Transgenic plants with enhanced growth characteristics

Abstract

transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

Description

RELATED APPLICATIONS

[0001]This application is a continuation-in-part of U.S. application Ser. No. 12/551,271, filed Aug. 31, 2009, which application claims priority to U.S. Provisional Application No. 61/190,520 filed Aug. 29, 2008.

BACKGROUND OF THE INVENTION

[0003]As the human population increases worldwide, and available farmland continues to be destroyed or otherwise compromised, the need for more effective and sustainable agriculture systems is of paramount interest to the human race. Improving crop yields, protein content, and plant growth rates represent major objectives in the development of agriculture systems that can more effectively respond to the challenges presented.

[0004]In recent years, the importance of improved crop production technologies has only increased as yields for many well-developed crops have tended to plateau. Many agricultural activities are time sensitive, with costs and returns being dependent upon rapid turnover of crops or upon time to market. Therefore, rapid plant growth is an economically important goal for many agricultural businesses that involve high-value crops such as grains, vegetables, berries and other fruits.

[0005]Genetic engineering has and continues to play an increasingly important yet controversial role in the development of sustainable agriculture technologies. A large number of genetically modified plants and related technologies have been developed in recent years, many of which are in widespread use today (Factsheet: Genetically Modified Crops in the United States, Pew Initiative on Food and Biotechnology, August 2004, http://pewagbiotech.org/resources/factsheets). The adoption of transgenic plant varieties is now very substantial and is on the rise, with approximately 250 million acres planted with transgenic plants in 2006.

[0006]While acceptance of transgenic plant technologies may be gradually increasing, particularly in the United States, Canada and Australia, many regions of the World remain slow to adopt genetically modified plants in agriculture, notably Europe. Therefore, consonant with pursuing the objectives of responsible and sustainable agriculture, there is a strong interest in the development of genetically engineered plants that do not introduce toxins or other potentially problematic substances into plants and/or the environment. There is also a strong interest in minimizing the cost of achieving objectives such as improving herbicide tolerance, pest and disease resistance, and overall crop yields. Accordingly, there remains a need for transgenic plants that can meet these objectives.

[0007]The goal of rapid plant growth has been pursued through numerous studies of various plant regulatory systems, many of which remain incompletely understood. In particular, the plant regulatory mechanisms that coordinate carbon and nitrogen metabolism are not fully elucidated. These regulatory mechanisms are presumed to have a fundamental impact on plant growth and development.

[0008]The metabolism of carbon and nitrogen in photosynthetic organisms must be regulated in a coordinated manner to assure efficient use of plant resources and energy. Current understanding of carbon and nitrogen metabolism includes details of certain steps and metabolic pathways which are subsystems of larger systems. In photosynthetic organisms, carbon metabolism begins with CO₂ fixation, which proceeds via two major processes, termed C-3 and C-4 metabolism. In plants with C-3 metabolism, the enzyme ribulose bisphosphate carboxylase (RuBisCo) catalyzes the combination of CO₂ with ribulose bisphosphate to produce 3-phosphoglycerate, a three carbon compound (C-3) that the plant uses to synthesize carbon-containing compounds. In plants with C-4 metabolism, CO₂ is combined with phosphoenol pyruvate to form acids containing four carbons (C-4), in a reaction catalyzed by the enzyme phosphoenol pyruvate carboxylase. The acids are transferred to bundle sheath cells, where they are decarboxylated to release CO₂, which is then combined with ribulose bisphosphate in the same reaction employed by C-3 plants.

[0009]Numerous studies have found that various metabolites are important in plant regulation of nitrogen metabolism. These compounds include the organic acid malate and the amino acids glutamate and glutamine. Nitrogen is assimilated by photosynthetic organisms via the action of the enzyme glutamine synthetase (GS) which catalyzes the combination of ammonia with glutamate to form glutamine. GS plays a key role in the assimilation of nitrogen in plants by catalyzing the addition of ammonium to glutamate to form glutamine in an ATP-dependent reaction (Miflin and Habash, 2002, Journal of Experimental Botany, Vol. 53, No. 370, pp. 979-987). GS also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS enzymes may be divided into two general classes, one representing the cytoplasmic form (GS1) and the other representing the plastidic (i.e., chloroplastic) form (GS2).

[0010]Previous work has demonstrated that increased expression levels of GS1 result in increased levels of GS activity and plant growth, although reports are inconsistent. For example, Fuentes et al. reported that CaMV S35 promoter-driven overexpression of Alfalfa GS1 (cytoplasmic form) in tobacco resulted in increased levels of GS expression and GS activity in leaf tissue, increased growth under nitrogen starvation, but no effect on growth under optimal nitrogen fertilization conditions (Fuentes et al., 2001, J. Exp. Botany 52: 1071-81). Temple et al. reported that transgenic tobacco plants overexpressing the full length Alfalfa GS1 coding sequence contained greatly elevated levels of GS transcript, and GS polypeptide which assembled into active enzyme, but did not report phenotypic effects on growth (Temple et al., 1993, Molecular and General Genetics 236: 315-325). Corruzi et al. have reported that transgenic tobacco overexpressing a pea cytosolic GS1 transgene under the control of the CaMV S35 promoter show increased GS activity, increased cytosolic GS protein, and improved growth characteristics (U.S. Pat. No. 6,107,547). Unkefer et al. have more recently reported that transgenic tobacco plants overexpressing the Alfalfa GS1 in foliar tissues, which had been screened for increased leaf-to-root GS activity following genetic segregation by selfing to achieve increased GS1 transgene copy number, were found to produce increased 2-hydroxy-5-oxoproline levels in their foliar portions, which was found to lead to markedly increased growth rates over wildtype tobacco plants (see, U.S. Pat. Nos. 6,555,500; 6,593,275; and 6,831,040).

[0011]Unkefer et al. have further described the use of 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) to improve plant growth (U.S. Pat. Nos. 6,555,500; 6,593,275; 6,831,040). In particular, Unkefer et al. disclose that increased concentrations of 2-hydroxy-5-oxoproline in foliar tissues (relative to root tissues) triggers a cascade of events that result in increased plant growth characteristics. Unkefer et al. describe methods by which the foliar concentration of 2-hydroxy-5-oxoproline may be increased in order to trigger increased plant growth characteristics, specifically, by applying a solution of 2-hydroxy-5-oxoproline directly to the foliar portions of the plant and over-expressing glutamine synthetase preferentially in leaf tissues.

[0012]A number of transaminase and hydrolyase enzymes known to be involved in the synthesis of 2-hydroxy-5-oxoproline in animals have been identified in animal liver and kidney tissues (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197: 304). In plants, the biochemical synthesis of 2-hydroxy-5-oxoproline has been known but has been poorly characterized. Moreover, the function of 2-hydroxy-5-oxoproline in plants and the significance of its pool size (tissue concentration) are unknown. Finally, the art provides no specific guidance as to precisely what transaminase(s) or hydrolase(s) may exist and/or be active in catalyzing the synthesis of 2-hydroxy-5-oxoproline in plants, and no such plant transaminases have been reported, isolated or characterized.

SUMMARY OF THE INVENTION

[0013]The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically Perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants. Similarly, flower and fruit or pod yields are also tremendously improved, with T0 generation lines typically showing 50% to 70% increases over their wild type counterparts, and in some cases showing a 100% increase. Transgenic plants exhibiting such enhanced growth phenotypic characteristics have been successfully generated across a spectrum of individual plant species, using various transformation methodologies, different expression vectors and promoters, and heterologous and homologous transgene sequences from a variety of species, as exemplified by the numerous working examples provided herein. This invention, therefore, provides a fundamental break-though technology that has the potential to transform virtually all areas of agriculture.

[0014]Applicants have identified the enzyme glutamine phenylpyruvate transaminase (GPT) as a catalyst of 2-hydroxy-5-oxoproline (2-oxoglutaramate) synthesis in plants. 2-oxoglutaramate is a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism. The invention provides isolated nucleic acid molecules encoding GPT, and discloses the novel finding that the encoded enzyme is directly involved in the synthesis of 2-hydroxy-5-oxoproline. This aspect of the invention is exemplified herein by the disclosure of GPT polynucleotides encoding GPTs from several species, including Arabidopsis, Grape, Rice, Soybean, Barley, Bamboo and a non-plant homolog from Zebra fish, most of which have been expressed as recombinant GPTs and confirmed as having GPT activity.

[0015]The invention further provides transgenic plants which express both a GPT transgene and a GS transgene. The expression of these two transgenes in such "double-transgene" plants results in a substantially increased rate of carbon dioxide fixation and an extremely potent growth enhancing effect, as these plants exhibit very significantly and sometimes tremendously enhanced growth rates and flower/fruit/pod/seed yields. Methods for the generation of such growth-enhanced transgenic plants are provided.

[0016]By preferentially increasing the concentration of the signal metabolite 2-oxoglutaramate (i.e., in foliar tissues), the transgenic plants of the invention are capable of producing higher overall yields over shorter periods of time, and therefore may provide agricultural industries with enhanced productivity across a wide range of crops. Importantly, unlike many transgenic plants described to date, the invention utilizes natural plant genes encoding a natural plant enzyme. The enhanced growth characteristics of the transgenic plants of the invention is achieved essentially by introducing additional GPT and GS capacity into the plant. Thus, the transgenic plants of the invention do not express any toxic substances, growth hormones, viral or bacterial gene products, and are therefore free of many of the concerns that have heretofore impeded the adoption of transgenic plants in certain parts of the World.

[0017]In one embodiment, the invention provides a transgenic plant comprising a GPT transgene and a GS transgene, wherein said GPT transgene and said GS transgene are operably linked to a plant promoter. In a specific embodiment, the GS transgene is a GS1 transgene. In another specific embodiment, the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49 and has GPT activity. In yet another specific embodiment, the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 4, SEQ ID NO: 7 from residue 11 and SEQ ID NO: 41, and (b) an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 41. In some embodiments, the GPT and GS transgenes are incorporated into the genome of the plant.

[0018]In a particular aspect of the invention, the GPT transgene and a GS transgene construct is incorporated into the genome of a plant selected from the group consisting of: maize, rice, sugar cane, wheat, oats, sorghum, switch grass, soya bean, tubers (such as potatoes), canola, lupins or cotton.

[0019]The invention also provides progeny of any generation of the transgenic plants of the invention, wherein said progeny comprises a GPT transgene and a GS transgene, as well as a seed of any generation of the transgenic plants of the invention, wherein said seed comprises said GPT transgene and said GS transgene. The transgenic plants of the invention may display one or more enhanced growth characteristics rate when compared to an analogous wild-type or untransformed plant, including without limitation increased growth rate, biomass yield, seed yield, flower or flower bud yield, fruit or pod yield, larger leaves, and may also display increased levels of GPT and/or GS activity, and/or increased levels of 2-oxoglutaramate. In some embodiments, the transgenic plants of the invention display increased nitrogen use efficiency or increased tolerance to salt or saline conditions.

[0020]In a further aspect of the invention there is provided a transplastomic plant or cell line carrying a GPT transgene and a GS transgene expression cassette, said expression cassette being flanked by sequences from the plant or plant cell's plastome.

[0021]Further still, the invention provides a method for preparing a transplastomic plant or cell line carrying a GPT transgene and a GS transgene construct, said method comprising the steps of: (a) inserting into at least one expression cassette at least a GPT transgene and a GS transgene, wherein said expression cassette is flanked by sequences from the plant or plant cell's plastome.

[0022]Methods for producing the transgenic plants of the invention and seeds thereof are also provided, including methods for producing a plant having enhanced growth properties, increased nitrogen use efficiency and increased tolerance to germination or growth in salt or saline conditions, relative to an analogous wild type or untransformed plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]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.

[0024]FIG. 1. Nitrogen assimilation and 2-oxoglutaramate biosynthesis: schematic of metabolic pathway.

[0025]FIG. 2. Photograph showing comparison of transgenic tobacco plants over-expressing either GS1 or GPT, compared to wild type tobacco plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See Examples 3 and 5, infra.

[0026]FIG. 3. Photograph showing comparison of transgenic Micro-Tom tomato plants over-expressing either GS1 or GPT, compared to wild type tomato plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See Examples 4 and 6, infra.

[0027]FIG. 4. Photographs showing comparisons of leaf sizes between wild type and GS1 or GPT transgenic tobacco plants. A: Comparison between leaves from GS1 transgenic tobacco (bottom leaf) and wild type (top leaf). B: Comparison between leaves from GPT transgenic tobacco (bottom leaf) and wild type (top leaf).

[0028]FIG. 5. Photographs showing comparisons of transgenic tobacco plants generated from various crosses between GS1 and GPT transgenic tobacco lines with wild type and single transgene plants. A-C: Cross 2, 3 and 7, respectively. See Example 7, infra.

[0029]FIG. 6. Photographs showing comparisons of leaf sizes between wild type and crosses between GS1 and GPT transgenic tobacco plants. A: Comparison between leaves from GSXGPT Cross 3 (bottom leaf) and wild type (top leaf). B: Comparison between leaves from GSXGPT Cross 7 (bottom leaf) and wild type (top leaf). See Example 7, infra.

[0030]FIG. 7. Photograph of transgenic pepper plant (right) and wild type control pepper plant (left), showing larger pepper fruit yield in the transgenic plant relative to the wild type control plant. See Example 8, infra.

[0031]FIG. 8. Transgenic bean plants compared to wild type control bean plants (several transgenic lines expressing Arabidopsis GPT and GS transgenes). Upper Left: plant heights on various days; Upper right: flower bud numbers; Lower left: flower numbers; Lower right: bean pod numbers. Wildtype is the control, and lines 2A, 4A and 5B are all transgenic plant lines. See Example 9, infra.

[0032]FIG. 9. Photograph of transgenic bean plant (right) and wild type control bean plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Arabidopsis GPT and GS transgenes. See Example 9, infra.

[0033]FIG. 10. Transgenic bean plants pods, flowers and flower buds compared to wild type control bean plants (transgenic line expressing grape GPT and Arabidopsis GS transgenes). See Example 10, infra.

[0034]FIG. 11. Photograph of transgenic bean plant (right) and wild type control bean plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Grape GPT and Arabidopsis GS transgenes. See Example 10, infra.

[0035]FIG. 12. Transgenic Cowpea Line A plants compared to wild type control Cowpea plants (transgenic line expressing Arabidopsis GPT and GS transgenes), showing that the transgenic plants grow faster and flower and set pods sooner than wild type control plants. (A) Relative height and longest leaf measurements as of May 21, (B) Relative trifolate leafs and flower buds as of June 18, (C) Relative numbers of flowers, flower buds and pea pods as of June 22. See Example 11, infra.

[0036]FIG. 13. Photograph of transgenic Cowpea Line A plant (right) and wild type control Cowpea plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Arabidopsis GPT and GS transgenes. See Example 11, infra.

[0037]FIG. 14. Transgenic Cowpea Line G plants compared to wild type control Cowpea plants (transgenic line expressing Grape GPT and Arabidopsis GS transgenes), showing that the transgenic plants grow faster and flower and set pods sooner than wild type control plants. (A) plant heights, (B) flowers and pea pod numbers, (C) leaf bud and trifolate numbers. See Example 12, infra.

[0038]FIG. 15. Photograph of transgenic Cowpea Line G plant (right) and wild type control Cowpea plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Grape GPT and Arabidopsis GS transgenes. See Example 12, infra.

[0039]FIG. 16. Photograph of transgenic Cantaloupe plant (right) and wild type control Cantaloupe plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Arabidopsis GPT and GS transgenes. See Example 14, infra.

[0040]FIG. 17. Photograph of transgenic Pumpkin plants (right) and wild type control Pumpkin plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. Transgenic lines expressing Arabidopsis GPT and GS transgenes. See Example 15, infra.

[0041]FIG. 18. Photograph of transgenic Arabidopsis plants (right) and wild type control Arabidopsis plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. Transgenic lines expressing Arabidopsis GPT and GS transgenes. See Example 16, infra.

[0042]FIG. 19. Transgenic tomato plants expressing Arabidopsis GPT and GS transgenes compared to control tomato plants. (A) Photograph of transgenic tomato plant leaves (right) vs. wild type control leaves (left) showing larger leaves in the transgenic plant. (B) Photograph of transgenic tomato plants (right) and wild type control plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. See Example 17, infra.

[0043]FIG. 20. Photograph of transgenic Camelina plant (right) and wild type control Camelina plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Arabidopsis GPT and GS transgenes. See Example 18, infra.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0044]Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed., Humana Press, 1^st edition, 2004); and, Agrobacterium Protocols (Wan, ed., Humana Press, 2^nd edition, 2006). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

[0045]Each document, reference, patent application or patent cited in this text is expressly incorporated herein in its entirety by reference, and each should be read and considered as part of this specification. That the document, reference, patent application or patent cited in this specification is not repeated herein is merely for conciseness.

[0046]The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form. Unless specifically limited, the term "polynucleotide" encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0047]The term "promoter" refers to anucleic acid control sequence or sequences that direct transcription of an operably linked nucleic acid. As used herein, a "plant promoter" is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0048]The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0049]The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0050]Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0051]The term "plant" includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, reproductive organs, embryos and parts thereof, etc.), seedlings, seeds and plant cells and progeny thereof. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.

[0052]The terms "GPT polynucleotide" and "GPT nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a polypeptide involved in catalyzing the synthesis of 2-oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GPT coding sequence" refers to the part of the gene which is transcribed and encodes a GPT protein. The term "targeting sequence" refers to the amino terminal part of a protein which directs the protein into a subcellular compartment of a cell, such as a chloroplast in a plant cell. GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PCR products derived therefrom.

[0053]A "GPT transgene" is a nucleic acid molecule comprising a GPT polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide. More particularly, the exogenous GPT transgene will be heterogeneous with any GPT polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the GPT transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.

[0054]The terms "GS polynucleotide" and "GS nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GS coding sequence" refers to the part of the gene which is transcribed and encodes a GS protein. The terms "GS1 polynucleotide" and "GS1 nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase isoform 1 protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GS1 coding sequence" refers to the part of the gene which is transcribed and encodes a GS1 protein.

[0055]A "GS transgene" is a nucleic acid molecule comprising a GS polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GS polynucleotide. A "GS1 transgene" is a nucleic acid molecule comprising a GS1 polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GS1 polynucleotide. More particularly, the exogenous GS or GS1 transgene will be heterogeneous with any GS or GS1 polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the GS or GS1 transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.

[0056]Exemplary GPT polynucleotides of the invention are presented herein, and include GPT coding sequences for Arabidopsis, Rice, Barley, Bamboo, Soybean, Grape, Clementine orange and Zebra Fish GPTs.

[0057]Partial length GPT polynucleotides include polynucleotide sequences encoding N- or C-terminal truncations of GPT, mature GPT (without targeting sequence) as well as sequences encoding domains of GPT. Exemplary GPT polynucleotides encoding N-terminal truncations of GPT include Arabidopsis-30, -45 and -56 constructs, in which coding sequences for the first 30, 45, and 56, respectively, amino acids of the full length GPT structure of SEQ ID NO: 2 are eliminated.

[0058]In employing the GPT polynucleotides of the invention in the generation of transformed cells and transgenic plants, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived, as further defined below. The term "GPT polynucleotide" specifically encompasses such substantially identical variants. Similarly, one of skill will recognize that because of codon degeneracy, a number of polynucleotide sequences will encode the same polypeptide, and all such polynucleotide sequences are meant to be included in the term GPT polynucleotide. In addition, the term specifically includes those sequences substantially identical (determined as described below) with an GPT polynucleotide sequence disclosed herein and that encode polypeptides that are either mutants of wild type GPT polypeptides or retain the function of the GPT polypeptide (e.g., resulting from conservative substitutions of amino acids in a GPT polypeptide). The term "GPT polynucleotide" therefore also includes such substantially identical variants.

[0059]The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0060]As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0061]The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0062]Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3^rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

[0063]The term "isolated" refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term "isolated" is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu. An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0064]The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0065]The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.

[0066]When percentage of sequence identity is used in reference to polypeptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

[0067]For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0068]A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0069]A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0070]The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0071]The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30° C. below the Tm. Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.

[0072]Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

[0073]Genomic DNA or cDNA comprising GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide sequences disclosed here. For this purpose, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37° C., and at least one wash in 0.2×SSC at a temperature of at least about 50° C., usually about 55° C. to about 60° C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency.

[0074]A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.

Transgenic Plants:

[0075]The invention provides novel transgenic plants exhibiting substantially enhanced agronomic characteristics, including faster growth, greater mature plant fresh weight and total biomass, earlier and more abundant flowering, and greater fruit, pod and seed yields. The transgenic plants of the invention are generated by introducing into a plant one or more expressible genetic constructs capable of driving the expression of one or more polynucleotides encoding glutamine synthetase (GS) and glutamine phenylpyruvate transaminase (GPT). In an exemplary embodiment, single-transgene parental lines carrying either a GPT or GS1 transgene coding sequence are generated, preferably selfed until homozygous for the transgene, then crossed to generate progeny plants containing both transgenes.

[0076]The transgenic plants of the invention may be any vascular plant of the phylum Tracheophyta, including angiosperms and gymnosperms. Angiosperms may be a monocotyledonous (monocot) or a dicotyledonous (dicot) plant. Important monocots include those of the grass families, such as the family Poaceae and Gramineae, including plants of the genus Avena (Avena sativa, oats), genus Hordeum (i.e., Hordeum vulgare, Barley), genus Oryza (i.e., Oryza sativa, rice, cultivated rice varieties), genus Panicum (Panicum spp., Panicum virgatum, Switchgrass), genus Phleum (Phleum pratense, Timothy-grass), genus Saccharum (i.e., Saccharum officinarum, Saccharum spontaneum, hybrids thereof, Sugarcane), genus Secale (i.e., Secale cereale, Rye), genus Sorghum (Sorghum vulgare, Sorghum), genus Triticum (wheat, various classes, including T. aestivum and T. durum), genus Fagopyrum (buckwheat, including F. esculentum), genus Triticosecale (Triticale, various hybrids of wheat and rye), genus Chenopodium (quinoa, including C. quinoa), genus Zea (i.e., Zea mays, numerous varieties) as well as millets (i.e., Pennisetum glaucum) including the genus Digitaria (D. exilis).

