 Sep. 2012 13 36.19 12.32 pGPPn2/1-9 25 Jun. 2012 28 Aug. 2012 15 34.72 11.84 pGPPn2/2-1 28 Aug. 2012 15 Nov. 2012 14 35.34 5.66 pGPPn2/2-2 28 Aug. 2012 6 Nov. 2012 21 40.48 7.38 pGPPn2/2-3 11 Sep. 2012 15 Nov. 2012 14 34.17 13.24 pGPPn2/2-4 11 Sep. 2012 19 Nov. 2012 13 32.28 7.92 pGPPn2/2-5 11 Sep. 2012 23 Nov. 2012 11 38.3 11.38 pGPPn2/2-6 11 Sep. 2012 23 Nov. 2012 14 28.16 8.61 pGPPn2/2-7 11 Sep. 2012 28 Nov. 2012 9 28.4 9.94 pGPPn2/2-8 11 Sep. 2012 23 Nov. 2012 10 33.29 9.40 pGUMn3/1-1 10 Sep. 2012 19 Nov. 2012 11 27.36 6.14 pGUMn3/1-3 14 Sep. 2012 26 Nov. 2012 12 34.5 6.70 pGUMn3/1-4 14 Sep. 2012 26 Nov. 2012 13 32.94 8.43 pGUMn3/1-6 14 Sep. 2012 30 Nov. 2012 20 30.08 4.07 pGUMn3/1-7 14 Sep. 2012 6 Dec. 2012 18 30.61 3.03 pGUMn3/1-8 14 Sep. 2012 14 Nov. 2012 9 21.1 7.98 pGUPn4/1-1 10 Sep. 2012 18 Nov. 2012 11 22.71 3.28 pGUPn4/1-2 10 Sep. 2012 19 Nov. 2012 7 22.05 3.69 pGUPn4/1-4 11 Sep. 2012 12 Nov. 2012 8 18.54 3.31 pGUPn4/1-5 11 Sep. 2012 13 Nov. 2012 10 29.62 11.08 pGUPn4/1-6 11 Sep. 2012 19 Nov. 2012 9 29.8 12.74 pGUPn4/1-7 14 Sep. 2012 21 Dec. 2012 9 20.8 3.05 pGUPn4/1-8 14 Sep. 2012 25 Nov. 2012 6 20.93 2.07 pGUPn4/1-9 14 Sep. 2012 30 Nov. 2012 16 33.09 2.88 pGUPn4/1-10 14 Sep. 2012 30 Nov. 2012 13 31.06 7.68 pGUPn4/1-11 14 Sep. 2012 19 Nov. 2012 12 24.93 3.20 pGUPn4/1-12 14 Sep. 2012 19 Nov. 2012 7 20.1 3.10 pGUPn4/1-13 14 Sep. 2012 18 Nov. 2012 15 33.12 5.47
(91) Thus, transgenic plants expressing a gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism under tissue-specific promoter exhibited higher biomass and seed weight than plants expressing the same gene or nucleotide sequence under a constitutive promoter, in the presence of an adequate supply of nitrogen.
(92) This indicates that transgenic Oryza sativa plants ectopically expressing an enzyme involved in nitrogen assimilation or metabolism are capable of optimising the utilization of available nitrogen under a range of environmental conditions thereby resulting in an increase in plant biomass, seed yield or a combination thereof.
(93) T1 plant lines were obtained and average biomass and seed-weight was determined for 6 plants per transgenic plant lines. The results are shown in
(94) Collectively the above results indicate that transgenic plants ectopically expressing an enzyme involved in nitrogen assimilation or metabolism are capable of optimising the utilization of available nitrogen under a range of environmental conditions thereby resulting in an increase in plant biomass, seed weight, root tap growth, root growth and root length or a combination thereof.
(95) The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims
(96) All citations are hereby incorporated by reference.
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