[0077]Important dicots include those of the family Solanaceae, such as plants of the genus Lycopersicon (Lycopersicon esculentum, tomato), genus Capiscum (Capsicum annuum, peppers), genus Solanum (Solanum tuberosum, potato, S. lycopersicum, tomato); genus Manihot (cassaya, M. esculenta), genus Ipomoea (sweet potato, I. batatas), genus Olea (olives, including O. europaea); plants of the Gossypium family (i.e., Gossypium spp., G. hirsutum, G. herbaceum, cotton); the Legumes (family Fabaceae), such as peas (Pisum spp, P. sativum), beans (Glycine spp., Glycine max(soybean); Phaseolus vulgaris, common beans, Vigna radiata, mung bean), chickpeas (Cicer arietinum)), lentils (Lens culinaris), peanuts (Arachis hypogaea); coconuts (Cocos nucifera) as well as various other important crops such as camelina (Camelina sativa, family Brassicaceae), citrus (Citrus spp, family Rutaceae), coffee (Coffea spp, family Rubiaceae), melon (Cucumis spp, family Cucurbitaceae), squash (Cucurbita spp, family Cucurbitaceae), roses (Rosa spp, family Rosaceae), sunflower (Helianthus annuus, family Asteraceae), sugar beets (Beta spp, family Amaranthaceae), including sugarbeet, B. vulgaris), genus Daucus (carrots, including D. carota), genus Pastinaca (parsnip, including P. sativa), genus Raphanus (radish, including R. sativus), genus Dioscorea (yams, including D. rotundata and D. cayenensis), genus Armoracia (horseradish, including A. rusticana), genus Elaeis (Oil palm, including E. guineensis), genus Linum (flax, including L. usitatissimum), genus Carthamus (safflower, including C. tinctorius L.), genus Sesamum (sesame, including S. indicum), genus Vitis (grape, including Vitis vinifera), and plants of the genus Brassica (family Brassicaceae, i.e., broccoli, brussel sprouts, cabbage, swede, turnip, rapeseed B. napus, and cauliflower).

[0078]Other specific plants which may be transformed to generate the transgenic plants of the invention include various other fruits and vegetables, such as apples, asparagus, avocado, banana, blackberry, blueberry, brussel sprout, cabbage, cotton, canola, carrots, radish, cucumbers, cherries, cranberries, cantaloupes, eggplant, grapefruit, lemons, limes, nectarines, oranges, peaches, pineapples, pears, plums, tangelos, tangerines, papaya, mango, strawberry, raspberry, lettuce, onion, grape, kiwi fruit, okra, parsnips, pumpkins, and spinach. In addition various flowering plants, trees and ornamental plants may be used to generate transgenic varietals, including without limitation lily, carnation, chrysanthemum, petunia, geranium, violet, gladioli, lupine, orchid and lilac.

[0079]In stable transformation embodiments of the invention, one or more copies of the expressible genetic construct become integrated into the host plant genome, thereby providing increased GS and GPT enzyme capacity into the plant, which serves to mediate increased synthesis of 2-oxoglutaramate, which in turn signals metabolic gene expression, resulting in increased plant growth and the enhancement other agronomic characteristics. 2-oxoglutaramate is a metabolite which is an extremely potent effector of gene expression, metabolism and plant growth (U.S. Pat. No. 6,555,500), and which may play a pivotal role in the coordination of the carbon and nitrogen metabolism systems (Lancien et al., 2000, Enzyme Redundancy and the Importance of 2-Oxoglutarate in Higher Plants Ammonium Assimilation, Plant Physiol. 123: 817-824). See, also, the schematic of the 2-oxoglutaramate pathway shown in FIG. 1.

[0080]In one aspect of the invention, applicants have isolated a nucleic acid molecule encoding the Arabidopsis glutamine phenylpyruvate transaminase (GPT) enzyme (see Example 1, infra), and have demonstrated for the first time that the expressed recombinant enzyme is active and capable of catalyzing the synthesis of the signal metabolite, 2-oxoglutaramate (Example 2, infra). Further, applicants have demonstrated for the first time that over-expression of the Arabidopsis glutamine transaminase gene in a transformed heterologous plant results in enhanced CO₂ fixation rates and increased growth characteristics (Example 3, infra).

[0081]Applicants' previous work demonstrated that over-expression of Alfalfa GS1 gene under the control of a strong constitutive promoter results in transgenic tobacco plants with higher levels of GS activity in the leaves. These plants outgrow their wild-type counterparts, fix CO₂ faster, contain increased concentrations of total protein, as well as increased concentrations of glutamine and 2-oxoglutaramate, and show increased rates of uptake of nitrate through their roots.

[0082]As disclosed herein (see Example 3, infra), over-expression of a transgene comprising the full-length Arabidopsis GPT coding sequence in transgenic tobacco plants also results in faster CO₂ fixation, and increased levels of total protein, glutamine and 2-oxoglutaramate. These transgenic plants also grow faster than wild-type plants (FIG. 2). Similarly, in preliminary studies conducted with tomato plants (see Example 4, infra), tomato plants transformed with the Arabidopsis GPT transgene showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants (FIG. 3 and Example 4, infra).

[0083]In one particular embodiment, exemplified herein by way of Examples 3, 5 and 7, infra, a first set of parental single-transgene tobacco plant lines carrying the Alfalfa GS1 gene, including 5' and 3' untranslated regions, were generated using Agrobacterium mediated gene transformation, under selective pressure, together with screening for the fastest growing phenotype, and selfing to transgene/phenotype homozygosity (see Example 5, infra). A second set of parental single-transgene tobacco plant lines carrying the full length coding sequence of Arabidopsis GPT were generated in the same manner (Example 3, infra). High growth rate performing plants from each of the parental lines were then sexually crossed to yield progeny lines (Example 7, infra).

[0084]The resulting progeny from multiple crosses of Arabidopsis GS1 and GPT transgenic tobacco plants produce far better and quite surprising increases in growth rates over the single-transgene parental lines as well as wildtype plants. FIG. 5 shows photographs of double-transgene progeny from single-transgene GS1×GPT plant crosses, relative to wild type and single-transgene parental plants. FIG. 6 shows photographs comparing leaf sizes of double-transgene progeny and wild type plants. Experimentally observed growth rates in these double transgenic plants ranged between 200% and 300% over wild-type plants (Example 7, infra). Moreover, total biomass levels increased substantially in the double-transgene plants, with whole plant fresh weights typically being about two to three times the wild-type plant weights. Similarly, seed yields showed similar increases in the double-transgene plants, with seed pod production typically two to three times the wild type average, and overall seed yields exceeding wild-type plant yields by 300-400%.

[0085]In addition to the transgenic tobacco plants referenced above, various other species of transgenic plants comprising GPT and GS transgenes are specifically exemplified herein. As exemplified herein, transgenic plants showing enhanced growth characteristics have been generated in two species of Tomato (see Examples 4 and 17), Pepper (Example 8), Beans (Examples 9 and 10), Cowpea (Examples 11 and 12), Alfalfa (Example 13), Cantaloupe (Example 14), Pumpkin (Example 15), Arabidopsis (Example 16) and Camilena (Example 18). These transgenic plants of the invention were generated using a variety of transformation methodologies, including Agrobacterium-mediated callus, floral dip, seed inoculation, pod inoculation, and direct flower inoculation, as well as combinations thereof, and via sexual crosses of single transgene plants, as exemplified herein. Different GPT and GS transgenes were successfully employed in generating the transgenic plants of the invention, as exemplified herein.

[0086]The invention also provides methods of generating a transgenic plant having enhanced growth and other agronomic characteristics. In one embodiment, a method of generating a transgenic plant having enhanced growth and other agronomic characteristics comprises introducing into a plant cell an expression cassette comprising a nucleic acid molecule encoding a GPT transgene, under the control of a suitable promoter capable of driving the expression of the transgene, so as to yield a transformed plant cell, and obtaining a transgenic plant which expresses the encoded GPT. In another embodiment, a method of generating a transgenic plant having enhanced growth and other agronomic characteristics comprises introducing into a plant cell one or more nucleic acid constructs or expression cassettes comprising nucleic acid molecules encoding a GPT transgene and an GS transgene, under the control of one or more suitable promoters (and, optionally, other regulatory elements) capable of driving the expression of the transgenes, so as to yield a plant cell transformed thereby, and obtaining a transgenic plant which expresses the GPT and GS transgenes.

[0087]Based on the results disclosed herein, it is clear that any number of GPT and GS polynucleotides may be used to generate the transgenic plants of the invention. Both GS1 and GPT proteins are highly conserved among various plant species, and it is evident from the experimental data disclosed herein that closely-related non-plant GPTs may be used as well (e.g., Danio rerio GPT). With respect to GPT, numerous GPT polynucleotides derived from different species have been shown to be active and useful as GPT transgenes. Similarly, different GS polynucleotides may be used, including without limitation any plant GS1 encoding polynucleotide that generates GS activity in a host cell transformed with an expressible GS1 construct.

[0088]In a specific embodiment, the GPT transgene is a GPT polynucleotide encoding an Arabidopsis derived GPT, such as the GPT of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 30, and the GS transgene is a GS polynucleotide encoding an Alfalfa derived GS1 (i.e., SEQ ID NO: 4) or an Arabidopsis derived GS1 (SEQ ID NO: 7). The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 1; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 1, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity; and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity. The GS1 transgene may be encoded by the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 6 or a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 3 or SEQ ID NO: 6, and encoding a polypeptide having GPT activity; and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 4 or 7, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GS activity.

[0089]In another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Grape derived GPT, such as the Grape GPTs of SEQ ID NO: 9 and SEQ ID NO: 31, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 8; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 8, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 9 or SEQ ID NO: 31, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

[0090]In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Rice derived GPT, such as the Rice GPTs of SEQ ID NO: 11 and SEQ ID NO: 32, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 10; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 11 or SEQ ID NO: 32, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

[0091]In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Soybean derived GPT, such as the Soybean GPTs of SEQ ID NO: 13, SEQ ID NO: 33 or SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 12, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 33 or SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

[0092]In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Barley derived GPT, such as the Barley GPTs of SEQ ID NO: 15 and SEQ ID NO: 34, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 14; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 15 or SEQ ID NO: 34, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

[0093]In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Zebra fish derived GPT, such as the Zebra fish GPTs of SEQ ID NO: 17 and SEQ ID NO: 35, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 16; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 16, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 17 or SEQ ID NO: 35, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

[0094]In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Bamboo derived GPT, such as the Bamboo GPT of SEQ ID NO: 36, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by a nucleotide sequence encoding the polypeptide of SEQ ID NO: 36, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

[0095]Other GPT polynucleotides suitable for use as GPT transgenes in the practice of the invention may be obtained by various means, as will be appreciated by one skilled in the art, tested for the ability to direct the expression of a GPT with GPT activity in a recombinant expression system (i.e., E. coli (see Examples 20-23), in a transient in planta expression system (see Example 19), or in a transgenic plant (see Examples 1-18).

Transgene Constructs/Expression Vectors

[0096]In order to generate the transgenic plants of the invention, the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct), which can direct the expression of the transgene sequence in transformed plant cells. Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into a plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors. Once introduced into the transformed plant cell, the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a transient or stable fashion. Stable expression is preferred, and is achieved by utilizing plant transformation vectors which are able to direct the chromosomal integration of the transgene construct. Once a plant cell has been successfully transformed, it may be cultivated to regenerate a transgenic plant.

[0097]A large number of expression vectors suitable for driving the constitutive or induced expression of inserted genes in transformed plants are known. In addition, various transient expression vectors and systems are known. To a large extent, appropriate expression vectors are selected for use in a particular method of gene transformation (see, infra). Broadly speaking, a typical plant expression vector for generating transgenic plants will comprise the transgene of interest under the expression regulatory control of a promoter, a selectable marker for assisting in the selection of transformants, and a transcriptional terminator sequence.

[0098]More specifically, the basic elements of a nucleic acid construct for use in generating the transgenic plants of the invention are: a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed plant cell, the transgene (s) (i.e., GPT coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and sometimes other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).

[0099]As Agrobacterium tumefaciens is the primary transformation system used to generate transgenic plants, there are numerous vectors designed for Agrobacterium transformation. For stable transformation, Agrobacterium systems utilize "binary" vectors that permit plasmid manipulation in both E. coli and Agrobacterium, and typically contain one or more selectable markers to recover transformed plants (Hellens et al., 2000, Technical focus: A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446-451). Binary vectors for use in Agrobacterium transformation systems typically comprise the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, and selectable marker and reporter genes.

[0100]So-called "super-binary" vectors provide higher transformation efficiencies, and generally comprise additional virulence genes from a Ti (Komari et al., 2006, Methods Mol. Biol. 343: 15-41). Super binary vectors are typically used in plants which exhibit lower transformation efficiencies, such as cereals. Such additional virulence genes include without limitation virB, virE, and virG (Vain et al., 2004, The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system. Transgenic Res. 13: 593-603; Srivatanakul et al., 2000, Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression. J. Plant Physiol. 157, 685-690; Park et al., 2000, Shorter T-DNA or additional virulence genes improve Agrobacterium-mediated transformation. Theor. Appl. Genet. 101, 1015-1020; Jin et al., 1987, Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol. 169: 4417-4425).

[0101]In the embodiments exemplified herein (see Examples, infra), expression vectors which place the inserted transgene(s) under the control of the constitutive CaMV 35S promoter and the RuBisCo promoter are employed. A number of expression vectors which utilize the CaMV 35S and RuBsCo promoter are known and/or commercially available and/or derivable using ordinary skill in the art. Additionally, numerous promoters suitable for directing the expression of the transgene are known and may be used in the practice of the invention, as further described, infra.

Plant Promoters

[0102]A large number of promoters which are functional in plants are known in the art. In constructing GPT and GS transgene constructs, the selected promoter(s) may be constitutive, non-specific promoters such as the Cauliflower Mosaic Virus 35S ribosomal promoter (CaMV 35S promoter), which is widely employed for the expression of transgenes in plants. Examples of other strong constitutive promoters include without limitation the rice actin 1 promoter, the CaMV 19S promoter, the Ti plasmid nopaline synthase promoter, the alcohol dehydrogenase promoter and the sucrose synthase promoter.

[0103]Alternatively, in some embodiments, it may be desirable to select a promoter based upon the desired plant cells to be transformed by the transgene construct, the desired expression level of the transgene, the desired tissue or subcellular compartment for transgene expression, the developmental stage targeted, and the like.

[0104]For example, when expression in photosynthetic tissues and compartments is desired, a promoter of the ribulose bisphosphate carboxylase (RuBisCo) gene may be employed. In the Examples which follow, expressible nucleic acid constructs comprising GPT and GS1 transgenes under the control of a tomato RuBisCo promoter were prepared and used in the generation of transgenic plants or to assay for GPT activity in planta or in E. coli.

[0105]When the expression in seeds is desired, promoters of various seed storage protein genes may be employed. For expression in fruits, a fruit-specific promoter such as tomato 2A11 may be used. Examples of other tissue specific promoters include the promoters encoding lectin (Vodkin et al., 1983, Cell 34:1023-31; Lindstrom et al., 1990, Developmental Genetics 11:160-167), corn alcohol dehydrogenase 1 (Vogel et al, 1989, J. Cell. Biochem. (Suppl. 0) 13:Part D; Dennis et al., 1984, Nucl. Acids Res., 12(9): 3983-4000), corn light harvesting complex (Simpson, 1986, Science, 233: 34-38; Bansal et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 3654-3658), corn heat shock protein (Odell et al., 1985, Nature, 313: 810-812; Rochester et al., 1986, EMBO J., 5: 451-458), pea small subunit RuBP carboxylase (Poulsen et al., 1986, Mol. Gen. Genet., 205(2): 193-200; Cashmore et al., 1983, Gen. Eng. Plants, Plenum Press, New York, pp 29-38), Ti plasmid mannopine synthase and Ti plasmid nopaline synthase (Langridge et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 3219-3223), petunia chalcone isomerase (Van Tunen et al., 1988, EMBO J. 7(5): 1257-1263), bean glycine rich protein 1 (Keller et al., 1989, EMBO J. 8(5): 1309-1314), truncated CaMV 35s (Odell et al., 1985, supra), potato patatin (Wenzler et al., 1989, Plant Mol. Biol. 12: 41-50), root cell (Conkling et al., 1990, Plant Physiol. 93: 1203-1211), maize zein (Reina et al., 1990, Nucl. Acids Res. 18(21): 6426; Kriz et al., 1987, Mol. Gen. Genet. 207(1): 90-98; Wandelt and Feix, 1989, Nuc. Acids Res. 17(6): 2354; Langridge and Feix, 1983, Cell 34: 1015-1022; Reina et al., 1990, Nucl. Acids Res. 18(21): 6426), globulin-1 (Belanger and Kriz, 1991, Genetics 129: 863-872), α-tubulin (Carpenter et al., 1992, Plant Cell 4(5): 557-571; Uribe et al., 1998, Plant Mol. Biol. 37(6): 1069-1078), cab (Sullivan, et al., 1989, Mol. Gen. Genet. 215(3): 431-440), PEPCase (Hudspeth and Grula, 1989, Plant Mol. Biol. 12: 579-589), R gene complex (Chandler et al., 1989, The Plant Cell 1: 1175-1183), chalcone synthase (Franken et al., 1991, EMBO J. 10(9): 2605-2612) and glutamine synthetase promoters (U.S. Pat. No. 5,391,725; Edwards et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3459-3463; Brears et al., 1991, Plant J. 1(2): 235-244).

[0106]In addition to constitutive promoters, various inducible promoter sequences may be employed in cases where it is desirable to regulate transgene expression as the transgenic plant regenerates, matures, flowers, etc. Examples of such inducible promoters include promoters of heat shock genes, protection responding genes (i.e., phenylalanine ammonia lyase; see, for example Bevan et al., 1989, EMBO J. 8(7): 899-906), wound responding genes (i.e., cell wall protein genes), chemically inducible genes (i.e., nitrate reductase, chitinase) and dark inducible genes (i.e., asparagine synthetase; see, for example U.S. Pat. No. 5,256,558). Also, a number of plant nuclear genes are activated by light, including gene families encoding the major chlorophyll a/b binding proteins (cab) as well as the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) (see, for example, Tobin and Silverthorne, 1985, Annu. Rev. Plant Physiol. 36: 569-593; Dean et al., 1989, Annu. Rev. Plant Physiol. 40: 415-439.).

[0107]Other inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al., 1993, Plant J. 4(3): 423-432), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988, Genetics 119(1): 185-197); the MPI proteinase inhibitor promoter (Cordero et al., 1994, Plant J. 6(2): 141-150), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995, Plant Mol. Biol. 29(6): 1293-1298; Quigley et al., 1989, J. Mol. Evol. 29(5): 412-421; Martinez et al., 1989, J. Mol. Biol. 208(4): 551-565) and light inducible plastid glutamine synthetase gene from pea (U.S. Pat. No. 5,391,725; Edwards et al., 1990, supra).

[0108]For a review of plant promoters used in plant transgenic plant technology, see Potenza et al., 2004, In Vitro Cell. Devel. Biol-Plant, 40(1): 1-22. For a review of synthetic plant promoter engineering, see, for example, Venter, M., 2007, Trends Plant Sci., 12(3): 118-124.

Glutamine Phenylpyruvate Transaminase (GPT) Transgene

[0109]The present invention discloses for the first time that plants contain a glutamine phenylpyruvate transaminase (GPT) enzyme which is directly functional in the synthesis of the signal metabolite 2-hydroxy-5-oxoproline. Until now, no plant glutamine phenylpyruvate transaminase with a defined function has been described. Applicants have isolated and tested GPT polynucleotide coding sequences derived from several plant and animal species, and have successfully incorporated the gene into heterologous transgenic host plants which exhibit markedly improved growth characteristics, including faster growth, higher foliar protein content, increased glutamine synthetase activity in foliar tissue, and faster CO₂ fixation rates.

[0110]In the practice of the invention, the GPT gene functions as one of at least two transgenes incorporated into the transgenic plants of the invention, the other being the glutamine sythetase gene (see infra).

[0111]It is expected that all plant species contain a GPT which functions in the same metabolic pathway, involving the biosynthesis of the signal metabolite 2-hydroxy-5-oxoproline. Thus, in the practice of the invention, any plant gene encoding a GPT homolog or functional variants thereof may be useful in the generation of transgenic plants of this invention. Moreover, given the structural similarity between various plant GPT protein structures and the putative (and biologically active) GPT homolog from Danio rerio (Zebra fish) (see Example 22), other non-plant GPT homologs may be used in preparing GPT transgenes for use in generating the transgenic plants of the invention.

[0112]When individually compared (by BLAST alignment) to the Arabidopsis mature protein sequence provided in SEQ ID NO: 30, the following sequence identities and homologies (BLAST "positives", including similar amino acids) were obtained for the following mature GPT protein sequences:

TABLE-US-00001 [SEQ ID] or FIG. NO. ORIGIN % IDENTITY % POSITIVE [31] Grape 84 93 [32] Rice 83 91 [33] Soybean 83 93 [34] Barley 82 91 [35] Zebra fish 83 92 [36] Bamboo 81 90 FIG. 2 Corn 79 90 FIG. 2 Castor 84 93 FIG. 2 Poplar 85 93

[0113]Underscoring the conserved nature of the structure of the GPT protein across most plant species, the conservation seen within the above plant species extends to the non-human putative GPTs from Zebra fish and Chlamydomonas. In the case of Zebra fish, the extent of identity is very high (83% amino acid sequence identity with the mature Arabidopsis GPT of SEQ ID NO: 30, and 92% homologous taking similar amino acid residues into account). The Zebra fish mature GPT was confirmed by expressing it in E. coli and demonstrating biological activity (synthesis of 2-oxoglutaramate).

[0114]In order to determine whether putative GPT homologs would be suitable for generating the growth-enhanced transgenic plants of the invention, one need initially express the coding sequence thereof in E. coli or another suitable host and determine whether the 2-oxoglutaramate signal metabolite is synthesized at increased levels (see Examples 19-23). Where such an increase is demonstrated, the coding sequence may then be introduced into both homologous plant hosts and heterologous plant hosts, and growth characteristics evaluated. Any assay that is capable of detecting 2-oxoglutaramate with specificity may be used for this purpose, including without limitation the NMR and HPLC assays described in Example 2, infra. In addition, assays which measure GPT activity directly may be employed, such as the GPT activity assay described in Example 7.

[0115]Any plant GPT with 2-oxoglutaramate synthesis activity may be used to transform plant cells in order to generate transgenic plants of the invention. There appears to be a high level of structural homology among plant species, which appears to extend beyond plants, as evidenced by the close homology between various plant GPT proteins and the putative Zebra fish GPT homolog. Therefore, various plant GPT genes may be used to generate growth-enhanced transgenic plants in a variety of heterologous plant species. In addition, GPT transgenes expressed in a homologous plant would be expected to result in the desired enhanced-growth characteristics as well (i.e., rice glutamine transaminase over-expressed in transgenic rice plants), although it is possible that regulation within a homologous cell may attenuate the expression of the transgene in some fashion that may not be operable in a heterologous cell.

Glutamine Synthetase (GS) Transgene:

[0116]In the practice of the invention, the glutamine synthetase (GS) gene functions as one of at least two transgenes incorporated into the transgenic plants of the invention (GPT being the other of the two).

[0117]Glutamine synthetase plays a key role in nitrogen metabolism in plants, as well as in animals and bacteria. The GS enzyme catalyzes the addition of ammonium to glutamate to synthesize glutamine in an ATP-dependent reaction. GS enzymes from assorted species show highly conserved amino acid residues considered to be important for active site function, indicating that GS enzymes function similarly (for review, see Eisenberg et al., Biochimica et Biophysica Acta, 1477:122 145, 2000).

[0118]GS is distributed in different subcellular locations (chloroplast and cytoplasm) and is found in various plant tissues, including leaf, root, shoot, seeds and fruits. There are two major isoforms of plant GS: the cystolic isoform (GS1) and the plastidic (chloroplastic) isoform (GS2). GS2 is principally found in leaf tissue and functions in the assimilation of ammonia produced by photorespiration or by nitrate reduction. GS1 is mainly found in leaf and root tissue, typically exists in a number of different isoforms in higher plants, and functions to assimilate ammonia produced by all other physiological processes (Coruzzi, 1991, Plant Science 74: 145-155; McGrath and Coruzzi, 1991, Plant J. 1(3): 275-280; Lam et al., 1996, Ann. Rev. Plant Physiol. Plant Mol. Biol. 47: 569-593; Stitt, 1999, Curr. Op. Plant Biol. 2: 178-186; Oliveira et al., 2001, Brazilian J. Med. Biol. Res. 34: 567-575). Multiple GS genes are associated with a complex promoter repertoire which enable the expression of GS in an organ and tissue specific manner, as well as in an environmental factor-dependent manner.

[0119]Plant glutamine synthetase consists of eight subunits, and the native enzyme in plants has a molecular mass ranging from 320 to 380 kD, each subunit having a molecular mass of between 38 and 45 kD. The GS1 genes of several plants, especially legumes, have been cloned and sequenced (Tischer et al., 1986, Mol Gen Genet. 203: 221-229; Gebhardt et al., 1986, EMBO J. 5: 1429-1435; Tingey et al., 1987, EMBO J. 6: 1-9; Tingey et al., 1988, J Biol. Chem. 263: 9651-9657; Bennett et al., 1989, Plant Mol. Biol. 12: 553-565; Boron and Legocki, 1993, Gene 136: 95-102; Roche et al., 1993, Plant Mol. Biol. 22: 971-983; Marsolier et al., 1995, Plant Mol. Biol. 27: 1-15; Temple et al., 1995, Mol Plant-Microbe Interact. 8: 218-227). All have been found to be encoded by nuclear genes (for review, see, Morey et al., 2002, Plant Physiol. 128(1): 182-193).

[0120]Chloroplastic GS2 appears to be encoded by a single gene, while various cystoloic GS1 isoforms are encoded within multigene families (Tingey et al., 1987, supra; Sakamoto et al., 1989, Plant Mol. Biol. 13: 611-614; Brears et al, 1991, supra; Li et al., 1993, Plant Mol. Biol., 23:401-407; Dubois et al., 1996, Plant Mol. Biol., 31:803-817; Lam et al., 1996, supra). GS1 multigene families appear to encode different subunits which may combine to form homo- or hetero-octamers, and the different members show a unique expression pattern suggesting that the gene members are differentially regulated, which may relate to the various functional roles of glutamine synthetase plays in overall nitrogen metabolism (Gebhardt et al., 1986, supra; Tingey et al., 1987, supra; Bennett et al., 1989, supra; Walker and Coruzzi, 1989, supra; Peterman and Goodman, 1991, Mol Gen Genet. 1991; 330:145-154; Marsolier et al., 1995, supra; Temple et al., 1995, supra; Dubois et al., 1996, supra).

[0121]In one embodiment, a GS1 gene coding sequence is employed to generate GS transgene constructs. In particular embodiments, further described in the Examples, infra, the Alfalfa or Arabidopsis GS1 gene coding sequence is used to generate a transgene construct that may be used to generate a transgenic plant expressing the GS1 transgene. As an example, such a construct may be used to transform Agrobacteria. The transformed Agrobacteria are then used to generate T₀ transgenic plants. Example 5 demonstrates the generation of T₀ GS1 transgenic tobacco plants using this approach. Similarly, Examples 6 and 17 demonstrates the generation of T₀ GS1 transgenic tomato plants, Example 8 demonstrates the generation of T₀ GS1 transgenic pepper plants, Examples 9 and 10 demonstrate the generation of T₀ GS1 transgenic bean plants, Examples 11 and 12 demonstrate the generation of T₀ GS1 transgenic cowpea plants, Example 13 demonstrates the generation of T₀ GS1 transgenic alfalfa plants, Example 14 demonstrates the generation of T₀ GS1 transgenic cantaloupe plants, Example 15 demonstrates the generation of T₀ GS1 transgenic pumpkin plants, Example 16 demonstrates the generation of T₀ GS1 transgenic Arabidopsis plants, and Example 18 demonstrates the generation of T₀ GS1 transgenic Cantaloupe plants.

Transcription Terminators:

[0122]In preferred embodiments, a 3' transcription termination sequence is incorporated downstream of the transgene in order to direct the termination of transcription and permit correct polyadenylation of the mRNA transcript. Suitable transcription terminators are those which are known to function in plants, including without limitation, the nopaline synthase (NOS) and octopine synthase (OCS) genes of Agrobacterium tumefaciens, the T7 transcript from the octopine synthase gene, the 3' end of the protease inhibitor I or II genes from potato or tomato, the CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator. In addition, a gene's native transcription terminator may be used. In specific embodiments, described by way of the Examples, infra, the nopaline synthase transcription terminator is employed.

Selectable Markers:

[0123]Selectable markers are typically included in transgene expression vectors in order to provide a means for selecting transformants. While various types of markers are available, various negative selection markers are typically utilized, including those which confer resistance to a selection agent that inhibits or kills untransformed cells, such as genes which impart resistance to an antibiotic (such as kanamycin, gentamycin, anamycin, hygromycin and hygromycinB) or resistance to a herbicide (such as sulfonylurea, gulfosinate, phosphinothricin and glyphosate). Screenable markers include, for example, genes encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405), genes encoding luciferase (Ow et al., 1986, Science 234: 856-859) and various genes encoding proteins involved in the production or control of anthocyanin pigments (See, for example, U.S. Pat. No. 6,573,432). The E. coli glucuronidase gene (gus, gusA or uidA) has become a widely used selection marker in plant transgenics, largely because of the glucuronidase enzyme's stability, high sensitivity and ease of detection (e.g., fluorometric, spectrophotometric, various histochemical methods). Moreover, there is essentially no detectable glucuronidase in most higher plant species.

Transformation Methodologies and Systems:

[0124]Various methods for introducing the transgene expression vector constructs of the invention into a plant or plant cell are well known to those skilled in the art, and any capable of transforming the target plant or plant cell may be utilized.

[0125]Agrobacterium-mediated transformation is perhaps the most common method utilized in plant transgenics, and protocols for Agrobacterium-mediated transformation of a large number of plants are extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2^nd edition, 2006). Agrobacterium tumefaciens is a Gram negative soil bacteria that causes tumors (Crown Gall disease) in a great many dicot species, via the insertion of a small segment of tumor-inducing DNA ("T-DNA", `transfer DNA`) into the plant cell, which is incorporated at a semi-random location into the plant genome, and which eventually may become stably incorporated there. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a `binary vector` system.

[0126]Agrobacterium transformation may be used for stably transforming dicots, monocots, and cells thereof (Rogers et al., 1986, Methods Enzymol., 118: 627-641; Hernalsteen et al., 1984, EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et al., 1984, Nature, 311: 763-764; Grimsley et al., 1987, Nature 325: 167-1679; Boulton et al., 1989, Plant Mol. Biol. 12: 31-40; Gould et al., 1991, Plant Physiol. 95: 426-434). Various methods for introducing DNA into Agrobacteria are known, including electroporation, freeze/thaw methods, and triparental mating. The most efficient method of placing foreign DNA into Agrobacterium is via electroporation (Wise et al., 2006, Three Methods for the Introduction of Foreign DNA into Agrobacterium, Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, N.J., pp. 43-53). In addition, given that a large percentage of T-DNAs do not integrate, Agrobacterium-mediated transformation may be used to obtain transient expression of a transgene via the transcriptional competency of unincorporated transgene construct molecules (Helens et al., 2005, Plant Methods 1:13).

[0127]A large number of Agrobacterium transformation vectors and methods have been described (Karimi et al., 2002, Trends Plant Sci. 7(5): 193-5), and many such vectors may be obtained commercially (for example, Invitrogen, Carlsbad, Calif.). In addition, a growing number of "open-source" Agrobacterium transformation vectors are available (for example, pCambia vectors; Cambia, Canberra, Australia). See, also, subsection herein on TRANSGENE CONSTRUCTS, supra. In a specific embodiment described further in the Examples, a pMON316-based vector was used in the leaf disc transformation system of Horsch et. al. (Horsch et al., 1995, Science 227:1229-1231) to generate growth enhanced transgenic tobacco and tomato plants.

[0128]Other commonly used transformation methods that may be employed in generating the transgenic plants of the invention include, without limitation microprojectile bombardment, or biolistic transformation methods, protoplast transformation of naked DNA by calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al., 1984, EMBO J. 3: 2727-2722; Potrykus et al., 1985, Mol. Gen. Genet. 199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82: 5824-5828; Shimamoto et al., 1989, Nature, 338: 274-276.

[0129]Biolistic transformation involves injecting millions of DNA-coated metal particles into target cells or tissues using a biolistic device (or "gene gun"), several kinds of which are available commercially. Once inside the cell, the DNA elutes off the particles and a portion may be stably incorporated into one or more of the cell's chromosomes (for review, see Kikkert et al., 2005, Stable Transformation of Plant Cells by Particle Bombardment/Biolistics, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Pena, Humana Press Inc., Totowa, N.J.).

[0130]Electroporation is a technique that utilizes short, high-intensity electric fields to permeabilize reversibly the lipid bilayers of cell membranes (see, for example, Fisk and Dandekar, 2005, Introduction and Expression of Transgenes in Plant Protoplasts, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Pena, Humana Press Inc., Totowa, N.J., pp. 79-90; Fromm et al., 1987, Electroporation of DNA and RNA into plant protoplasts, in Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press, London, UK, pp. 351-366; Joersbo and Brunstedt, 1991, Electroporation: mechanism and transient expression, stable transformation and biological effects in plant protoplasts. Physiol. Plant. 81, 256-264; Bates, 1994, Genetic transformation of plants by protoplast electroporation. Mol. Biotech. 2: 135-145; Dillen et al., 1998, Electroporation-mediated DNA transfer to plant protoplasts and intact plant tissues for transient gene expression assays, in Cell Biology, Vol. 4, ed., Celis, Academic Press, London, UK, pp. 92-99). The technique operates by creating aqueous pores in the cell membrane, which are of sufficiently large size to allow DNA molecules (and other macromolecules) to enter the cell, where the transgene expression construct (as T-DNA) may be stably incorporated into plant genomic DNA, leading to the generation of transformed cells that can subsequently be regenerated into transgenic plants.

[0131]Newer transformation methods include so-called "floral dip" methods, which offer the promise of simplicity, without requiring plant tissue culture, as is the case with all other commonly used transformation methodologies (Bent et al., 2006, Arabidopsis thaliana Floral Dip Transformation Method, Methods Mol Biol, vol. 343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, N.J., pp. 87-103; Clough and Bent, 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16: 735-743). However, with the exception of Arabidopsis, these methods have not been widely used across a broad spectrum of different plant species. Briefly, floral dip transformation is accomplished by dipping or spraying flowering plants in with an appropriate strain of Agrobacterium tumefaciens. Seeds collected from these T₀ plants are then germinated under selection to identify transgenic T₁ individuals. Example 16 demonstrated floral dip inoculation of Arabidopsis to generate transgenic Arabidopsis plants.

[0132]Other transformation methods include those in which the developing seeds or seedlings of plants are transformed using vectors such as Agrobacterium vectors. For example, as exemplified in Example 8, such vectors may be used to transform developing seeds by injecting a suspension or mixture of the vector (i.e., Agrobacteria) directly into the seed cavity of developing pods (i.e., pepper pods, bean pods, pea pods and the like). Seedlings may be transformed as described for Alfalfa in Example 13. Germinating seeds may be transformed as described for Camelina in Example 18. Intra-fruit methods, in which the vector is injected into fruit or developing fruit, may be used as described for Cantaloupe melons in Example 14 and pumpkins in Example 15.

[0133]Still other transformation methods include those in which the flower structure is targeted for vector inoculation, such as the flower inoculation methods described for beans in Examples 9 and 10, peas in Examples 11 and 12 and tomatoes in Example 17.

[0134]In addition, although transgenes are most commonly inserted into the nuclear DNA of plant cells, trangenes may also be inserted into plastidic DNA (i.e., into the plastome of the chloroplast). In most flowering plants, plastids do not occur in the pollen cells, and therefore transgenic DNA incorporated within a plastome will not be passed on through propagation, thereby restricting the trait from migrating to wild type plants. Plastid transformation, however, is more complex than cell nucleus transformation, due to the presence of many thousands of plastomes per cell (as high as 10,000).

[0135]Transplastomic lines are genetically stable only if all plastid copies are modified in the same way, i.e. uniformly. This is typically achieved through repeated regeneration under certain selection conditions to eliminate untransformed plastids, by segregating transplastomic and untransformed plastids, resulting in the selection of homoplasmic cells carrying the transgene construct and the selectable marker stably integrated therein. Plastid transformation has been successfully performed in various plant species, including tobacco, potatoes, oilseed rape, rice and Arabidopsis.

[0136]To generate transplastomic lines carrying GPT and GS transgenes, the transgene expression cassette is inserted into flanking sequences from the plastome. Using homologous recombination, the transgene expression cassette becomes integrated into the plastome via a natural recombination process. The basic DNA delivery techniques for plastid transformation include particle bombardment of leaves or polyethylene glycol-mediated DNA transformation of protoplasts. Transplastomic plants carrying transgenes in the plastome may be expressed at very high levels, due to the fact that many plastids (i.e., chloroplasts) per cell, each carrying many copies of the plastome. This is particularly the case in foliar tissue, where a single mature leaf cell may contain over 10,000 copies of the plastome. Following a successful transformation of the plastome, the transplastomic events carry the transgene on every copy of the plastid genetic material. This can result in the transgene expression levels representing as much as half of the total protein produced in the cell.

[0137]Plastid transformation methods and vector systems are described, for example, in recent U.S. Pat. Nos. 7,528,292; 7,371,923; 7,235,711; and, 7,193,131. See also U.S. Pat. Nos. 6,680,426 and 6,642,053.

[0138]The foregoing plant transformation methodologies may be used to introduce transgenes into a number of different plant cells and tissues, including without limitation, whole plants, tissue and organ explants including chloroplasts, flowering tissues and cells, protoplasts, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells, tissue cultured cells of any of the foregoing, any other cells from which a fertile regenerated transgenic plant may be generated. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation.

[0139]Methods of regenerating individual plants from transformed plant cells, tissues or organs are known and are described for numerous plant species.

[0140]As an illustration, transformed plantlets (derived from transformed cells or tissues) are cultured in a root-permissive growth medium supplemented with the selective agent used in the transformation strategy (i.e., and antibiotic such as kanamycin). Once rooted, transformed plantlets are then transferred to soil and allowed to grow to maturity. Upon flowering, the mature plants are preferably selfed (self-fertilized), and the resultant seeds harvested and used to grow subsequent generations. Examples 3-6 describe the regeneration of transgenic tobacco and tomato plants.

[0141]T₀ transgenic plants may be used to generate subsequent generations (e.g., T₁, T₂, etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed). For example, as described in Example 7, infra, individual plants over expressing the Alfalfa GS1 gene and outperforming wildtype plants were crossed with individual plants over-expressing the Arabidopsis GPT gene and outperforming wildtype plants, by simple sexual crossing using manual pollen transfer. Reciprocal crosses were made such that each plant served as the male in a set of crosses and each plant served as the female in a second set of crosses. During the mature plant growth stage, the plants are typically examined for growth phenotype, CO₂ fixation rate, etc. (see following subsection).

Selection of Growth-Enhanced Transgenic Plants:

[0142]Transgenic plants may be selected, screened and characterized using standard methodologies. The preferred transgenic plants of the invention will exhibit one or more phenotypic characteristics indicative of enhanced growth and/or other desirable agronomic properties. Transgenic plants are typically regenerated under selective pressure in order to select transformants prior to creating subsequent transgenic plant generations. In addition, the selective pressure used may be employed beyond T₀ generations in order to ensure the presence of the desired transgene expression construct or cassette.

[0143]T₀ transformed plant cells, calli, tissues or plants may be identified and isolated by selecting or screening for the genetic composition of and/or the phenotypic characteristics encoded by marker genes contained in the transgene expression construct used for the transformation. For example, selection may be conducted by growing potentially-transformed plants, tissues or cells in a growth medium containing a repressive amount of antibiotic or herbicide to which the transforming genetic construct can impart resistance. Further, the transformed plant cells, tissues and plants can be identified by screening for the activity of marker genes (i.e., β-glucuronidase) which may be present in the transgene expression construct.

[0144]Various physical and biochemical methods may be employed for identifying plants containing the desired transgene expression construct, as is well known. Examples of such methods include Southern blot analysis or various nucleic acid amplification methods (i.e., PCR) for identifying the transgene, transgene expression construct or elements thereof, Northern blotting, S1 RNase protection, reverse transcriptase PCR (RT-PCR) amplification for detecting and determining the RNA transcription products; and protein gel electrophoresis, Western blotting, immunoprecipitation, enzyme immunoassay, and the like may be used for identifying the protein encoded and expressed by the transgene.

[0145]In another approach, expression levels of genes, proteins and/or metabolic compounds that are know to be modulated by transgene expression in the target plant may be used to identify transformants. In one embodiment of the present invention, increased levels of the signal metabolite 2-oxoglutaramate may be used to screen for desirable transformants, as exemplified in the Examples. Similarly, increased levels of GPT and/or GS activity may be assayed, as exemplified in the Examples.

[0146]Ultimately, the transformed plants of the invention may be screened for enhanced growth and/or other desirable agronomic characteristics. Indeed, some degree of phenotypic screening is generally desirable in order to identify transformed lines with the fastest growth rates, the highest seed yields, etc., particularly when identifying plants for subsequent selfing, cross-breeding and back-crossing. Various parameters may be used for this purpose, including without limitation, growth rates, total fresh weights, dry weights, seed and fruit yields (number, weight), seed and/or seed pod sizes, seed pod yields (e.g., number, weight), leaf sizes, plant sizes, increased flowering, time to flowering, overall protein content (in seeds, fruits, plant tissues), specific protein content (i.e., GS), nitrogen content, free amino acid, and specific metabolic compound levels (i.e., 2-oxoglutaramate). Generally, these phenotypic measurements are compared with those obtained from a parental identical or analogous plant line, an untransformed identical or analogous plant, or an identical or analogous wild-type plant (i.e., a normal or parental plant). Preferably, and at least initially, the measurement of the chosen phenotypic characteristic(s) in the target transgenic plant is done in parallel with measurement of the same characteristic(s) in a normal or parental plant. Typically, multiple plants are used to establish the phenotypic desirability and/or superiority of the transgenic plant in respect of any particular phenotypic characteristic.

[0147]Preferably, initial transformants are selected and then used to generate T₁ and subsequent generations by selfing (self-fertilization), until the transgene genotype breeds true (i.e., the plant is homozygous for the transgene). In practice, this is accomplished by screening at each generation for the desired traits and selfing those individuals, often repeatedly (i.e., 3 or 4 generations). As exemplified herein, transgenic plant lines propagated through at least one sexual generation (Tobacco, Arabidopsis, Tomato) demonstrated higher transgene product activities compared to lines that did not have the benefit of sexual reproduction and the concomitant increase in transgene copy number.

[0148]Stable transgenic lines may be crossed and back-crossed to create varieties with any number of desired traits, including those with stacked transgenes, multiple copies of a transgene, etc. Various common breeding methods are well know to those skilled in the art (see, e.g., Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)). Additionally, stable transgenic plants may be further modified genetically, by transforming such plants with further transgenes or additional copies of the parental transgene. Also contemplated are transgenic plants created by single transformation events which introduce multiple copies of a given transgene or multiple transgenes.

[0149]In another aspect, the invention provides transgenic plants characterized by increased nitrogen use efficiency. Nitrogen use efficiency may be expressed as plant yield per given amount of nitrogen. In the Examples provided herein, the transgene and control plants all received the same nutrient solutions in the same amounts. The transgenic plants were consistently characterized by higher yields, and thus have higher nitrogen use efficiencies.

[0150]In yet another aspect, the invention provides transgenic plants and seeds thereof with increased tolerance to high salt growth conditions. This aspect of the invention is exemplified by Example 24, which describes the germination of transgenic tobacco plant seeds in very high salt conditions (200 mM NaCl). While counterpart wild type tobacco seeds germinated at a rate of only about 10%, on average, the transgenic tobacco seeds achieved nearly the same rate of germination obtained under no salt conditions for both transgenic and wild type seeds, or about 92%.

EXAMPLES

[0151]Various aspects of the invention are further described and illustrated by way of the several examples which follow, none of which are intended to limit the scope of the invention.

Example 1

Isolation of Arabidopsis Gluamine Phenylpyruvate Transaminase (GPT) Gene

[0152]In an attempt to locate a plant enzyme that is directly involved in the synthesis of the signal metabolite 2-oxoglutaramate, applicants hypothesized that the putative plant enzyme might bear some degree of structural relationship to a human protein that had been characterized as being involved in the synthesis of 2-oxoglutaramate. The human protein, glutamine transaminase K (E.C. 2.6.1.64) (also referred in the literature as cysteine conjugate β-lyase, kyneurenine aminotransferase, glutamine phenylpyruvate transaminase, and other names), had been shown to be involved in processing of cysteine conjugates of halogenated xenobiotics (Perry et al., 1995, FEBS Letters 360:277-280). Rather than having an activity involved in nitrogen assimilation, however, human cysteine conjugate β-lyase has a detoxifying activity in humans, and in animals (Cooper and Meister, 1977, supra). Nevertheless, the potential involvement of this protein in the synthesis of 2-oxoglutaramate was of interest.

[0153]Using the protein sequence of human cysteine conjugate β-lyase, a search against the TIGR Arabidopsis plant database of protein sequences identified one potentially related sequence, a polypeptide encoded by a partial sequence at the Arabidopsis gene locus at At1q77670, sharing approximately 36% sequence homology/identity across aligned regions.

[0154]The full coding region of the gene was then amplified from an Arabidopsis cDNA library (Stratagene) with the following primer pair:

TABLE-US-00002 [SEQ ID NO: 37] 5'-CCCATCGATGTACC TGGACATAAATGGTGTGATG-3' [SEQ ID NO: 38] 5'-GATGGTACCTCAGACTTTTCTCTTAAGCTTCTGCTTC-3'

[0155]These primers were designed to incorporate Cla I (ATCGAT) and Kpn I (GGTACC) restriction sites to facilitate subsequent subcloning into expression vectors for generating transgenic plants. Takara ExTaq DNA polymerase enzyme was used for high fidelity PCR using the following conditions: initial denaturing 94° C. for 4 minutes, 30 cycles of 94° C. 30 second, annealing at 55° C. for 30 seconds, extension at 72° C. for 90 seconds, with a final extension of 72° C. for 7 minutes. The amplification product was digested with Cla I and Kpn I restriction enzymes, isolated from an agarose gel electrophoresis and ligated into vector pMon316 (Rogers, et. al. 1987 Methods in Enzymology 153:253-277) which contains the cauliflower mosaic virus (CaMV, also CMV) 35S constitutive promoter and the nopaline synthase (NOS) 3' terminator. The ligation product was transformed into DH5a cells and transformants sequenced to verify the insert.

[0156]A 1.3 kb cDNA was isolated and sequenced, and found to encode a full length protein of 440 amino acids in length, including a putative chloroplast signal sequence.

Example 2

Production of Biologically Active Recombinant Arabidopsis Glutamine Phenylpyruvate Transaminase (GPT)

[0157]To test whether the protein encoded by the cDNA isolated as described in Example 1, supra, is capable of catalyzing the synthesis of 2-oxoglutaramate, the cDNA was expressed in E. coli, purified, and assayed for its ability to synthesize 2-oxoglutaramate using a standard method.

NMR Assay for 2-Oxoglutaramate:

[0158]Briefly, the resulting purified protein was added to a reaction mixture containing 150 mM Tris-HCl, pH 8.5, 1 mM beta mercaptoethanol, 200 mM glutamine, 100 mM glyoxylate and 200 μM pyridoxal 5'-phosphate. The reaction mixture without added test protein was used as a control. Test and control reaction mixtures were incubated at 37° C. for 20 hours, and then clarified by centrifugation to remove precipitated material. Supernatants were tested for the presence and amount of 2-oxoglutaramate using ¹³C NMR with authentic chemically synthesized 2-oxoglutaramate as a reference. The products of the reaction are 2-oxoglutaramate and glycine, while the substrates (glutamine and glyoxylate) diminish in abundance. The cyclic 2-oxoglutaramate gives rise to a distinctive signal allowing it to be readily distinguished from the open chain glutamine precursor.

HPLC Assay for 2-Oxoglutaramate:

[0159]An alternative assay for GPT activity uses HPLC to determine 2-oxoglutaramate production, following a modification of Calderon et al., 1985, J Bacteriol 161(2): 807-809. Briefly, a modified extraction buffer consisting of 25 mM Tris-HCl pH 8.5, 1 mM EDTA, 20 μM FAD, 10 mM Cysteine, and ˜1.5% (v/v) Mercaptoethanol. Tissue samples from the test material (i.e., plant tissue) are added to the extraction buffer at approximately a 1/3 ratio (w/v), incubated for 30 minutes at 37° C., and stopped with 200 μl of 20% TCA. After about 5 minutes, the assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8 mm ID×30 cm L column, with a mobile phase in 0.01N H₂SO₄, a flow rate of approximately 0.2 ml/min, at 40° C. Injection volume is approximately 20 and retention time between about 38 and 39 minutes. Detection is achieved with 210 nm UV light.

Results Using NMR Assay:

[0160]This experiment revealed that the test protein was able to catalyze the synthesis of 2-oxoglutaramate. Therefore, these data indicate that the isolated cDNA encodes a glutamine phenylpyruvate transaminase that is directly involved in the synthesis of 2-oxoglutaramate in plants. Accordingly, the test protein was designated Arabidopsis glutamine phenylpyruvate transaminase, or "GPT".

[0161]The nucleotide sequence of the Arabidopsis GPT coding sequence is shown in the Table of Sequences, SEQ ID NO. 1. The translated amino acid sequence of the GPT protein is shown in SEQ ID NO. 2.

Example 3

Creation of Transgenic Tobacco Plants Over-Expressing Arabidopsis GPT

[0162]Generation of Plant Expression Vector pMON-PJU:

[0163]Briefly, the plant expression vector pMon316-PJU was constructed as follows. The isolated cDNA encoding Arabidopsis GPT (Example 1) was cloned into the ClaI-KpnI polylinker site of the pMON316 vector, which places the GPT gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) transcriptional terminator. A kanamycin resistance gene was included to provide a selectable marker.

Agrobacterium-Mediated Plant Transformations:

[0164]pMON-PJU and a control vector pMon316 (without inserted DNA) were transferred to Agrobacterium tumefaciens strain pTiTT37ASE using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing the antibiotics spectinomycin (100 micro gm/ml) and kanamycin (50 micro gm/ml). Antibiotic resistant colonies of Agrobacterium were examined by PCR to assure that they contained plasmid.

[0165]Nicotiana tabacum cv. Xanthi plants were transformed with pMON-PJU transformed Agrobacteria using the leaf disc transformation system of Horsch et. al. (Horsch et al., 1995, Science 227:1229-1231). Briefly, sterile leaf disks were inoculated and cultured for 2 days, then transferred to selective MS media containing 100 μg/ml kanamycin and 500 μg/ml clafaran. Transformants were confirmed by their ability to form roots in the selective media.

Generation of GPT Transgenic Tobacco Plants:

[0166]Sterile leaf segments were allowed to develop callus on Murashige & Skoog (M&S) media from which the transformant plantlets emerged. These plantlets were then transferred to the rooting-permissive selection medium (M&S medium with kanamycin as the selection agent). The healthy, and now rooted, transformed tobacco plantlets were then transferred to soil and allowed to grow to maturity and upon flowering the plants were selfed and the resultant seeds were harvested. During the growth stage the plants had been examined for growth phenotype and the CO₂ fixation rate was measured for many of the young transgenic plants.

Production of T1 and T2 Generation GPT Transgenic Plants:

[0167]Seeds harvested form the T₀ generation of the transgenic tobacco plants were germinated on M&S media containing kanamycin (100 mg/L) to enrich for the transgene. At least one fourth of the seeds did not germinate on this media (kanamycin is expected to inhibit germination of the seeds without resistance that would have been produced as a result of normal genetic segregation of the gene) and more than half of the remaining seeds were removed because of demonstrated sensitivity (even mild) to the kanamycin.

[0168]The surviving plants (T₁ generation) were thriving and these plants were then selfed to produce seeds for the T₂ generation. Seeds from the T₁ generation were germinated on MS media supplemented for the transformant lines with kanamycin (10 mg/liter). After 14 days they were transferred to sand and provided quarter strength Hoagland's nutrient solution supplemented with 25 mM potassium nitrate. They were allowed to grow at 24° C. with a photoperiod of 16 h light and 8 hr dark with a light intensity of 900 micromoles per meter squared per second. They were harvested 14 days after being transferred to the sand culture.

Characterization of GPT Transgenic Plants:

[0169]Harvested transgenic plants (both GPT transgenes and vector control transgenes) were analyzed for glutamine sythetase activity in root and leaf, whole plant fresh weight, total protein in root and leaf, and CO₂ fixation rate (Knight et al., 1988, Plant Physiol. 88: 333). Non-transformed, wild-type A. tumefaciens plants were also analyzed across the same parameters in order to establish a baseline control.

[0170]Growth characteristic results are tabulated below in Table I. Additionally, a photograph of the GPT transgenic plant compared to a wild type control plant is shown in FIG. 2 (together with GS1 transgenic tobacco plant, see Example 5). Across all parameters evaluated, the GPT transgenic tobacco plants showed enhanced growth characteristics. In particular, the GPT transgenic plants exhibited a greater than 50% increase in the rate of CO₂ fixation, and a greater than two-fold increase in glutamine synthetase activity in leaf tissue, relative to wild type control plants. In addition, the leaf-to-root GS ratio increased by almost three-fold in the transaminase transgenic plants relative to wild type control. Fresh weight and total protein quantity also increased in the transgenic plants, by about 50% and 80% (leaf), respectively, relative to the wild type control. These data demonstrate that tobacco plants overexpressing the Arabidopsis GPT transgene achieve significantly enhanced growth and CO₂ fixation rates.

TABLE-US-00003 TABLE I Leaf Root Protein mg/gram fresh weight Wild type - control 8.3 2.3 Line PN1-8 a second control 8.9 2.98 Line PN9-9 13.7 3.2 Glutamine Synthetase activity, micromoles/min/mg protein Wild type (Ratio of leaf:root = 4.1:1) 4.3 1.1 PN1-8 (Ratio of leaf:root = 4.2:1) 5.2 1.3 PN9-9 (Ratio of leaf:root = 10.9:1) 10.5 0.97 Whole Plant Fresh Weight, g Wild type 21.7 PN1-8 26.1 PN9-9 33.1 CO₂ Fixation Rate, umole/m2/sec Wild type 8.4 PN1-8 8.9 PN9-9 12.9 Data = average of three plants Wild type - Control plants; not regenerated or transformed. PN1 lines were produced by regeneration after transformation using a construct without inserted gene. A control against the processes of regeneration and transformation. PN 9 lines were produced by regeneration after transformation using a construct with the Arabidopsis GPT gene.

Example 4

Generation of Transgenic Tomato Plants Carrying Arabidopsis GPT Transgene

[0171]Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the Arabidopsis GPT transgene were generated using the vectors and methods described in Example 3. T₀ transgenic tomato plants were generated and grown to maturity. Initial growth characteristic data of the GPT transgenic tomato plants is presented in Table II. The transgenic plants showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants. In addition, the transgenic plants developed multiple main stems, whereas wild type plants developed with a single main stem. A photograph of a GPT transgenic tomato plant compared to a wild type plant is presented in FIG. 3 (together with GS1 transgenic tomato plants, see Example 6).

TABLE-US-00004 TABLE II Growth Wildtype GPT Transgenic Characteristics Tomato Tomato Stem height, cm 6.5 18, 12, 11 major stems Stems 1 3 major, 0 other Buds 2 16 Flowers 8 12 Fruit 0 3

Example 5

Generation of Transgenic Tobacco Plants Overexpressing Alfalfa Gs1

[0172]Generation of Plant Expression Vector pGS111:

[0173]Transgenic tobacco plants overexpressing the Alfalfa GS1 gene were generated as previously described (Temple et al., 1993, Mol. Gen. Genetics 236: 315-325). Briefly, the plant expression vector pGS111 was constructed by inserting the entire coding sequence together with extensive regions of both the 5' and 3' untranslated regions of the Alfalfa GS1 gene [SEQ ID NO: 3] (DasSarma at al., 1986, Science, Vol 232, Issue 4755, 1242-1244) into pMON316 (Rogers et al., 1987, supra), placing the transgene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) transcriptional terminator. A kanamycin resistance gene was included to provide a selectable marker.

Generation of GS1 Transformants:

[0174]pGS111 was transferred to Agrobacterium tumefaciens strain pTiTT37ASE using triparental mating as described (Rogers et al., 1987, supra; Unkefer et al., U.S. Pat. No. 6,555,500). Nicotiana tabacum cv. Xanthi plants were transformed with pGS111 transformed Agrobacteria using the leaf disc transformation system of Horsch et. al. (Horsch et al., 1995, Science 227:1229-1231). Transformants were selected and regenerated on MS medium containing 100 μg/ml kanamycin. Shoots were rooted on the same medium (with kanamycin, absent hormones) and transferred to potting soil:perlite:vermiculite (3:1:1), grown to maturity, and allowed to self. Seeds were harvested from this T₀ generation, and subsequent generations produced by selfing and continuing selection with kanamycin. The best growth performers were used to yield a T3 progeny for crossing with the best performing GPT over-expressing lines identified as described in Example 3. A photograph of the GS1 transgenic plant compared to a wild type control plant is shown in FIG. 2 (together with GPT transgenic tobacco plant, see Example 3)

Example 6

Generation of Transgenic Tomato Plants Carrying Alfalfa GS1 Transgene

[0175]Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the Alfalfa GS1 transgene were generated using the vector described in Example 5 and a transformation protocol essentially as described (Sun et al., 2006. Plant Cell Physiol. 46(3) 426-31). T₀ transgenic tomato plants were generated and grown to maturity. Initial growth characteristic data of the GPT transgenic tomato plants is presented in Table III. The transgenic plants showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants. In addition, the transgenic plants developed multiple main stems, whereas wild type plants developed with a single main stem. A photograph of a GS1 transgenic tomato plant compared to a wild type plant is presented in FIG. 3 (together with GPT transgenic tomato plant, see Example 4).

TABLE-US-00005 TABLE III Growth Wildtype GS1 Transgenic Characteristics Tomato Tomato Stem height, cm 6.5 16, 7, 5 major stems Stems 1 3 major, 3 med, 1 sm Buds 2 2 Flowers 8 13 Fruit 0 4

Example 7

Generation of Double Transgenic Tobacco Plants Carrying GS1 and GPT Transgenes

[0176]In an effort to determine whether the combination of GS1 and GPT transgenes in a single transgenic plant might improve the extent to which growth and other agronomic characteristics may be enhanced, a number of sexual crosses between high producing lines of the single transgene (GS1 or GPT) transgenic plants were carried out. The results obtained are dramatic, as these crosses repeatedly generated progeny plants having surprising and heretofore unknown increases in growth rates, biomass yield, and seed production.

Materials and Methods:

[0177]Single-transgene, transgenic tobacco plants overexpressing GPT or GS1 were generated as described in Examples 3 and 4, respectively. Several of fastest growing T₂ generation GPT transgenic plant lines were crossed with the fastest growing T3 generation GS1 transgenic plant lines using reciprocal crosses. The progeny were then selected on kanamycin containing M&S media as described in Example 3, and their growth, flowering and seed yields examined.

[0178]Tissue extractions for GPT and GS activities: GPT activity was extracted from fresh plant tissue after grinding in cold 100 mM Tris-HCl, pH 7.6, containing 1 mm ethylenediaminetetraacetic, 200 mM pyridoxal phosphate and 6 mM mercaptoethanol in a ratio of 3 ml per gram of tissue. The extract was clarified by centrifugation and used in the assay. GS activity was extracted from fresh plant tissue after grinding in cold 50 mM Imidazole, pH 7.5 containing 10 mM MgCl2, and 12.5 mM mercaptoethanol in a ratio of 3 ml per gram of tissue. The extract was clarified by centrifugation and used in the assay. GPT activity was assayed as described in Calderon and Mora, 1985, Journal Bacteriology 161:807-809. GS activity was measured as described in Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910-922. Both assays involve an incubation with substrates and cofactor at the proper pH. Detection was by HPLC.

Results:

[0179]The results are presented in two ways. First, specific growth characteristics are tabulated in Tables IV.A and IV.B (biomass, seed yields, growth rate, GS activity, GPT activity, 2-oxoglutaramate activity, etc). Second, photographs of progeny plants and their leaves are shown in comparison to single-transgene and wild type plants and leaves are presented in FIG. 5 and FIG. 6, which show much larger whole plants, larger leaves, and earlier and/or more abundant flowering in comparison to the parental single-transgene plants and wild type control plants.

[0180]Referring to Table IV.A, double-transgene progeny plants form these crosses showed tremendous increases total biomass (fresh weight), with fresh weights ranging from 45-89 grams per individual progeny plant, compared to a range of only 19-24 grams per individual wild type plant, representing on average, about a two- to three-fold increase over wild type plants, and representing at the high end, an astounding four-fold increase in biomass over wild type plants. Taking the 24 individual double-transgene progeny plants evaluated, the average individual plant biomass was about 2.75 times that of the average wild type control plant. Four of the progeny lines showed approximately 2.5 fold greater average per plant fresh weights, while two lines showed over three-fold greater fresh weights in comparison to wild type plants.

[0181]In comparison to the single-transgene parental lines, the double-transgene progeny plants also showed far more than an additive growth enhancement. Whereas GPT single-transgene lines show as much as about a 50% increase over wild type biomass, and GS1 single-transgene lines as much as a 66% increase, progeny plants averaged almost a 200% increase over wild type plants.

[0182]Similarly, the double transgene progeny plants flowered earlier and more prolifically than either the wild type or single transgene parental lines, and produced a far greater number of seed pods as well as total number of seeds per plant. Referring again to Table IV.A, on average, the double-transgene progeny produced over twice the number of seed pods produced by wild type plants, with two of the high producer plants generating over three times the number of seed pods compared to wild type. Total seed yield in progeny plants, measured on a per plant weight basis, ranged from about double to nearly quadruple the number produced in wild type plants.

TABLE-US-00006 TABLE IV.A FRESH WEIGHT SEED PODS SEED YIELD GS ACTIVITY PLANT LINE g/whole plant #pods/plant g/plant LEAF ROOT L/R RATIO Wild Type Tobacco Wild type 1 18.73 26 0.967 Wild type 2 24.33 24 1.07 Wild type 3 23.6 32 0.9 Wild type 4 18.95 32 1.125 WT Average 21.4025 28.5 1.0155 7.75 1.45 5.34 Cross 1 X1L1a × PA9-9ff 1 59.21 62 2.7811 2 65.71 56 3 55.36 72 4 46.8 56 Cross 1 Average 56.77 61.5 +274% 14.98 1.05 14.27 Compared to WT +265% +216% +193% -28% +267% Cross 2 PA9-2 × L9 1 70.83 61 1.76 2 49.17 58 3.12 3 50.23 90 NA 4 45.77 Cross 2 Average 54 58.3 2.44 16.32 1.81 9.02 Compared to WT +252% +205% +240% +211% +125% +169% Cross 3 PA9-9ff × L1a 1 89.1 77 3.687 2 78.18 3 58.34 4 61.79 Cross 3 Average 71.85 77 (one plant) 3.678 (one plant) 15.92 1.38 11.54 Compared to WT +336% +270% +362% +205% -5% +216% Cross 5 PA9-10aa × L1a 1 65.34 45 2.947 2 53.28 64 3.3314 3 49.85 42 1.5667 4 44.63 42 2.5013 Cross 5 Average 53.275 48.25 2.86928 13.03 1.8 7.24 Compared to WT +244% +169% +283% +168% Cross 6 PA9-17b × L1a 1 56.7 64 2.492 2 55.05 66 2.162 3 51.51 59 1.8572 4 45.38 72 4.742 Cross 6 Average 52.16 65.25 2.8133 14.114.752 1.1.1124 13.29 Compared to WT +244% +229% +277% Cross 7 PA9-20aa × L1b 1 76.26 67 2.0535 2 66.27 42 1.505 3 72.26 72 2.3914 4 63.91 91 2.87 Cross 7 Average 69.675 68 2.204975 14.12 1.24 11.39 Compared to WT +326% +239% +217% Control PA9-9ff 1 32.18 N/A 2 32.64 N/A 3 34.67 N/A 4 25.18 N/A Average 31.17 N/A 11.57 1.14 10.15 Compared to WT +148% Control GS L1a 1 41.74 N/A 2 36.24 N/A 3 33.8 N/A 4 30.48 N/A Average 35.57 N/A 13.15 1.23 10.69 Compared to WT +166%

[0183]Table IV.B shows growth rate, biomass and yield, and biochemical characteristics of Line XX (Line 3 further selfed) compared to the single transgene line expressing GS1 and wild type control tobacco. All parameters are greatly increased in the double transgenic plant (Line XX). Notably, 2-oxoglutaramate activity was almost 17-fold higher, and seed yield and foliar biomass was three-fold higher, in Line XX plants versus control plants.

TABLE-US-00007 TABLE IV.B Specific Foliar Fruit/ GS Activity GPT Activity 2-oxoglu- Trans Plant Growth Rate Biomass Flowers/ Seed umol/min/ nmol/h/ taramate Gene Type mg/g/d FWt, g Buds Yield g gFWt gFWt nmol/gFWt Assay Wildtype, 228 21.40 28.5 1.02 7.75 16.9 68.9 No avg Line 1 GS 269 35.57 NM NM 11.6 NM 414 Yes Line XX 339 59.71 62.9 2.94 16.3 243.9 1,153.6 Yes NM Not Measured

Example 8

Generation of Double Transgenic Pepper Plants Carrying GS1 and GPT Transgenes

[0184]In this example, Big Jim chili pepper plants (New Mexico varietal) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter, using Agrobacterium-mediated transfer to seed pods. After 3 days, seeds were harvested and used to generate T0 plants and screened for transformants. The resulting double-transgenic plants showed higher pod yields, faster growth rates, and greater biomass yields in comparison to the control plants.

Materials and Methods:

[0185]Solanaceae Capisicum Pepper plants ("Big Jim" varietal) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON (see Example 3), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (Tomato rubisco rbcS3C promoter: Kyozulka et al., 1993, Plant Physiol. 103: 991-1000; SEQ ID NO: 22; vector construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer to seed pods.

[0186]For this and all subsequent examples, the Cambia 1201 or 1305.1 vectors were constructed according to standard cloning methods (Sambrook et al., 1989, supra, Saiki et al., 1988, Science 239: 487-491). The vector is supplied with a 35S CaMV promoter; that promoter was replaced with RcbS-3C promoter from tomato to control the expression of the target gene. The Cambia 1201 vectors contain bacterial chlorophenicol and plant hygromycin resistance selectable marker genes. The Cambia 1305.1 vectors contain bacterial chlorophenicol and hygromycin resistance selectable marker genes.

[0187]The transgene expression vectors pMON (GPT transgene) and pCambia 1201 (GS transgene) were transferred to separate Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159). Transformed Agrobacterium were selected on media containing 50 μg/ml of either streptamycin for pMON constructs or chloroamphenicol for the Cambia constructs. Transformed Agrobacterium cells were grown in LB culture media containing 25 μg/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.

[0188]Pepper plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrobacterium is injected directly into the seed cavity of developing pods. Briefly, developing pods were injected with the 200 ml mixture in order to inoculate immature seeds with the Agrobacteria essentially as described (Wang and Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). In order to induce Agrobacteria virulence and improve transformation efficiencies, 10 μg/ml acetosyringonone was added to the Agrobacteria cultures prior to pod inoculations (see, Sheikholeslam and Weeks, 1986, Plant Mol. Biol. 8: 291-298).

[0189]Using a syringe, pods were injected with a liberal quantity of the Agrobacterium vector mixture, and left to incubate for about 3 days. Seeds were then harvested and germinated, and developing plants observed for phenotypic characteristics including growth and antibiotic resistance. Plants carrying the transgenes were green, whereas untransformed plants showed signs of chlorosis in leaf tips. Vigorous growing transformants were further cultivated and compared to wild type pepper plants grown under identical conditions.

Results:

[0190]The results are presented in FIG. 7 and Table V. FIG. 7 shows a photograph of a GPT+GS double transgenic pepper plant compared to a control plant grown for the same time under identical conditions. This photograph shows tremendous pepper yield in the transgenic line compared to the control plant.

[0191]Table V presents biomass yield and GS activity, as well as transgene genotyping, in the transgenic lines compared to the wild type control. Referring to Table V, double-transgene progeny plants showed tremendous increases total biomass (fresh weight), with fresh weights, ranging from 393-662 grams per individual transgenic plant, compared to an average of 328 grams per wild type plant. Transgenic line A5 produced more than twice the total biomass of the controls. Moreover, pepper yields in the transgenic lines were greatly improved over wild type plants, and were 50% greater than control plants (on average). Notably, one of the transgene lines produced twice as many peppers as the control plant average.

TABLE-US-00008 TABLE V TRANSGENIC PEPPER GROWTH/BIOMASS AND REPRODUCTION Biomass, Yield GS activity Transgene Foliar Fresh Peppers, g Umoles/min/ Presence Plant type Wt, g DWt gFWt Assay Wildtype, avg 328.2 83.7 1.09 Negative Line A2 457.3 184.2 1.57 GPT - Yes Line A5 661.7 148.1 1.8 GPT - Yes Line B1 493.4 141.0 1.3 GPT - Yes Line B4 393.1 136.0 1.6 GPT - Yes Line C1 509.4 152.9 1.55 GPT - Yes FWt Fresh Weight; DWt Dry Weight

Example 9

Generation of Double Transgenic Bean Plants Carrying Arabidopsis GS1 and GPT Transgenes

[0192]In this example, yellow wax bean plants (Phaseolus vulgaris) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pCambia 1201, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into flowers.

Materials and Methods:

[0193]The transgene expression vectors pCambia 1201-GPT (including construct of SEQ ID NO: 27) and pCambia 1201-GS (including construct of SEQ ID NO: 6) were transferred to separate Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159). Transformed Agrobacterium were selected on media containing 50 μg/ml of chloroamphenicol. Transformed Agrobacterium cells were grown in LB culture media containing 25 μg/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.

[0194]Bean plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrobacteria is injected directly into the flower structure (Yasseem, 2009, Plant Mol. Biol. Reporter 27: 20-28). In order to induce Agrobacteria virulence and improve transformation efficiencies, 10 μg/ml acetosyringonone was added to the Agrobacteria cultures prior to flower inoculation. Briefly, once flowers bloomed, the outer structure encapsulating the reproductive organs was gently opened with forceps in order to permit the introduction of the Agrobacteria mixture, which was added to the flower structure sufficient to flood the anthers.

[0195]Plants were grown until bean pods developed, and seeds were harvested and used to generate transgenic plants. Transgenic plants were then grown together with control bean plants under identical conditions, photographed and phenotypically characterized. Growth rates were measured for both transgenic and control plants. In this and all examples, Glutamine synthetase (GS) activity was assayed according to the methods in Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910-922; and, Glutamine phenylpyruvate transaminase (GPT) activity was assayed according to the methods in Calderon et al., 1985, J. Bacteriol. 161: 807-809. See details in Example 7, Methods, supra.

Results:

[0196]The results are presented in FIG. 8, FIG. 9 and Table VI.

[0197]FIG. 8 shows GPT+GS transgenic bean line A growth rate data relative to control plants, including plant heights on various days into cultivation, as well as numbers of flower buds, flowers, and bean pods. These data show that the GPT+GS double transgenic bean plants outgrew their counterpart control plants. The transgenic plants grew taller, flowered earlier and produced more flower buds and flowers, and developed bean pods and produced more bean pods that the wild type control plants.

TABLE-US-00009 TABLE VI TRANSGENIC BEANS LINE A GS Activity Bean Pod GPT Activity umoles/min/ Antibiotic Plant Type Yield FWt, g nmoles/h/gFWt gFWt Resistance Wildtype, avg 126.6 101.9 25.2 Negative 2A 211.5 NM NM + 4A 207.7 NM NM + 5B 205.7 984.7 101.3 + WT Wildtype; FWt Fresh Weight; NM Not Measured

[0198]Table VI presents bean pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table VI, double-transgene progeny plants showed substantial bean pod biomass increases (fresh pod weight) in comparison to the control plants, with bean pod biomass yields consistently above 200 grams per individual transgenic plant, compared to an average of 127 grams per wild type plant, representing an over 60% increase in pod yield in the double transgene lines relative to control plant(s).

[0199]Lastly, FIG. 9 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions, showing increased growth in the transgenic plant.

Example 10

Generation of Double Transgenic Bean Plants Carrying Arabidopsis GS1 and Grape GPT Transgenes

[0200]In this example, yellow wax bean plants (Phaseolus vulgaris) were transformed with the Grape GPT full length coding sequence included in SEQ ID NO: 8 under the control of the RuBisCo promoter within the expression vector pCambia 1305.1, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into developing pods.

Materials and Methods:

[0201]The transgene expression vectors pCambia 1201-GPT(grape) (including construct of SEQ ID NO: 8) and pCambia 1201-GS (including construct of SEQ ID NO: 6) were transferred to separate Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159). Transformed Agrobacterium were selected on media containing 50 μg/ml of chloroamphenicol. Transformed Agrobacterium cells were grown in LB culture media containing 25 μg/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.

[0202]Bean plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrobacteria is injected directly into the flower structure. In order to induce Agrobacteria virulence and improve transformation efficiencies, 10 μg/ml acetosyringonone was added to the Agrobacteria cultures prior to flower inoculation. Briefly, once flowers bloomed, the outer structure encapsulating the reproductive organs was gently opened with forceps in order to permit the introduction of the Agrobacteria mixture, which was added to the flower structure sufficient to flood the anthers.

[0203]Plants were grown until bean pods developed, and seeds were harvested and used to generate transgenic plants. Transgenic plants were then grown together with control bean plants under identical conditions, photographed and phenotypically characterized. Growth rates were measured for both transgenic and control plants.

Results:

[0204]The results are presented in FIG. 10, FIG. 11 and Table VII.

[0205]FIG. 10 shows GPT+GS transgenic bean line G growth rate data relative to control plants, specifically including numbers of flower buds, flowers, and bean pods. These data show that the GPT+GS double transgenic bean plants outgrew their counterpart control plants. Notably, the transgenic plants produced substantially more bean pods that the wild type control plants.

TABLE-US-00010 TABLE VII TRANSGENIC BEANS LINE G: POD YIELDS Plant Type Bean Pod Yield FWt, g Antibiotic Resistance Wild type, avg 157.9 Negative G1 200.5 + G2 178.3 + WT Wildtype; FWt Fresh Weight; NM Not Measured

[0206]Table VII presents bean pod yield and antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table VII, double-transgene progeny plants showed substantial bean pod biomass increases (fresh pod weight) in comparison to the control plants, with bean pod biomass yields of 200.5 (line G1) and 178 grams (line G2) per individual transgenic plant, compared to an average of 158 grams per individual wild type plant, representing approximately a 27% increase in pod yield in the double transgene lines relative to control plants.

[0207]Lastly, FIG. 11 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions. The transgenic plant shows substantially increased size and biomass, larger leaves and a more mature flowering compared to the control plant.

Example 11

Generation of Double Transgenic Cowpea Plants Carrying Arabidopsis GS1 and GPT Transgenes

[0208]In this example, common Cowpea plants were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into flowers. Materials and methods were as in Example 9, supra.

Results:

[0209]The results are presented in FIGS. 12 and 13, and Table VI. FIG. 12 shows relative growth rates for the GPT+GS transgenic Cowpea line A and wild type control Cowpea at several intervals during cultivation, including (FIG. 12A) height and longest leaf measurements, (FIG. 12B) trifolate leafs and flower buds, and (FIG. 12C) flowers, flower buds and pea pods. These data show that the GPT+GS double transgenic Cowpea plants outgrew their counterpart control plants. The transgenic plants grew faster and taller, had longer leaves, and set flowers and pods sooner than wild type control plants.

TABLE-US-00011 TABLE VIII TRANSGENIC COWPEA LINE A Pea Pod Yield, GPT Activity GS Activity Antibiotic Plant Type FWt, g nmoles/h/gFWt umol/min/gFWt Resistance Wildtype, avg 74.7 44.4 28.3 Negative 4A 112.8 NM 41.3 + 8B 113.8 736.2 54.9 + WT Wildtype; FWt Fresh Weight; NM Not Measured

[0210]Table VIII presents pea pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table VIII, double-transgene progeny plants showed substantial pea pod biomass increases (fresh pod weight) in comparison to the control plants, with average transgenic plant pea pod biomass yields nearly 52% greater than the yields measured in control plant(s).

[0211]Lastly, FIG. 13 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions, showing increased biomass and pod yield in the transgenic plant relative to the wild type control plant.

Example 12

Generation of Double Transgenic Cowpea Plants Carrying Arabidopsis GS1 and Grape GPT Transgenes

[0212]In this example, common Cowpea plants were transformed with the Grape GPT full length coding sequence included in SEQ ID NO: 8 under the control of the RuBisCo promoter within the expression vector pCambia 1305.1 (including construct of SEQ ID NO: 8), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer into flowers. Materials and methods were as in Example 11, supra.

Results:

[0213]The results are presented in FIGS. 14 and 15, and Table IX.

[0214]FIG. 14 shows relative growth rates for the GPT+GS transgenic Cowpea line G and wild type control Cowpea. These data show that the transgenic plants are consistently higher (FIG. 14A), produce substantially more flowers, flower buds and pea pods (FIG. 14B), and develop trifolates and leaf buds faster (FIG. 14C).

TABLE-US-00012 TABLE IX TRANSGENIC COWPEA LINE G Pod Yield, GPT Activity GS Activity Antibiotic Plant Type FWt, g nmoles/h/gFWT umol/min/gFWt Resistance Wildtype, 59.7 44.4 26.7 Negative avg G9 102.0 555.6 34.5 + WT Wildtype; FWt Fresh Weight; NM Not Measured

[0215]Table IX presents pea pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table IX, double-transgene progeny plants showed substantial pea pod biomass increases (fresh pod weight) in comparison to the control plants, with average pea pod biomass yields 70% greater in the transgenic plants compared to control plant(s).

[0216]Lastly, FIG. 15 shows a photograph of a GPT+GS double transgenic pea plant compared to a control plant grown for the same time under identical conditions, showing increased height, biomass and leaf size in the transgenic plant relative to the wild type control plant.

Example 13

Generation of Double Transgenic Alfalfa Plants Carrying Arabidopsis GS1 and GPT Transgenes

[0217]In this example, Alfalfa plants (Medicago sativa, var Ladak) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer into seedling plants. Agrobacterium vectors and mixtures were prepared for seedling inoculations as described in Example 11, supra.

Seedling Inoculations:

[0218]When Alfalfa seedlings were still less than about 1/2 inch tall, they were soaked in paper toweling that had been flooded with the Agrobacteria mixture containing both transgene constructs. The seedlings were left in the paper toweling for two to three days, removed and then planted in potting soil. Resulting T0 and control plants were then grown for the first 30 days in a growth chamber, thereafter cultivated in a greenhouse, and then harvested 42 days after sprouting. At this point, only the transgenic Alfalfa line displayed flowers, as the wild type plants only displayed immature flower buds. The plants were characterized as to flowering status and total biomass.

Results:

[0219]The results are presented in Table X. The data shows that the transgenic Alfalfa plants grew faster, flowered sooner, and yielded on average about a 62% biomass increase relative to the control plants.

TABLE-US-00013 TABLE X TRANSGENIC ALFALFA VS. CONTROL Plant Type Biomass at Sacrifice, g Flowering Stage Wildtype, avg 6.03 Small defined buds No buds swelling. No flowers Transgene #5 10.38 4 Open flowers Transgene # 11 9.03 Flower buds swelling Transgene #13 9.95 Flower buds swelling

Example 14

Generation of Double Transgenic Cantaloupe Plants Carrying Arabidopsis GS1 and GPT Transgenes

[0220]In this example, Cantaloupe plants (Cucumis melo var common) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer via injection into developing melons. Agrobacterium vectors and mixtures were prepared for intra-melon inoculations as described in Example 8, supra. Inoculations into developing melons were carried out essentially as described in

Example 8

[0221]The plants were characterized as to flowering status and total biomass relative to control melon plants grown under identical conditions.

[0222]The results are presented in FIG. 16 and Table XI. Referring to Table XI, the transgenic plants showed substantial foliar plant biomass increases in comparison to the control plants, with an average increase in biomass of 63%. Moreover, a tremendous increase in flower and flower bud yields was observed in all five transgenic lines. Control plants displayed no flowers and only 5 buds at sacrifice, on average. In sharp contrast, the transgenic plants displayed between 2 and 5 flowers per plant, and between 21 and 30 flower buds, per plant, indicating a substantially higher growth rate and flower yield. Increased flower yield would be expected to translate into correspondingly higher melon yields in the transgenic plants. Referring to FIG. 16 (a photograph comparing transgenic Cantaloupe plants to control Cantaloupe plants), the transgenic Cantaloupe plants show dramatically increased height, overall biomass and flowering status relative to the control plants.

TABLE-US-00014 TABLE XI TRANGENIC CANTALOUPE VERSUS CONTROL Biomass Flowers/Flower Antibiotic Plant Type Foliar FWt, g Buds at Sacrifice Resistance Wildtype, avg 22.8 0/5 Negative Line 1 37.0 3/21 + Line 2 35.0 2/30 + Line 3 37.1 3/27 + Line 4 40.6 5/26 + Line 5 35.7 4/30 + FWt Fresh Weight

Example 15

Generation of Double Transgenic Pumpkin Plants Carrying Arabidopsis GS1 and GPT Transgenes

[0223]In this example, common Pumpkin plants (Cucurbita maxima) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer via injection into developing pumpkins, essentially as described in Example 14, supra. The transgenic and control pumpkin plants were grown under identical conditions until the emergence of flower buds in the control plants, then all plants were characterized as to flowering status and total biomass.

[0224]The results are presented in FIG. 17 and Table XII. Referring to Table XII, the transgenic plants showed substantial foliar plant biomass increases in comparison to the control plants, with an increase in average biomass yield of 67% over control plants. Moreover, an increase in flower bud yields was observed in four of the five transgenic lines in comparison to control. Control plants displayed only 4 buds at sacrifice (average). In contrast, four transgenic plant lines displayed between 8 and 15 flowers buds per plant, representing a two- to nearly four-fold yield increase.

TABLE-US-00015 TABLE XII TRANGENIC PUMPKIN VERSUS CONTROL Biomass Flower Buds at Antibiotic Plant Type Foliar FWt, g Sacrifice Resistance Wildtype, avg 47.7 4.2 Negative Line 1 (Photo) 82.3 8 Line 2 74.3 8 + Line 3 80.3 9 + Line 4 (Photo) 77.8 4 + Line 5 84.5 15 + FWt Fresh Weight;

[0225]Referring to FIG. 17 (a photograph comparing transgenic pumpkin plants to control plants), the transgenic pumpkin plants show substantially increased plant size, overall biomass and leaf sizes and numbers relative to the control plants.

Example 16

Generation of Double Transgenic Arabidopsis Plants Carrying Arabidopsis GS1 and GPT Transgenes

[0226]In this example, Arabidopsis thaliana plants were transformed with the truncated Arabidopsis GPT coding sequence of SEQ ID NO: 18 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and transgenic plants thereafter transformed with the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated "floral dip" transfer as described (Harrison et al., 2006, Plant Methods 2:19-23; Clough and Bent, 1998, Plant J. 16:735-743). Agrobacterium vectors pMON316 carrying GPT and pCambia 1201 carrying GS1 were prepared as described in Examples 3 and 11, respectively.

[0227]Transformation of two different cultures of Agrobacterium with either a pMon 316+ Arabidopsis GTP construct or with a Cambia 1201+ Arabidopsis GS construct was done by electroporation using the method of Weigel and Glazebrook 2002. The transformed Agrobacterium were then grown under antibiotic selection, collected by centrifugation resuspended in LB broth with antibiotic and used in the floral dip of Arabidopsis inflorescence. Floral dipped Arabidopsis plants were taken to maturity and self-fertilized and seeds were collected. Seeds from twice dipped plants were first geminated on a media containing 20 ug/ml of kanamycin and by following regular selection procedures surviving seedlings were transferred to media containing 20 ug of hygromycin. Plants (3) surviving the selection process on both antibiotics were self-fertilized and seeds were collected. Seeds from the T1 generation were germinated on MS media containing 20 ug/ml of hygromycin and surviving seedlings were taken to maturity, self-fertilized and seeds collected. This seed population the T2 generation was then used for subsequent growth studies.

[0228]The results are presented in FIG. 18 and Table XIII. Referring to Table XIII, which shows data from 6 wild type and 6 transgenic Arabidopsis plants (averaged), the transgenic plants displayed increased levels of both GPT and GS activity. GPT activity was over twenty-fold higher than the control plants. Moreover, the transgenic plant fresh foliar weight average was well over four-fold that of the wild type control plant average. A photograph of young transgene Arabidopsis plants in comparison to wild type control Arabidopsis plants grown under identical conditions is shown in FIG. 18, and reveals a consistent and very significant growth/biomass increase in transgenic plants relative to the control plants.

TABLE-US-00016 TABLE XIII TRANSGENIC ARABIDOPSIS VERSUS CONTROL Biomass, g GS Activity Fresh foliar GPT Activity umol/min/gF Antibiotic Plant type wt nmol/h/gFWt Wt Resistance Wildtype, avg 0.246 18.4 7.0 Negative Transgene 1.106 395.6 18.2 Positive

Example 17

Generation of Transgenic Tomato Plants Carrying Arabidopsis GPT and GS1 Transgenes

[0229]In this example, tomato plants (Solanum lycopersicon, "Money Maker" variety) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6). Single transgene (GPT) transgenic tomato plants were generated and grown to flowering essentially as described in Example 4. The Arabidopsis GS1 transgene was then introduced into the single-transgene T0 plants using Agrobacterium-mediated transfer via injection directly into flowers (as described in Example 8). The transgenic and control tomato plants were grown under identical conditions and characterized as to growth phenotype characteristics. Resulting T0 double-transgene plants were then grown to maturity, photographed along with control tomato plants, and phenotypically characterized.

[0230]The results are presented in FIG. 19 and in Table X. Referring to Table XIX, double-transgene tomato plants showed substantial foliar plant biomass increases in comparison to the control plants, with an increase in average biomass yield of 45% over control. Moreover, as much as a 70% increase in tomato fruit yield was observed in the transgenic lines compared to control plants (e.g., 51 tomatoes harvested from Line 4C, versus and average of approximately 30 tomatoes from control plants). A much higher level of GPT activity was observed in the transgenic plants (e.g., line 4C displaying an approximately 32-fold higher GPT activity in comparison to the average GPT activity measured in control plants). GS activity was also higher in the transgenic plants relative to control plants (almost double in Line 4C).

[0231]With respect to growth phenotype, and referring to FIG. 19, the transgenic tomato plants displayed substantially larger leaves compared to control plants (FIG. 19A). In addition, it can be seen that the transgenic tomato plants were substantially larger, taller and of a greater overall biomass (see FIG. 19B).

TABLE-US-00017 TABLE XIX TRANSGENIC TOMATO GROWTH AND REPRODUCTION Total Tomatoes GPT GS Biomass Harvested Activity Activity Transgene Foliar until nmoles/ umoles/ Presence Plant Type FWt, g Sacrifice h/gFWt min/gFWt Assay Wildtype, 891 30.2 287 14.27 Negative avg Line 6C 1288 43 9181 18.3 + Line 4C 1146 51 1718 26.4 +

Example 18

Generation of Transgenic Camilena Plants Carrying Arabidopsis GPT and GS1 Transgenes

[0232]In this example, Camelina plants (Camelina sativa, Var MT 303) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the RuBisCo promoter within the expression vector pCambia 1201, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into germinating seeds according to the method described in Chee et al., 1989, Plant Physiol. 91: 1212-1218. Agrobacterium vectors and mixtures were prepared for seed inoculations as described in Example 11, supra.

[0233]Transgenic and control Camelina plants were grown under identical conditions (30 days in a growth chamber and then moved to greenhouse cultivation) for 39 days, and characterized as to biomass, growth characteristics and flowering stage.

[0234]The results are presented in Table XX and FIG. 20. Referring to Table XX, it can be seen that total biomass in the transgenic plants was, on average, almost double control plant biomass. Canopy diameter was also significantly improved in the transgenic plants. FIG. 20 shows a photograph of transgenic Camelina compared to control. The transgenic plant is noticeably larger and displays more advanced flowering.

TABLE-US-00018 TABLE XX TRANSGENIC CAMELINA VERSUS CONTROL Height/Canopy Plant Type Diameter, inches Biomass G Flowering Stage Wildtype, avg 14/4 8.35 Partial flowering Transgene C-1 15.5/5 16.54 Full flowering Transgene C-3 14/7 14.80 Initial flowering

Example 19

Activity of Barley GPT Transgene in Planta

[0235]In this example, the putative coding sequence for Barley GPT was isolated and expressed from a transgene construct using an in planta transient expression assay. Biologically active recombinant Barley GPT was produced, and catalyzed the increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

[0236]The Barley (Hordeum vulgare) GPT coding sequence was determined and synthesized. The DNA sequence of the Barley GPT coding sequence used in this example is provided in SEQ ID NO: 14, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 15.

[0237]The coding sequence for Barley GPT was inserted into the 1305.1 cambia vector, and transferred to Agrobacterium tumefaciens strain LBA404 using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing hygromycin (50 micro gm/ml). Antibiotic resistant colonies of Agrobacterium were selected for analysis.

[0238]The transient tobacco leaf expression assay consisted of injecting a suspension of transformed Agrobacterium (1.5-2.0 OD 650) into rapidly growing tobacco leaves. Intradermal injections were made in a grid across the leaf surface to assure that a significant amount of the leaf surface would be exposed to the Agrobacterium. The plant was then allowed to grow for 3-5 days when the tissue was extracted as described for all other tissue extractions and the GPT activity measured.

[0239]GPT activity in the inoculated leaf tissue (1217 nanomoles/gFWt/h) was three-fold the level measured in the control plant leaf tissue (407 nanomoles/gFWt/h), indicating that the Hordeum GPT construct directed the expression of biologically active GPT in a transgenic plant.

Example 20

Isolation and Expression of Recombinant Rice GPT Gene Coding Sequence and Analysis of Biological Activity

[0240]In this example, the putative coding sequence for rice GPT was isolated and expressed in E. coli. Biologically active recombinant rice GPT was produced, and catalyzed the increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and Methods:

[0241]Rice GPT Coding Sequence and Expression in E. coli:

[0242]The rice (Oryza sativa) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25×106 cells were then assayed for biological activity using the NMR assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E. coli cells transformed with an empty vector.

[0243]The DNA sequence of the rice GPT coding sequence used in this example is provided in SEQ ID NO: 10, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 11.

HPLC Assay for 2-Oxoglutaramate:

[0244]HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, following a modification of Calderon et al., 1985, J Bacteriol 161(2): 807-809. Briefly, a modified extraction buffer consisting of 25 mM Tris-HCl pH 8.5, 1 mM EDTA, 20 μM Pyridoxal phosphate, 10 mM Cysteine, and ˜1.5% (v/v) Mercaptoethanol was used. Samples (lysate from E. coli cells, 25×106 cells) were added to the extraction buffer at approximately a 1/3 ratio (w/v), incubated for 30 minutes at 37° C., and stopped with 200 μl of 20% TCA. After about 5 minutes, the assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8 mm ID×30 cm L column, with a mobile phase in 0.01N H₂SO₄, a flow rate of approximately 0.2 ml/min, at 40° C. Injection volume is approximately 20 and retention time between about 38 and 39 minutes. Detection is achieved with 210 nm UV light.

[0245]NMR analysis comparison with authentic 2-oxoglutaramate was used to establish that the Arabidopisis full length sequence expresses a GPT with 2-oxoglutaramate synthesis activity. Briefly, authentic 2-oxoglutarmate (structure confirmed with NMR) made by chemical synthesis to validate the HPLC assay, above, by confirming that the product of the assay (molecule synthesized in response to the expressed GPT) and the authentic 2-oxoglutaramate elute at the same retention time. In addition, when mixed together the assay product and the authentic compound elute as a single peak. Furthermore, the validation of the HPLC assay also included monitoring the disappearance of the substrate glutamine and showing that there was a 1:1 molar stoechiometry between glutamine consumed to 2-oxoglutaramte produced. The assay procedure always included two controls, one without the enzyme added and one without the glutamine added. The first shows that the production of the 2-oxoglutaramate was dependent upon having the enzyme present, and the second shows that the production of the 2-oxoglutaramate was dependent upon the substrate glutamine.

Results:

[0246]Expression of the rice GPT coding sequence of SEQ ID NO: 10 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically, 1.72 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant rice GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, an 86-fold activity level increase over control.

Example 21

Isolation and Expression of Recombinant Soybean GPT Gene Coding Sequence and Analysis of Biological Activity

[0247]In this example, the putative coding sequence for soybean GPT was isolated and expressed in E. coli. Biologically active recombinant soybean GPT was produced, and catalyzed the increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and Methods:

[0248]Soybean GPT Coding Sequence and Expression in E. coli:

[0249]The soybean (Glycine max) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25×106 cells were then assayed for biological activity using the HPLC assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E. coli cells transformed with an empty vector.

[0250]The DNA sequence of the soybean GPT coding sequence used in this example is provided in SEQ ID NO: 12, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 13.

HPLC Assay for 2-Oxoglutaramate:

[0251]HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra.

Results:

[0252]Expression of the soybean GPT coding sequence of SEQ ID NO: 12 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically, 31.9 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant soybean GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, a nearly 1.600-fold activity level increase over control.

Example 22

Isolation and Expression of Recombinant Zebra Fish GPT Gene Coding Sequence and Analysis of Biological Activity

[0253]In this example, the putative coding sequence for Zebra fish GPT was isolated and expressed in E. coli. Biologically active recombinant Zebra fish GPT was produced, and catalyzed the increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and Methods:

[0254]Zebra Fish GPT Coding Sequence and Expression in E. coli:

[0255]The Zebra fish (Danio rerio) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25×106 cells were then assayed for biological activity using the HPLC assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E. coli cells transformed with an empty vector.

[0256]The DNA sequence of the Zebra fish GPT coding sequence used in this example is provided in SEQ ID NO: 16, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 17.

HPLC Assay for 2-Oxoglutaramate:

[0257]HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra.

Results:

[0258]Expression of the Zebra fish GPT coding sequence of SEQ ID NO: 16 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically, 28.6 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant Zebra fish GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, a more than 1.400-fold activity level increase over control.

Example 23

Generation and Expression of Recombinant Truncated Arabidopsis GPT Gene Coding Sequences and Analysis of Biological Activity

[0259]In this example, two different truncations of the Arabidopsis GPT coding sequence were designed and expressed in E. coli, in order to evaluate the activity of GPT proteins in which the putative chloroplast signal peptide is absent or truncated. Recombinant truncated GPT proteins corresponding to the full length Arabidopsis GPT amino acid sequence of SEQ ID NO: 2, truncated to delete either the first 30 amino-terminal amino acid residues, or the first 45 amino-terminal amino acid residues, were successfully expressed and showed biological activity in catalyzing the increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and Methods:

[0260]Truncated Arabidopsis GPT Coding Sequences and Expression in E. coli:

[0261]The DNA coding sequence of a truncation of the Arabidopsis thaliana GPT coding sequence of SEQ ID NO: 1 was designed, synthesized, inserted into a PET28 vector, and expressed in E. coli. The DNA sequence of the truncated Arabidopsis GPT coding sequence used in this example is provided in SEQ ID NO: 20 (-45 AA construct), and the corresponding truncated GPT protein amino acid sequence is provided in SEQ ID NO: 21. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25×10⁶ cells were then assayed for biological activity using HPLC as described in Example 20. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E. coli cells transformed with an empty vector.

[0262]Expression of the truncated-45 Arabidopsis GPT coding sequence of SEQ ID NO: 20 resulted in the over-expression of biologically active recombinant GPT protein (2-oxoglutaramate synthesis-catalyzing bioactivity). Specifically, 16.1 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the truncated-45 GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, a more than 800-fold activity level increase over control. For comparison, the full length Arabidopsis gene coding sequence expressed in the same E. coli assay generated 2.8 nanomoles of 2-oxoglutaramate activity, or roughly less than one-fifth the activity observed from the truncated recombinant GPT protein.

Example 24

GPT+GS Transgenic Tobacco Seed Germination Tolerates High Salt Concentrations

[0263]In this example, seeds form the double transgene tobacco line XX-3 (Cross 3 in Table 4, see Example 7) were tested in a seed germination assay designed to evaluate tolerance to high salt concentrations.

Materials and Methods:

[0264]Tobacco seeds from the wild type and XX-3 populations were surfaced sterilized (5% bleach solution for 5 minutes followed by a 10% ethanol wash for 3 minutes) and rinsed with sterile distilled water. The surface sterilized seeds were then spread on Murashige and Skoog media (10% agarose) without sucrose and containing either 0 or 200 mM NaCl. The seeds were allowed to germinate in darkness for 2 days followed by 6 days under a 16:8 photoperiod at 24° C. On day eight the rate of germination was determined by measuring the percentage of seeds from the control or transgene plants that had germinated.

Results:

[0265]The results are tabulated in Table XXI below. The rate of germination of the transgenic plant line seeds under zero salt conditions was the same as observed with wild type control plant seeds. In stark contrast, the germination rate of the transgenic plant line seeds under very high salt conditions far exceeded the rate seen in wild type control seeds. Whereas over 81% of the transgenic plant seeds had germinated under the high salt conditions, only about 9% of the wild type control plant seeds had germinated by the same time point. These data indicate that the transgenic seeds are capable of germinating very well under high salt concentrations, an important trait for plant growth in areas of increasingly high water and/or soil salinity.

TABLE-US-00019 TABLE XXI TRANSGENIC TOBACCO PLANTS GERMINATE AND TOLERATE HIGH SALT Control (0 mM NaCl) Test (200 mM NaCl)a Plant type % Germination % Germination Wild type 92, 87, 94 9, 11, 8 Transgene line XX-3 92, 91, 94 84, 82, 78

Example 25

Method for Generating Transgenic Maize Plants Carrying Hordeum GPT and GS1 Transgenes

[0266]This example provides a method for generating transgenic maize plants expressing GPT and GS1 transgenes. Maize (Zea mays, hybrid line Hi-II) type II callus is biolistically transformed with an expression cassette comprising the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression casette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of maize callus is achieved by particle bombardment.

Vector Constructs:

[0267]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, is cloned into the plasmid pAHC25 (Christensen and Quail, 1996, Transgenic Research 5:213-218) modified to include a bar gene conferring resistance to bialophos, or a similar vector, in order to generate the transgene expression vector.

Transformation and Regeneration:

[0268]The transgene expression vector is introduced into immature zygotic embryo source callus of parent maize hybrid line Hi-II (A188xB73 origin) (Armstrong et al., 1991, Maize Genetics Coop Newsletter 65:92-93) using particle bombardment, essentially as described (Frame et al., 2000, In Vitro Cell. Dev. Biol-Plant 36:21-29; this method was developed by and is routinely used at the Iowa State University Center for Plant Transformation).

[0269]More specifically, immature zygotic embryo source callus is prepared for transformation by serial culturing on a callus-initiating medium (N6E, Songstad et al., 1996, In vitro Cell Dev. Biol.-Plant 32:179-183). Washed gold particles are coated with the plasmid construct and used to bombard the callus with a PDS 1000/He biolistic gun as described (Sanford et al., 1993, Methods in Enzymology 217: 483-509). After 7-10 days on initiation medium, the callus is then transferred to selection medium containing bialophos (N6S, Songstad et al., 1996, supra) and allowed to grow. Following the development of bialophos resistant clones, callus pieces are transferred to a regeneration medium (Armstrong and Green, 1985, Planta 164:207-214) containing bialophos and allowed to grow for several weeks. Thereafter, the resulting plantlets are transferred to regeneration medium without the selection agent, and cultivated.

[0270]Transgenic corn plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event. Various phenotypic characteristics may be observed in T₀ events, as well as in T₁ and subsequent generations, and used to select seed sources for the development of subsequent generations. High performing lines may be selfed to achieve trait homozygosity and/or crossed.

Example 26

Method for Generating Transgenic Rice Plants Carrying Hordeum GPT and GS1 Transgenes

[0271]This example provides a method for generating transgenic rice plants expressing GPT and GS1 transgenes. Rice (Oryza sativa, Japonica cultivar Nipponbare) type II calus is transformed with the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation is achieved by Agrobacterium-mediated transformation.

Vector Constructs:

[0272]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, is cloned into base vector pTF101.1, using standard molecular cloning methodologies, to generate the transgene expression vector. Base vector pTF101.1 is a derivative of the pPZP binary vector (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994), which includes the right and left T-DNA border fragments from a nopaline strain of A. tumefaciens, a broad host origin of replication (pVS1) and a spectinomycin-resistant marker gene (aadA) for bacterial selection. The plant selectable marker gene cassette includes the phosphinothricin acetyl transferase (bar) gene from Streptomyces hygroscopicus that confers resistance to the herbicides glufosinate and bialophos. The soybean vegetative storage protein terminator (Mason et al., 1993) follows the 3' end of the bar gene.

Media:

[0273]YEP Medium: 5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl₂, 15 g/L Bacto-agar. pH to 6.8 with NaOH. After autoclaving, the appropriate antibiotics are added to the medium when it has cooled to 50° C.

[0274]Infection Medium: N6 salts and vitamins (Chu et al., 1975, Sci. Sinica 18: 659-668), 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.7 g/L L-proline, 68.4 g/L sucrose, and 36 g/L glucose (pH 5.2). This medium is filter-sterilized and stored at 4° C. Acetosyringone (AS, 100 μM) is added just prior to use (prepared from 100 μM stocks of filter-sterilized AS, dissolved in DMSO to 200 mM then diluted 1:1 with water).

[0275]Callus Induction Medium: N6 salts and vitamins, 300 mg/L casamino acids, 2.8 g/L L-proline, 30 g/L sucrose, and 4 g/L gelrite (pH 5.8). Filter sterilized N6 Vitamins and 2 mg/L 2,4-D, are added to this medium after autoclaving.

[0276]Co-cultivation Medium (make fresh): N6 salts and vitamins, 300 mg/L casamino acids, 30 g/L sucrose, 10 g/L glucose, and 4 g/L gelrite (pH 5.8). Filter sterilized N6 vitamins, acetosyringone (AS) 100 μM and 2 mg/L 2,4-D are added to this medium after autoclaving.

[0277]Selection Medium: N6 salts and vitamins, 300 mg/L casamino acids, 2.8 g/L L-proline, 30 g/L sucrose, and 4 g/L gelrite (pH 5.8). Filter sterilized N6 vitamins, 2 mg/L 2,4-D, 2 mg/L Bialaphos (Shinyo Sangyo, Japan) and 500 mg/L carbenicillin are added to this medium after autoclaving.

[0278]Regeneration Medium I: MS salts and vitamins (Murashige and Skoog, 1962), 2 g/L casamino acids, 30 g/L sucrose, 30 g/L sorbitol, and 4 g/L gelrite (pH 5.8). Filter sterilized MS vitamins, 100 mg/L cefotaxime, 100 mg/L vancomycin, 0.02 mg/L NAA (naphthaleneacetic acid), 2 mg/L kinetin (Toki, 1997, supra) and 2 mg/L Bialaphos are added to this medium after autoclaving.

[0279]Regeneration Medium II: MS Salts and vitamins, 100 mg/L myo-inositol, 30 g/L sucrose, 3 g/L gelrite, (pH 5.8).

Transformation and Regeneration:

[0280]Japonica rice cultivar Nipponbare is transformed with Agrobacterium tumefaciens strain EHA101 (Hood et al., 1986, J. Bacteriol. 168:1291-1301), transformed with the pTF101.1 transgene expression vector carrying the hordeum GS1+GPT expression cassette. The vector system pTF101.1 in EHA101 is maintained on YEP medium (An et al., 1988) containing 100 mg/L spectinomycin (for pTF101.1) and 50 mg/L kanamycin (for EHA101).

[0281]Briefly, callus tissue derived from the mature rice embryo is used as the starting material for transformation. Callus induction, co-cultivation, selection and regeneration I media are based on those of Hiei et al., 1994, The Plant Journal 6 (2):271-282.

[0282]More specifically, calli are induced as follows. First, 15-20 rice seeds are dehusked and rinsed in 10 ml of 70% Ethanol (50 ml conical tube) by vigorously shaking the tube for one minute, followed by rinsing once with sterile water. Then, 10 ml of 50% commercial bleach (5.25% hypochlorite) is added and placed on a shaker for 30 minutes (low setting). The bleach solution is then poured-off and the seeds rinsed five times with ˜10 ml of sterilized water each time. With a small portion of the final rinse, the seeds are poured onto sterilized filter paper (in a sterile petri plate) and then allowed to dry. Using sterile forceps, several (i.e., 5) seeds are transferred to the surface of individual sterile petri plates containing callus induction medium. The plates are wrapped with vent tape and incubated in the light (16:8 photoperiod) at 29° C. Seeds are observed every few days and those showing signs of contamination are discarded.

[0283]After two to three weeks, developing callus is visible on the scutellum of the mature seed. Calli are then subcultured to fresh induction medium and allowed to proliferate. Four days prior to infection, the callus tissue is cut into 2-4 mm pieces and transferred to fresh induction medium.

[0284]The selection medium uses modifications from Toki (Toki, 1997, Plant Molecular Biology Reporter 15:16-21) whereby bialophos (2 mg/L) is employed for plant selection and carbenicillin (500 mg/L) for counter selection against Agrobacterium. Regeneration II medium is as described (Armstrong and Green, 1985, Planta 164:207-214).

[0285]Agrobacterium culture is grown (i.e., for 3 days at 19° C., or 2 days at 28° C.) on YEP medium amended with spectinomycin (100 mg/L) and kanamycin (50 mg/L). An aliquot of the culture is then suspended in ˜15 ml of liquid infection medium supplemented with 100 μM AS in a 50 ml conical tube (no pre-induction). The optical density is adjusted to <0.1 (OD₅₅₀=0.06-0.08) before use.

[0286]For infection, rice calli are first placed into bacteria-free infection medium+AS (50 ml conical). This pre-wash is removed and replaced with 10 ml of the prepared Agrobacterium suspension (OD₅₅₀<0.1). Then, the conical is fastened onto a vortex shaker (low setting) for two minutes. After infection, calli are poured out of the conical onto a stack of sterile filter paper in a 100×15 petri dish to blot dry. Then, they are transferred off the filter paper and onto the surface of co-cultivation medium with sterile forceps. Co-cultivation plates are wrapped with vent tape and incubated in the dark at 25° C. for three days. After three days of co-cultivation, the calli are washed five times with 5 ml of the liquid infection medium (no AS) supplemented with carbenicillin (500 mg/L) and vancomycin (100 mg/L). Calli are blotted dry on sterile filter paper as before. Individual callus pieces are transferred off the paper and onto selection medium containing 2 mg/L bialaphos. Selection plates are wrapped with parafilm and placed in the light at 29° C.

[0287]For selection of stable transformation events, plant tissue is cultured onto fresh selection medium every two weeks. This should be done with the aid of a microscope to look for any evidence of Agrobacterium overgrowth. If overgrowth is noted, the affected calli should be avoided (contaminated calli should not be transferred). The remaining tissue is then carefully transferred, preferably using newly sterilized forceps for each calli. Putative clones begin to appear after six to eight weeks on selection. A clone is recognized as white, actively growing callus and is distinguishable from the brown, unhealthy non-transformed tissue. Individual transgenic events are identified and the white, actively growing tissue is transferred to individual plates in order to produce enough tissue to take to regeneration. Regeneration of transgenic plants is accomplished by selecting new lobes of growth from the callus tissue and transferring them onto Regeneration Medium I (light, 25° C.). After two to three weeks, the maturing tissue is transferred to Regeneration Medium II for germination (light, 25° C.). When the leaves are approximately 4-6 cm long and have developed good-sized roots, the plantlets may be transferred (on an individual basis, typically 7-14 days after germination begins) to soilless mix using sterile conditions.

[0288]Transgenic rice plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event. Various phenotypic characteristics may be observed in T₀ events, as well as in T₁ and subsequent generations, and used to select seed sources for the development of subsequent generations. High performing lines may be selfed to achieve trait homozygosity and/or crossed.

Example 27

Method for Generating Transgenic Sugarcane Plants Carrying Hordeum GPT and GS1 Transgenes

[0289]This example provides a method for generating transgenic sugarcane plants expressing GPT and GS1 transgenes. Sugarcane (Saccharum spp L) is biolistically transformed with an expression cassette comprising the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of sugarcane callus is achieved by particle bombardment.

Vector Constructs:

[0290]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, are cloned into a small plasmid well established for sugarcane expression, such as pAHC20 (Thomson et al., 1987, EMBO J. 6:2519-2523), using standard molecular cloning methodologies, to generate the transgene expression vector. The plasmid used contains a selectable marker against either the phospinothricin family of herbicides or the antibiotics geneticin or kanamycin, each of which have been shown effective (Ingelbrecht et al., 1999, Plant Physiology 119:1187-1197; Gallo-Maegher & Irvine, 1996, Crop Science 36:1367-1374).

Transformation and Regeneration:

[0291]The plasmid containing the expression cassette encoding the hordeum GS1 and GPT coding sequences is introduced into embryogenic callus prepared for transformation by the basic method of Gallo-Maegher and Irvine (Gallo-Maegher and Irvine, 1996, supra) and Ingelbrecht et al. (Ingelbrecht et al., 1999, supra) with the improved stimulation of shoot regeneration with thidiazuron (Gallo-Maegher et al., 2000, In vitro Cell Dev. Biol.-Plant 36:37-40). This particle bombardment method is effective in transforming sugarcane (see, for example, Gilbert et al., 2005, Crop Science 45:2060-2067; and see the foregoing references). Regenerable sugarcane varieties, such as the commercial varieties CP65-357 and CP72-1210, may be used to generate transgene events.

[0292]Briefly, 7- to 40-week old calli are bombarded with plasmid-coated tungsten or gold particles. Two days after bombardment the calli are transferred to selection medium. Four weeks later the resistant calli are transferred to shoot-induction medium containing the selection agent and sub-cultured every two weeks for approximately 12 weeks, at which time the shoots are transferred to Magenta boxes containing rooting medium with selection agent. The shoots are maintained on this medium for approximately 8 weeks, at which time those with good root development are transferred to potting mix and the adapted to atmospheric growth.

[0293]Transgenic sugarcane plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event. Various phenotypic characteristics may be observed in T₀ events, as well as in T₁ and subsequent generations, and used to select seed sources for the development of subsequent generations. High performing lines may be selfed to achieve trait homozygosity and/or crossed.

Example 28

Method for Generating Transgenic Wheat Plants Carrying Hordeum GPT And GS1 Transgenes

[0294]This example provides a method for generating transgenic wheat plants expressing

[0295]GPT and GS1 transgenes. Wheat (Triticum spp.) is biolistically transformed with an expression cassette comprising the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of wheat callus is achieved by particle bombardment.

Vector Constructs:

[0296]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn (maize) ubiquitin promoters, respectively, are cloned into a plasmid such as pAHC17, which contains the bar gene to provide the desired resistance to the phosphinothricin-class of herbicides for selection of transformants, using standard molecular cloning methodologies, to generate the transgene expression vector.

Transformation and Regeneration:

[0297]Wheat is transformed biolistically, and transgenic events regenerated, essentially as described (Weeks et al., 1993, Plant Physiology. 102:1077-1084; Blechl and Anderson, 1996, Nat. Biotech. 14:875-879; Okubara et. al., 2002, Theoretical and Applied Genetics. 106:74-83). These methods were developed and are routinely practiced at the US Department of Agriculture, Agricultural Research Service, Western Regional Research Center (Albany Calif.). The highly regenerable hexaploid spring wheat cultivar `Bobwhite` is used as the source of immature embryos for bombardment with plasmid-coated particles.

[0298]Bombarded embryos are cultured without selection for 1-3 weeks in the dark on MS media before transferring them to shoot induction medium (MS media plus hormones and selection agent bialophos (1, 1.5, 2, 3 mg/L) for 2-8 weeks with subculturing weekly (Blechl et al., 2007, J Cereal Science 45:172-183). Shoots that formed are transferred to rooting medium also containing the selection agent (bialophos 3 mg/L) (Weeks et al., 1993, supra). Well-rooted plantlets are transferred to potting media and adapted to atmospheric growth conditions.

[0299]Transgenic wheat plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event. Various phenotypic characteristics may be observed in T₀ events, as well as in T₁ and subsequent generations, and used to select seed sources for the development of subsequent generations. High performing lines may be selfed to achieve trait homozygosity and/or crossed.

Example 29

Method for Generating Transgenic Sorghum Plants Carrying Hordeum GPT and GS1 Transgenes

[0300]This example provides a method for generating transgenic sorghum plants expressing GPT and GS1 transgenes. Sorghum (Sorghum spp L) is transformed with Agrobacterium carrying an expression cassette encoding the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SE ID NO: 44.

Vector Constructs:

[0301]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, is cloned into a stable binary vector such as pZY101 (Vega et al 2008, Plant Cell Rep. 27:297-305), using standard molecular cloning methodologies, to generate the transgene expression vector.

Transformation and Regeneration:

[0302]Agrobacterium-mediated transformation and recovery Of transgenic sorghum plants is as described (Lu et al., 2009, Plant Cell Tissue Organ Culture 99:97-108). These methods are routinely used by the University of Missouri Plant Transformation Core Facility. The public sorghum line, P898012, is grown as described (Lu et al., 2009, supra) and transformed with Agrobacterium tumefaciens strain EHA101 (Hood et al., 1986, supra) transformed with the transgene expression vector.

[0303]More specifically, Agrobacterium (0.3-0.4 OD) harboring the transgene expression vector is used to inoculate immature sorghum embryos for 5 minutes. The embryos are then transferred onto filter paper on top of their co-cultivation medium, containing acetosyringone to enhance the effectiveness of the infection. Embryos are incubated for 3-5 days and then transferred for another 4 days on resting medium (containing carbenicillin) and then transferred onto callus induction medium (with selection agent PPT) with weekly transfers. Once somatic embyrogenic cells develop they are transferred onto shooting medium (with carbenicillin and PPT) until shoots (2-5 cm long) develop. Shoots are transferred to Magenta boxes with rooting medium (with PPT) and maintained in 16 h light and 8 h darkness until 8-20 cm tall well-rooted plantlets are produced. They are then transferred to potting mix and adapted to atmospheric conditions.

[0304]Transgenic sorghum plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event. Various phenotypic characteristics may be observed in T₀ events, as well as in T₁ and subsequent generations, and used to select seed sources for the development of subsequent generations. High performing lines may be selfed to achieve trait homozygosity and/or crossed.

Example 30

Method for Generating Transgenic Switchgrass Plants Carrying Hordeum GPT and GS1 Transgenes

[0305]This example provides a method for generating transgenic switchgrass plants expressing GPT and GS1 transgenes. Switchgrass (Panicum virgatum) is transformed with Agrobacterium carrying a transgene expression vector including an expression cassette encoding the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SE ID NO: 44.

Vector Constructs:

[0306]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn (maize) ubiquitin promoters, respectively, is cloned into a Cambia vector thirteen hundred series (i.e., 1305.1) containing the HPT gene which provides hygromycin resistance for selection of the Switchgrass events, using standard molecular cloning methodologies, to generate the transgene expression vector.

Transformation and Regeneration:

[0307]Agrobacterium-mediated transformation and recovery of transgenic switchgrass plants is essentially as described (Somleva et al., 2002, Crop Science 42:2080-2087; Somleva 2006, Switchgrass (Panicum virgatum L.) In Methods in Molecular Biology Vol 344. Agrobacterium Protocols 2/e, Volume 2. Ed K. Wang Humana Press Inc., Totowa, N.J.; Xi et al 2009, Bioengineering Research 2:275-283). These methods are routinely used by the Plant Biotechnology Resource and Outreach Center at Michigan State University.

[0308]Briefly, explants of embryonic callus from the mature caryopses of the public Switchgrass cv. Alamo are transformed with Agrobacterium tumefaciens strain EHA105 (Hood et al., 1986, supra) carrying the transgene expression vector. Agrobacterium (0.8-1.0 OD) harboring the transgene expression vector and pretreated with acetosynringone is used to inoculate the switchgrass callus for 10 minutes and then co-cultivated for 4-6 days in the dark. The explants are then washed free of the agrobacterium and placed on selection medium containing the antibiotic timentin and hygromycin; selection requires 2-6 months. Subculturing is carried out at 4-week intervals. Regeneration is accomplished in 4-8 weeks on media containing GA3, timentin and hygromycin under a photoperiod of 16 h light and 8 dark. The plantlets are then transferred to Magenta boxes with regeneration medium containing GA3, timentin and hygromycin for another 4 weeks as before. The plants are then transferred to soil and adapted to atmospheric growth.

[0309]Transgenic switchgrass plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event. Various phenotypic characteristics may be observed in T₀ events, as well as in T₁ and subsequent generations, and used to select seed sources for the development of subsequent generations. High performing lines may be selfed to achieve trait homozygosity and/or crossed.

Example 31

Method for Generating Transgenic Soybean Plants Carrying Arabidopsis GPT and GS1 Transgenes

[0310]This example provides a method for generating transgenic soybean plants expressing GPT and GS1 transgenes. Soybean (Glycine max) is transformed with Agrobacterium carrying a transgene expression vector including an expression cassette encoding the Arabidopsis glutamine synthetase (GS1) coding sequence of SEQ ID NO: 7 under the control of the tomato RuBisCo small subunit promoter of SEQ ID NO: 22 (expression cassette of SEQ ID NO: 47), and the Arabidopsis GPT coding sequence of SEQ ID NO: 1 under the control of the 35S cauliflower mosaic virus (CMV) promoter (expression cassette of SEQ ID NO: 27).

Vector Constructs:

[0311]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the tomato RuBisCo small subunit and 35S CMV promoters, respectively, is cloned into pTF101.1, using standard molecular cloning methodologies, to generate the transgene expression vector. pTF101.1 is a derivative of the pPZP binary vector (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994), which includes the right and left T-DNA border fragments from a nopaline strain of A. tumefaciens, a broad host origin of replication (pVS1) and a spectinomycin-resistant marker gene (aadA) for bacterial selection. The plant selectable marker gene cassette includes the phosphinothricin acetyl transferase (bar) gene from Streptomyces hygroscopicus that confers resistance to the herbicides glufosinate and bialophos. The soybean vegetative storage protein terminator (Mason et al., 1993) follows the 3' end of the bar gene.

Media:

[0312]YEP Solid Medium: 5 g/L Yeast extract, 10 g/L Peptone, 5 g/L NaCl₂, 12 g/L Bacto-agar. pH to 7.0 with NaOH. Appropriate antibiotics should be added to the medium after autoclaving. Pour into sterile 100×15 plates (˜25 ml per plate).

[0313]YEP Liquid Medium: 5 g/L Yeast extract, 10 g/L Peptone, 5 g/L NaCl₂. pH to 7.0 with NaOH. Appropriate antibiotics should be added to the medium prior to inoculation.

[0314]Co-cultivation Medium: 1/10× B5 major salts, 1/10× B5 minor salts, 2.8 mg/L Ferrous, 3.8 mg/L NaEDTA, 30 g/L Sucrose, 3.9 g/L MES, and 4.25 g/L Noble agar (pH 5.4). Filter sterilized 1× B5 vitamins, GA3 (0.25 mg/L), BAP (1.67 mg/L), Cysteine (400 mg/L), Dithiothrietol (154.2 mg/L), and 40 mg/L acetosyringone are added to this medium after autoclaving. Pour into sterile 100×15 mm plates (˜88 plates/L). When solidified, overlay the co-cultivation medium with sterile filter paper to reduce bacterial overgrowth during co-cultivation (Whatman #1, 70 mm).

[0315]Infection Medium: 1/10× B5 major salts, 1/10× B5 minor salts, 2.8 mg/L Ferrous, 3.8 mg/L NaEDTA, 30 g/L Sucrose, 3.9 g/L MES (pH 5.4). Filter sterilized 1× B5 vitamins, GA3 (0.25 mg/L), BAP (1.67 mg/L), and 40 mg/L acetosyringone are added to this medium after autoclaving.

[0316]Shoot Induction Washing Medium: 1× B5 major salts, 1× B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose, and 0.59 g/L MES (pH 5.7). Filter sterilized 1× B5 vitamins, BAP (1.11 mg/L), Timentin (100 mg/L), Cefotaxime (200 mg/L), and Vancomycin (50 mg/L) are added to this medium after autoclaving.

[0317]Shoot Induction Medium I: 1× B5 major salts, 1× B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose, 0.59 g/L MES, and 7 g/L Noble agar (pH 5.7). Filter sterilized 1× B5 vitamins, BAP (1.11 mg/L), Timentin (50 mg/L), Cefotaxime (200 mg/L), and Vancomycin (50 mg/L) are added to this medium after autoclaving. Pour into sterile 100×20 mm plates (26 plates/L).

[0318]Shoot Induction Medium II: 1× B5 major salts, 1× B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose, 0.59 g/L MES, and 7 g/L Noble agar (pH 5.7). Filter sterilized 1× B5 vitamins, BAP (1.11 mg/L), Timentin (50 mg/L), Cefotaxime (200 mg/L), Vancomycin (50 mg/L) and Glufosinate (6 mg/L) are added to this medium after autoclaving. Pour into sterile 100×20 mm plates (26 plates/L).

[0319]Shoot Elongation Medium: 1×MS major salts, 1×MS minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose, 0.59 g/L MES, and 7 g/L Noble agar (pH 5.7). Filter sterilized 1× B5 vitamins, Asparagine (50 mg/L), L-Pyroglutamic Acid (100 mg/L), IAA (0.1 mg/L), GA3 (0.5 mg/L), Zeatin-R (1 mg/L), Timentin (50 mg/L), Cefotaxime (200 mg/L), Vancomycin (50 mg/L), and Glufosinate (6 mg/L) are added to this medium after autoclaving. Pour into sterile 100×25 mm plates (22 plates/L).

[0320]Rooting Medium: 1× MS major salts, 1×MS minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 20 g/L Sucrose, 0.59 g/L MES, and 7 g/L Noble agar (pH 5.6). Filter sterilized 1× B5 vitamins, Asparagine (50 mg/L), and L-Pyroglutamic Acid (100 mg/L) are added to this medium after autoclaving. Pour into sterile 150×25 mm vial (10 ml/vial).

Transformation and Regeneration:

[0321]Agrobacterium cultures are prepared for infecting seed explants as follows. The vector system, pTF102 in EHA101, is cultured on YEP medium (An et al., 1988) containing 100 mg/L spectinomycin (for pTF102), 50 mg/L kanamycin (for EHA101), and 25 mg/L chloramphenicol (for EHA101). 24 hours prior to infection a 2 ml culture of Agrobacterium is started by inoculating a loop of bacteria from the fresh YEP plate in YEP liquid medium amended with antibiotics. This culture is allowed to grow to saturation (8-10 hours) at 28° C. in a shaker incubator (-250 rpm). Then 0.2 ml of starter culture is transferred to a 1 L flask containing 250 ml of YEP medium amended with antibiotics. The culture is allowed to grow overnight at 28° C., 250 rpm to log phase (OD650=0.3-0.6 for EHA105) or late log phase (OD₆₅₀=1.0-1.2 for EHA101). The Agrobacterium culture is then pelleted at 3,500 rpm for 10 minutes at 20° C., and the pellet resuspended in infection medium by pipetting through the pellet. Bacterial cell densities are adjusted to a final OD650=0.6 (for EHA105) or OD650=0.6 to 1.0 (for EHA101). Agrobacteria-containing infection medium is shaken at 60 rpm for at least 30 minutes before use.

[0322]Explants are prepared for inoculation as follows. Seeds are sterilized, ideally with a combination of bleach solution and exposure to chlorine gas. Prior to infection, (-20 hours), sees are imbibed with deionized sterile water in the dark. Imbibed soybean seeds are transferred to a sterile 100×15 petri plate for dissection. Using a scalpel (i.e., #15 blade), longitudinal cuts are made along the hilum to separate the cotyledons and remove the seed coat. The embryonic axis found at the nodal end of the cotyledons is excised, and any remaining axial shoots/buds attached to the cotyledonary node are also removed.

[0323]Agrobacterium-mediated transformation is conducted as follows. Half-seed explants are dissected into a 100×25 mm petri plate and 30 ml Agrobacterium-containing infection media added thereto, such that the explants are completely covered by the infection media. Explants are allowed to incubate at room temperature for a short period of time (i.e., 30 minutes), preferably with occasional gentle agitation.

[0324]After infection, the explants are transferred to co-cultivation medium, preferably so that the flat, axial side is touching the filter paper. These plates are typically wrapped in parafilm, and cultivated for 5 days at 24° C. under an 18:6 photoperiod. Following this co-cultivation, shoot growth is induced by first washing the explants in shoot induction washing medium at room temperature, followed by placing the explants in shoot induction medium I, such that the explants are oriented with the nodal end of the cotyledon imbedded in the medium and the regeneration region flush to the surface with flat side up (preferably at a 30-45° angle). Explants are incubated at 24° C., 18:6 photoperiod, for 14 days. Explants are thereafter transferred to shoot induction medium II and maintained under the same conditions for another 14 days.

[0325]Following shoot induction, explants are transferred to shoot elongation medium, as follows. First, cotyledons are removed from the explants. A fresh cut at the base of the shoot pad flush to the medium is made, and the explants transferred to shoot elongation medium (containing glufosinate) and incubated at 24° C., 18:6 photoperiod, for 2-8 weeks. Preferably, explant tissue is transferred to fresh shoot elongation medium every 2 weeks, and at transfer, a fresh horizontal slice at the base of the shoot pad is made.

[0326]When shoots surviving the glufosinate selection have reached ˜3 cm length, they are excised from the shoot pad, briefly dipped in indole-3-butyric acid (1 mg/ml, 1-2 minutes), then transferred to rooting medium for acclimatization (i.e., in 150×25 mm glass vials with the stems of the shoots embedded approximately 1/2 cm into the media). When well rooted, the shoots are transferred to soil and plantlets grown at 24° C., 18:6 photoperiod, for at least one week, watering as needed. When the plantlets have at least two healthy trifoliates, an herbicide paint assay may be applied to confirm resistance to glufosinate. Briefly, using a cotton swab, Liberty herbicide (150 mg l-1) is applied to the upper leaf surface along the midrib of two leaves on two different trifoliates. Painted plants are transferred to the greenhouse and covered with a humidome. Plantlets are scored 3-5 days after painting. Resistant plantlets may be transplanted immediately to larger pots (i.e., 2 gal).

Example 32

Method for Generating Transgenic Potato Plants Carrying Arabidopsis GPT and GS1 Transgenes

[0327]This example provides a method for generating transgenic potato plants expressing GPT and GS1 transgenes. Potato (Solanum tuberosum, cultivar Desiree) is transformed with Agrobacterium carrying a transgene expression vector including an expression cassette encoding the Arabidopsis glutamine synthetase (GS1) coding sequence of SEQ ID NO: 7 under the control of the tomato RuBisCo small subunit promoter of SEQ ID NO: 22 (expression cassette of SEQ ID NO: 47), and the Arabidopsis GPT coding sequence of SEQ ID NO: 1 under the control of the 35S cauliflower mosaic virus (CMV) promoter (expression cassette of SEQ ID NO: 27).

Vector Constructs:

[0328]An expression cassette comprising the hordeum GS1 and GPT genes, under the control of the tomato RuBisCo small subunit and 35S CMV promoters, respectively, is cloned into the Cambia 2201 vector which provides kanamycin resistance.

Transformation and Regeneration:

[0329]A suitable Agrobacterium tumefaciens strain such as UC-Riverside Agro-1 strain is employed and used for infecting potato explant tissue (see, Narvaez-Vasquez et al., 1992, Plant Mo. Biol. 20:1149-1157). Cultures are maintained at 28° C. in liquid medium containing 10 g/L Yeast extract, 10 g/L Peptone, 5 g/L NaCl₂, 10 mg/L kanamycin, 30 mg/L tetracycline, and 9.81 g/L Acetosyringone (50 mM). Overnight cultures are diluted with liquid MS medium (4.3 g/L MS salts, 20 g/L sucrose, 1 mg/L thiamine, 100 mg/L inositol and 7 g/L phytoagar, pH to 5.8.) to 10⁸ Agrobacterium cells/ml for the infection of plant tissues (co-cultivation).

[0330]Potato leaf discs or tuber discs may be used as the explants to be inoculated. Discs are pre-conditioned by incubation on feeder plates for two to three days at 25° C. under dark conditions. Pre-conditioned explants are infected with Agrobacterium by soaking in 20 ml of sterile liquid MS medium (supra), containing 10⁸ Agrobacterium cells/ml for about 20 minutes. Before or during the co-cultivation, the explants are carefully punched with a syringe needle, or scalpel blade. Then, the explants are blotted dry with sterile filter paper, and incubated again in feeder plates for another two days. Explants are then transferred to liquid medium with transgene-transformed Agrobacterium, and incubated for three days at 28° C. under dark conditions for calli and shoot development (development (2-4 cm) in the presence of kanamycin (100 mg/L).

[0331]Following co-cultivation, supra, the explants are washed three times with sterile liquid medium and finally rinsed with the same medium containing 500 mg/l of cefotaxime. The explants are blotted dry with sterile filter paper and placed on shoot induction medium (4.3 g/L MS salts, 10 mg/L thiamine, 1 mg/L nicotinic acid, 1 mg/L pyridxine, 100 mg/L inositol, 30 g/L sucrose, 1 mg/L zeatin, 0.5 mg/L IAA, 7 g/L phytoagar, 250 mg/L Cefotaxime, 500 mg/L Carbenicillin, 100 mg/L Kanamycin) for 4-6 weeks. Thereafter, plantlets are transferred to rooting medium (4.3 g/L MS salts, 10 mg/L thiamine, 1 mg/L nicotinic acid, 1 mg/L pyridxine, 100 mg/L inositol, 20 g/L sucrose, 50 μg/L IAA, 7 g/L phytoagar, 50 mg/L Kanamycin and 500 mg/L Vancomycin) for 3-4 weeks.

[0332]All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0333]The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

TABLE-US-00020 SEQ ID NO: 1 Arabidopsis glutamine phenylpyruvate transaminase DNA coding sequence: ATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTAGCTTCAAAGCCTCTC TTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAAAATCCATCGTCCTAT CGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTCTACTCAAAAACCCGT CCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATGAG CATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTC GACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAA ACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGC GGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTAC ATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGG TGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCTCTA TGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCATCC CTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTATGA ACACTCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAAACC ATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTATACG ATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTATGTA TGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGATG GAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAAG CACACTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGTTG CAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAATG TGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTTCC CATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGAGA ACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGATCC CAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTTTG CGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGCAG AAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 2 Arabidopsis GPT amino acid sequence MYLDINGVMIKQFSFKASLLPFSSNFRQSSAKIHRPIGATMTTVSTQNESTQKPVQV AKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEAAIQAIKDGKNQYARG YGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAMLGLINPGDEVILFAPFY DSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHNPTGKMFTRE ELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSLTG WKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDYNVK KETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCEYLIEEVGVVAIPTSVF YLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 3 Alfalfa GS1 DNA coding sequence (upper case) with 5' and 3' untranslated sequences (indicated in lower case). atttccgttttcgttttcatttgattcattgaatcaaatcgaatcgaatctttaggattcaatacag attccttagattttactaagtttgaaaccaaaaccaaaacATGTCTCTCCTTTCAGAT CTTATCAACCTTGACCTCTCCGAAACCACCGAGAAAATCATCGCCG AATACATATGGATTGGTGGATCTGGTTTGGACTTGAGGAGCAAAGC AAGGACTCTACCAGGACCAGTTACTGACCCTTCACAGCTTCCCAAG TGGAACTATGATGGTTCCAGCACAGGTCAAGCTCCTGGAGAAGAT AGTGAAGTTATTATCTACCCACAAGCCATTTTCAAGGACCCATTTA GAAGGGGTAACAATATCTTGGTTATGTGTGATGCATACACTCCAGC TGGAGAGCCCATTCCCACCAACAAGAGACATGCAGCTGCCAAGAT TTTCAGCCATCCTGATGTTGTTGCTGAAGTACCATGGTATGGTATT GAGCAAGAATACACCTTGTTGCAGAAAGACATCAATTGGCCTCTTG GTTGGCCAGTTGGTGGTTTTCCTGGACCTCAGGGACCATACTATTG TGGAGCTGGTGCTGACAAGGCATTTGGCCGTGACATTGTTGACTC ACATTACAAAGCCTGTCTTTATGCCGGCATCAACATCAGTGGAATC AATGGTGAAGTGATGCCTGGTCAATGGGAATTCCAAGTTGGTCCCT CAGTTGGTATCTCTGCTGGTGATGAGATATGGGTTGCTCGTTACAT TTTGGAGAGGATCACTGAGGTTGCTGGTGTGGTGCTTTCCTTTGAC CCAAAACCAATTAAGGGTGATTGGAATGGTGCTGGTGCTCACACAA ATTACAGCACCAAGTCTATGAGAGAAGATGGTGGCTATGAAGTCAT CTTGAAAGCAATTGAGAAGCTTGGGAAGAAGCACAAGGAGCACAT TGCTGCTTATGGAGAAGGCAACGAGCGTAGATTGACAGGGCGACA TGAGACAGCTGACATTAACACCTTCTTATGGGGTGTTGCAAACCGT GGTGCGTCGATTAGAGTTGGAAGGGACACAGAGAAAGCAGGGAAA GGTTATTTCGAGGATAGGAGGCCATCATCTAACATGGATCCATATG TTGTTACTTCCATGATTGCAGACACCACCATTCTCTGGAAACCATA Agccaccacacacacatgcattgaagtatttgaaagtcattgttgattccgcattagaatttgg tcattgttttttctaggatttggatttgtgttattgttatggttcacactttgtttgtttgaatttgaggc cttgttataggtttcatatttctttctcttgttctaagtaaatgtcagaataataatgtaat SEQ ID NO: 4 Alfalfa GS1 amino acid sequence MSLLSDLINLDLSETTEKIIAEYIWIGGSGLDLRSKARTLPGPVTDPSQLPKWNYDGS STGQAPGEDSEVIIYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKIFSH PDVVAEVPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGAGADKAFGRDI VDSHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDEIWVARYILERITEVA GVVLSFDPKPIKGDWNGAGAHTNYSTKSMREDGGYEVILKAIEKLGKKHKEHIAAYG EGNERRLTGRHETADINTFLWGVANRGASIRVGRDTEKAGKGYFEDRRPSSNMDP YVVTSMIADTTILWKP SEQ ID NO: 5 Alfalfa GS1 DNA coding sequence (upper case) with 5' and 3' untranslated sequences (indicated in lower case) and vector sequences from ClaI to SmaI/SspI and SspI/SmaI to SalI/XhoI (lower case, underlined). atcgatgaattcgagctcggtacccatttccgttttcgttttcatttgattcattgaatcaaatcga atcgaatctttaggattcaatacagattccttagattttactaagtttgaaaccaaaaccaaaa cATGTCTCTCCTTTCAGATCTTATCAACCTTGACCTCTCCGAAACCA CCGAGAAAATCATCGCCGAATACATATGGATTGGTGGATCTGGTTT GGACTTGAGGAGCAAAGCAAGGACTCTACCAGGACCAGTTACTGA CCCTTCACAGCTTCCCAAGTGGAACTATGATGGTTCCAGCACAGGT CAAGCTCCTGGAGAAGATAGTGAAGTTATTATCTACCCACAAGCCA TTTTCAAGGACCCATTTAGAAGGGGTAACAATATCTTGGTTATGTG TGATGCATACACTCCAGCTGGAGAGCCCATTCCCACCAACAAGAG ACATGCAGCTGCCAAGATTTTCAGCCATCCTGATGTTGTTGCTGAA GTACCATGGTATGGTATTGAGCAAGAATACACCTTGTTGCAGAAAG ACATCAATTGGCCTCTTGGTTGGCCAGTTGGTGGTTTTCCTGGACC TCAGGGACCATACTATTGTGGAGCTGGTGCTGACAAGGCATTTGG CCGTGACATTGTTGACTCACATTACAAAGCCTGTCTTTATGCCGGC ATCAACATCAGTGGAATCAATGGTGAAGTGATGCCTGGTCAATGGG AATTCCAAGTTGGTCCCTCAGTTGGTATCTCTGCTGGTGATGAGAT ATGGGTTGCTCGTTACATTTTGGAGAGGATCACTGAGGTTGCTGGT GTGGTGCTTTCCTTTGACCCAAAACCAATTAAGGGTGATTGGAATG GTGCTGGTGCTCACACAAATTACAGCACCAAGTCTATGAGAGAAGA TGGTGGCTATGAAGTCATCTTGAAAGCAATTGAGAAGCTTGGGAAG AAGCACAAGGAGCACATTGCTGCTTATGGAGAAGGCAACGAGCGT AGATTGACAGGGCGACATGAGACAGCTGACATTAACACCTTCTTAT GGGGTGTTGCAAACCGTGGTGCGTCGATTAGAGTTGGAAGGGACA CAGAGAAAGCAGGGAAAGGTTATTTCGAGGATAGGAGGCCATCAT CTAACATGGATCCATATGTTGTTACTTCCATGATTGCAGACACCAC CATTCTCTGGAAACCATAAgccaccacacacacatgcattgaagtatttgaaagtc attgttgattccgcattagaatttggtcattgttttttctaggatttggatttgtgttattgttatggttc acactttgtttgtttgaatttgaggccttgttataggtttcatatttctttctcttgttctaagtaaatg tcagaataataatgtaatggggatcctctagagtcgag SEQ ID NO: 6 Arabidopsis GS1 coding sequence Cambia 1201 vector + rbcS3C + arabidopsis GS1Bold ATG is the start site, AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA ACCAATTATTTCAGCA TCTCTGCTCTCAGATCTCGTTAACCTCAACCTCA CCGATGCCACCGGGAAAATCATCGCCGAATACATATGGATCGGTGGATCTGGA ATGGATATCAGAAGCAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCA AAGCTTCCCAAGTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGA AGACAGTGAAGTCATTCTATACCCTCAGGCAATATTCAAGGATCCCTTCAGGAAA GGCAACAACATCCTGGTGATGTGTGATGCTTACACACCAGCTGGTGATCCTATT CCAACCAACAAGAGGCACAACGCTGCTAAGATCTTCAGCCACCCCGACGTTGC CAAGGAGGAGCCTTGGTATGGGATTGAGCAAGAATACACTTTGATGCAAAAGGA TGTGAACTGGCCAATTGGTTGGCCTGTTGGTGGCTACCCTGGCCCTCAGGGAC CTTACTACTGTGGTGTGGGAGCTGACAAAGCCATTGGTCGTGACATTGTGGATG CTCACTACAAGGCCTGTCTTTACGCCGGTATTGGTATTTCTGGTATCAATGGAGA AGTCATGCCAGGCCAGTGGGAGTTCCAAGTCGGCCCTGTTGAGGGTATTAGTT CTGGTGATCAAGTCTGGGTTGCTGGATACCTTCTCGAGAGGATCACTGAGATCT CTGGTGTAATTGTCAGCTTCGACCCGAAACCAGTCCCGGGTGACTGGAATGGA GCTGGAGCTCACTGCAACTACAGCACTAAGACAATGAGAAACGATGGAGGATTA GAAGTGATCAAGAAAGCGATAGGGAAGCTTCAGCTGAAACACAAAGAACACATT GCTGCTTACGGTGAAGGAAACGAGCGTCGTCTCACTGGAAAGCACGAAACCGC AGACATCAACACATTCTCTTGGGGAGTCGCGAACCGTGGAGCGTCAGTGAGAG

TGGGACGTGACACAGAGAAGGAAGGTAAAGGGTACTTCGAAGACAGAAGGCCA GCTTCTAACATGGATCCTTACGTTGTCACCTCCATGATCGCTGAGACGACCATA CTCGGTTGA SEQ ID NO: 7 Arabidopsis GS1 amino acid sequence Vector sequences at N-terminus in italics MVDLRNRRTSMSLLSDLVNLNLTDATGKIIAEYIWIGGSGMDIRSKARTLPGPVTDPS KLPKWNYDGSSTGQAAGEDSEVILYPQAIFKDPFRKGNNILVMCDAYTPAGDPIPTN KRHNAAKIFSHPDVAKEEPWYGIEQEYTLMQKDVNWPIGWPVGGYPGPQGPYYC GVGADKAIGRDIVDAHYKACLYAGIGISGINGEVMPGQWEFQVGPVEGISSGDQVW VARYLLERITEISGVIVSFDPKPVPGDWNGAGAHCNYSTKTMRNDGGLEVIKKAIGK LQLKHKEHIAAYGEGNERRLTGKHETADINTFSWGVANRGASVRVGRDTEKEGKG YFEDRRPASNMDPYVVTSMIAETTILG SEQ ID NO: 8 Grape GPT coding DNA sequence Showing Cambia 1305.1 with (3' end of) rbcS3C + Vitis vinifera GPT (Grape). Bold ATG is the start site, parentheses are the catI intron and the underlined actagt is the speI cloning site used to splice in the GPT gene. AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA ACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA ATGCAGCTCTCTCAATGTACCTGGACATTCCCAGAGTTGC TTAAAAGACCAGCCTTTTTAAGGAGGAGTATTGATAGTATTTCGAGTAGAAGTAG GTCCAGCTCCAAGTATCCATCTTTCATGGCGTCCGCATCAACGGTCTCCGCTCC AAATACGGAGGCTGAGCAGACCCATAACCCCCCTCAACCTCTACAGGTTGCAAA GCGCTTGGAGAAATTCAAAACAACAATCTTTACTCAAATGAGCATGCTTGCCATC AAACATGGAGCAATAAACCTTGGCCAAGGGTTTCCCAACTTTGATGGTCCTGAG TTTGTCAAAGAAGCAGCAATTCAAGCCATTAAGGATGGGAAAAACCAATATGCTC GTGGATATGGAGTTCCTGATCTCAACTCTGCTGTTGCTGATAGATTCAAGAAGG ATACAGGACTCGTGGTGGACCCCGAGAAGGAAGTTACTGTTACTTCTGGATGTA CAGAAGCAATTGCTGCTACTATGCTAGGCTTGATAAATCCTGGTGATGAGGTGA TCCTCTTTGCTCCATTTTATGATTCCTATGAAGCCACTCTATCCATGGCTGGTGC CCAAATAAAATCCATCACTTTACGTCCTCCGGATTTTGCTGTGCCCATGGATGAG CTCAAGTCTGCAATCTCAAAGAATACCCGTGCAATCCTTATAAACACTCCCCATA ACCCCACAGGAAAGATGTTCACAAGGGAGGAACTGAATGTGATTGCATCCCTCT GCATTGAGAATGATGTGTTGGTGTTTACTGATGAAGTTTACGACAAGTTGGCTTT CGAAATGGATCACATTTCCATGGCTTCTCTTCCTGGGATGTACGAGAGGACCGT GACTATGAATTCCTTAGGGAAAACTTTCTCCCTGACTGGATGGAAGATTGGTTG GACAGTAGCTCCCCCACACCTGACATGGGGAGTGAGGCAAGCCCACTCATTCC TCACGTTTGCTACCTGCACCCCAATGCAATGGGCAGCTGCAACAGCCCTCCGG GCCCCAGACTCTTACTATGAAGAGCTAAAGAGAGATTACAGTGCAAAGAAGGCA ATCCTGGTGGAGGGATTGAAGGCTGTCGGTTTCAGGGTATACCCATCAAGTGG GACCTATTTTGTGGTGGTGGATCACACCCCATTTGGGTTGAAAGACGATATTGC GTTTTGTGAGTATCTGATCAAGGAAGTTGGGGTGGTAGCAATTCCGACAAGCGT TTTCTACTTACACCCAGAAGATGGAAAGAACCTTGTGAGGTTTACCTTCTGTAAA GACGAGGGAACTCTGAGAGCTGCAGTTGAAAGGATGAAGGAGAAACTGAAGCC TAAACAATAGGGGCACGTGA SEQ ID NO: 9 Grape GPT amino acid sequence MVDLRNRRTSMQLSQCTWTFPELLKRPAFLRRSIDSISSRSRSSSKYPSFMASAST VSAPNTEAEQTHNPPQPLQVAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGP EFVKEAAIQAIKDGKNQYARGYGVPDLNSAVADRFKKDTGLVVDPEKEVTVTSGCT EAIAATMLGLINPGDEVILFAPFYDSYEATLSMAGAQIKSITLRPPDFAVPMDELKSAI SKNTRAILINTPHNPTGKMFTREELNVIASLCIENDVLVFTDEVYDKLAFEMDHISMAS LPGMYERTVTMNSLGKTFSLTGWKIGWTVAPPHLTWGVRQAHSFLTFATCTPMQW AAATALRAPDSYYEELKRDYSAKKAILVEGLKAVGFRVYPSSGTYFVVVDHTPFGLK DDIAFCEYLIKEVGVVAIPTSVFYLHPEDGKNLVRFTFCKDEGTLRAAVERMKEKLKP KQ SEQ ID NO: 10 Rice GPT DNA coding sequence Rice GPT codon optimized for E. coli expression; untranslated sequences shown in lower case atgtggATGAACCTGGCAGGCTTTCTGGCAACCCCGGCAACCGCAACCGCAACCC GTCATGAAATGCCGCTGAACCCGAGCAGCAGCGCGAGCTTTCTGCTGAGCAGC CTGCGTCGTAGCCTGGTGGCGAGCCTGCGTAAAGCGAGCCCGGCAGCAGCAG CAGCACTGAGCCCGATGGCAAGCGCAAGCACCGTGGCAGCAGAAAACGGTGC AGCAAAAGCAGCAGCAGAAAAACAGCAGCAGCAGCCGGTGCAGGTGGCGAAA CGTCTGGAAAAATTTAAAACCACCATTTTTACCCAGATGAGCATGCTGGCGATTA AACATGGCGCGATTAACCTGGGCCAGGGCTTTCC GAACTTTGATGGCCCGGATTTTGTGAAAGAAGCGGCGATTCAGGCGATTAACGC GGGCAAAAACCAGTATGCGCGTGGCTATGGCGTGCCGGAACTGAACAGCGCGA TTGCGGAACGTTTTCTGAAAGATAGCGGCCTGCAGGTGGATCCGGAAAAAGAA GTGACCGTGACCAGCGGCTGCACCGAAGCGATTGCGGCGACCATTCTGGGCCT GATTAACCCGGGCGATGAAGTGATTCTGTTTGCGCCGTTTTATGATAGCTATGA AGCGACCCTGAGCATGGCGGGCGCGAACGTGAAAGCGATTACCCTGCGTCCG CCGGATTTTAGCGTGCCGCTGGAAGAACTGAAAGCGGCCGTGAGCAAAAACAC CCGTGCGATTATGATTAACACCCCGCATAACCCGACCGGCAAAATGTTTACCCG TGAAGAACTGGAATTTATTGCGACCCTGTGCAAAGAAAACGATGTGCTGCTGTT TGCGGATGAAGTGTATGATAAACTGGCGTTTGAAGCGGATCATATTAGCATGGC GAGCATTCCGGGCATGTATGAACGTACCGTGACCATGAACAGCCTGGGCAAAA CCTTTAGCCTGACCGGCTGGAAAATTGGCTGGGCGATTGCGCCGCCGCATCTG ACCTGGGGCGTGCGTCAGGCACATAGCTTTCTGACCTTTGCAACCTGCACCCC GATGCAGGCAGCCGCCGCAGCAGCACTGCGTGCACCGGATAGCTATTATGAAG AACTGCGTCGTGATTATGGCGCGAAAAAAGCGCTGCTGGTGAACGGCCTGAAA GATGCGGGCTTTATTGTGTATCCGAGCAGCGGCACCTATTTTGTGATGGTGGAT CATACCCCGTTTGGCTTTGATAACGATATTGAATTTTGCGAATATCTGATTCGTG AAGTGGGCGTGGTGGCGATTCCGCCGAGCGTGTTTTATCTGAACCCGGAAGAT GGCAAAAACCTGGTGCGTTTTACCTTTTGCAAAGATGATGAAACCCTGCGTGCG GCGGTGGAACGTATGAAAACCAAACTGCGTAAAAAAAAGCTTgcggccgcactcgagc accaccaccaccaccactga SEQ ID NO: 11 Rice GPT amino acid sequence Includes amino terminal amino acids MW for cloning and His tag sequences from pet28 vector in italics. MWMNLAGFLATPATATATRHEMPLNPSSSASFLLSSLRRSLVASLRKASPAAAAAL SPMASASTVAAENGAAKAAAEKQQQQPVQVAKRLEKFKTTIFTQMSMLAIKHGAINL GQGFPNFDGPDFVKEAAIQAINAGKNQYARGYGVPELNSAIAERFLKDSGLQVDPE KEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPPDFS VPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIATLCKENDVLLFADEVYDKL AFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFL TFATCTPMQAAAAAALRAPDSYYEELRRDYGAKKALLVNGLKDAGFIVYPSSGTYF VMVDHTPFGFDNDIEFCEYLIREVGVVAIPPSVFYLNPEDGKNLVRFTFCKDDETLR AAVERMKTKLRKKKLAAALEHHHHHH SEQ ID NO: 12 Soybean GPT DNA coding sequence TOPO 151D WITH SOYBEAN for E. coli expression From starting codon. Vector sequences are italicized ATGCATCATCACCATCACCATGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTC GATTCTACGGAAAACCTGTATTTTCAGGGAATTGATCCCTTCACCGCGAAACGT CTGGAAAAATTTCAGACCACCATTTTTACCCAGATGAGCCTGCTGGCGATTAAAC ATGGCGCGATTAACCTGGGCCAGGGCTTTCCGAACTTTGATGGCCCGGAATTT GTGAAAGAAGCGGCGATTCAGGCGATTCGTGATGGCAAAAACCAGTATGCGCG TGGCTATGGCGTGCCGGATCTGAACATTGCGATTGCGGAACGTTTTAAAAAAGA TACCGGCCTGGTGGTGGATCCGGAAAAAGAAATTACCGTGACCAGCGGCTGCA CCGAAGCGATTGCGGCGACCATGATTGGCCTGATTAACCCGGGCGATGAAGTG ATTATGTTTGCGCCGTTTTATGATAGCTATGAAGCGACCCTGAGCATGGCGGGC GCGAAAGTGAAAGGCATTACCCTGCGTCCGCCGGATTTTGCGGTGCCGCTGGA AGAACTGAAAAGCACCATTAGCAAAAACACCCGTGCGATTCTGATTAACACCCC GCATAACCCGACCGGCAAAATGTTTACCCGTGAAGAACTGAACTGCATTGCGAG CCTGTGCATTGAAAACGATGTGCTGGTGTTTACCGATGAAGTGTATGATAAACT GGCGTTTGATATGGAACATATTAGCATGGCGAGCCTGCCGGGCATGTTTGAACG TACCGTGACCCTGAACAGCCTGGGCAAAACCTTTAGCCTGACCGGCTGGAAAAT TGGCTGGGCGATTGCGCCGCCGCATCTGAGCTGGGGCGTGCGTCAGGCGCAT GCGTTTCTGACCTTTGCAACCGCACATCCGTTTCAGTGCGCAGCAGCAGCAGCA CTGCGTGCACCGGATAGCTATTATGTGGAACTGAAACGTGATTATATGGCGAAA CGTGCGATTCTGATTGAAGGCCTGAAAGCGGTGGGCTTTAAAGTGTTTCCGAGC AGCGGCACCTATTTTGTGGTGGTGGATCATACCCCGTTTGGCCTGGAAAACGAT GTGGCGTTTTGCGAATATCTGGTGAAAGAAGTGGGCGTGGTGGCGATTCCGAC CAGCGTGTTTTATCTGAACCCGGAAGAAGGCAAAAACCTGGTGCGTTTTACCTT TTGCAAAGATGAAGAAACCATTCGTAGCGCGGTGGAACGTATGAAAGCGAAACT GCGTAAAGTCGACTAA SEQ ID NO: 13 Soybean GPT amino acid sequence Translated protein product, vector sequences italicized

MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTAKRLEKFQTTIFTQMSLLAIKHGAI NLGQGFPNFDGPEFVKEAAIQAIRDGKNQYARGYGVPDLNIAIAERFKKDTGLVVDP EKEITVTSGCTEAIAATMIGLINPGDEVIMFAPFYDSYEATLSMAGAKVKGITLRPPDF AVPLEELKSTISKNTRAILINTPHNPTGKMFTREELNCIASLCIENDVLVFTDEVYDKL AFDMEHISMASLPGMFERTVTLNSLGKTFSLTGWKIGWAIAPPHLSWGVRQAHAFL TFATAHPFQCAAAAALRAPDSYYVELKRDYMAKRAILIEGLKAVGFKVFPSSGTYFV VVDHTPFGLENDVAFCEYLVKEVGVVAIPTSVFYLNPEEGKNLVRFTFCKDEETIRS AVERMKAKLRKVD SEQ ID NO: 14 Barley GPT DNA coding sequence Coding sequence from start with intron removed TAGATCTGAGGAACCGACGA ATGGCATCCGCCCCCGCCTCCGC CTCCGCGGCCCTCTCCACCGCCGCCCCCGCCGACAACGGGGCCGCCAAGCCC ACGGAGCAGCGGCCGGTACAGGTGGCTAAGCGATTGGAGAAGTTCAAAACAAC AATTTTCACACAGATGAGCATGCTCGCAGTGAAGCATGGAGCAATAAACCTTGG ACAGGGGTTTCCCAATTTTGATGGCCCTGACTTTGTCAAAGATGCTGCTATTGA GGCTATCAAAGCTGGAAAGAATCAGTATGCAAGAGGATATGGTGTGCCTGAATT GAACTCAGCTGTTGCTGAGAGATTTCTCAAGGACAGTGGATTGCACATCGATCC TGATAAGGAAGTTACTGTTACATCTGGGTGCACAGAAGCAATAGCTGCAACGAT ATTGGGTCTGATCAACCCTGGGGATGAAGTCATACTGTTTGCTCCATTCTATGAT TCTTATGAGGCTACACTGTCCATGGCTGGTGCGAATGTCAAAGCCATTACACTC CGCCCTCCGGACTTTGCAGTCCCTCTTGAAGAGCTAAAGGCTGCAGTCTCGAA GAATACCAGAGCAATAATGATTAATACACCTCACAACCCTACCGGGAAAATGTTC ACAAGGGAGGAACTTGAGTTCATTGCTGATCTCTGCAAGGAAAATGACGTGTTG CTCTTTGCCGATGAGGTCTACGACAAGCTGGCGTTTGAGGCGGATCACATATCA ATGGCTTCTATTCCTGGCATGTATGAGAGGACCGTCACTATGAACTCCCTGGGG AAGACGTTCTCCTTGACCGGATGGAAGATCGGCTGGGCGATAGCACCACCGCA CCTGACATGGGGCGTAAGGCAGGCACACTCCTTCCTCACATTCGCCACCTCCA CGCCGATGCAATCAGCAGCGGCGGCGGCCCTGAGAGCACCGGACAGCTACTT TGAGGAGCTGAAGAGGGACTACGGCGCAAAGAAAGCGCTGCTGGTGGACGGG CTCAAGGCGGCGGGCTTCATCGTCTACCCTTCGAGCGGAACCTACTTCATCATG GTCGACCACACCCCGTTCGGGTTCGACAACGACGTCGAGTTCTGCGAGTACTT GATCCGCGAGGTCGGCGTCGTGGCCATCCCGCCAAGCGTGTTCTACCTGAACC CGGAGGACGGGAAGAACCTGGTGAGGTTCACCTTCTGCAAGGACGACGACACG CTAAGGGCGGCGGTGGACAGGATGAAGGCCAAGCTCAGGAAGAAATGA SEQ ID NO: 15 Barley GPT amino acid sequence Translated sequence from start site (intron removed) MVDLRNRRTSMASAPASASAALSTAAPADNGAAKPTEQRPVQVAKRLEKFKTTIFT QMSMLAVKHGAINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYARGYGVPELNSAVA ERFLKDSGLHIDPDKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAG ANVKAITLRPPDFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIADLCKE NDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPP HLTWGVRQAHSFLTFATSTPMQSAAAAALRAPDSYFEELKRDYGAKKALLVDGLKA AGFIVYPSSGTYFIMVDHTPFGFDNDVEFCEYLIREVGVVAIPPSVFYLNPEDGKNLV RFTFCKDDDTLRAAVDRMKAKLRKK SEQ ID NO: 16 Zebra fish GPT DNA coding sequence Danio rerio sequence designed for expression in E coli. Bold, italicized nucleotides added for cloning or from pET28b vector. GTGGCGAAACGTCTGGAAAAATTTAAAACCACCATTTTTACCCAGATGA GCATGCTGGCGATTAAACATGGCGCGATTAACCTGGGCCAGGGCTTTCCGAAC TTTGATGGCCCGGATTTTGTGAAAGAAGCGGCGATTCAGGCGATTCGTGATGGC AACAACCAGTATGCGCGTGGCTATGGCGTGCCGGATCTGAACATTGCGATTAG CGAACGTTATAAAAAAGATACCGGCCTGGCGGTGGATCCGGAAAAAGAAATTAC CGTGACCAGCGGCTGCACCGAAGCGATTGCGGCGACCGTGCTGGGCCTGATT AACCCGGGCGATGAAGTGATTGTGTTTGCGCCGTTTTATGATAGCTATGAAGCG ACCCTGAGCATGGCGGGCGCGAAAGTGAAAGGCATTACCCTGCGTCCGCCGG ATTTTGCGCTGCCGATTGAAGAACTGAAAAGCACCATTAGCAAAAACACCCGTG CGATTCTGCTGAACACCCCGCATAACCCGACCGGCAAAATGTTTACCCCGGAAG AACTGAACACCATTGCGAGCCTGTGCATTGAAAACGATGTGCTGGTGTTTAGCG ATGAAGTGTATGATAAACTGGCGTTTGATATGGAACATATTAGCATTGCGAGCCT GCCGGGCATGTTTGAACGTACCGTGACCATGAACAGCCTGGGCAAAACCTTTA GCCTGACCGGCTGGAAAATTGGCTGGGCGATTGCGCCGCCGCATCTGACCTGG GGCGTGCGTCAGGCGCATGCGTTTCTGACCTTTGCAACCAGCAACCCGATGCA GTGGGCAGCAGCAGTGGCACTGCGTGCACCGGATAGCTATTATACCGAACTGA AACGTGATTATATGGCGAAACGTAGCATTCTGGTGGAAGGCCTGAAAGCGGTG GGCTTTAAAGTGTTTCCGAGCAGCGGCACCTATTTTGTGGTGGTGGATCATACC CCGTTTGGCCATGAAAACGATATTGCGTTTTGCGAATATCTGGTGAAAGAAGTG GGCGTGGTGGCGATTCCGACCAGCGTGTTTTATCTGAACCCGGAAGAAGGCAA AAACCTGGTGCGTTTTACCTTTTGCAAAGATGAAGGCACCCTGCGTGCGGCGGT GGATCGTATGAAAGAAAAACTGCGTAAA SEQ ID NO: 17 Zebra fish GPR amino acid sequence Amino acid sequence of Danio rerio cloned and expressed in E. coli (bold, italicized amino acids are added from vector/cloning and His tag on C-terminus) VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGNNQ YARGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGDEVIVF APFYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPHNPTGKMF TPEELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVTMNSLGKTFSL TGWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAAAVALRAPDSYYTELKRDY MAKRSILVEGLKAVGFKVFPSSGTYFVVVDHTPFGHENDIAFCEYLVKEVGVVAIPT SVFYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRK SEQ ID NO: 18 Arabidopsis truncated GPT-30 construct DNA sequence Arabidopsis GPT coding sequence with 30 amino acids removed from the targeting sequence. ATGGCCAAAATCCATCGTCCTATCGGAGCCACCATGACCACAGTTTCGACTCAG AACGAGTCTACTCAAAAACCCGTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAG ACTACTATTTTCACTCAAATGAGCATATTGGCAGTTAAACATGGAGCGATCAATT TAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAAAGAAGCTGCGA TCCAAGCTATTAAAGATGGTAAAAACCAGTATGCTCGTGGATACGGCATTCCTCA GCTCAACTCTGCTATAGCTGCGCGGTTTCGTGAAGATACGGGTCTTGTTGTTGA TCCTGAGAAAGAAGTTACTGTTACATCTGGTTGCACAGAAGCCATAGCTGCAGC TATGTTGGGTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTGCACCGTTTTAT GATTCCTATGAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAAAGGAATCACTT TACGTCCACCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTA ACAAGACTCGAGCCATCCTTATGAACACTCCGCACAACCCGACCGGGAAGATGT TCACTAGGGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGAAAACGATGTGC TTGTGTTCTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATGGATCACATTTC TATAGCTTCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGAATTCCCTGGGA AAGACTTTCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCAT CTGACTTGGGGAGTTCGACAAGCACACTCTTACCTCACATTCGCCACATCAACA CCAGCACAATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAA GAGCTGAAAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAG GAAGTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGAT CACACTCCATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTATCTTATTGAAG AAGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAG GGAAGAATTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACGTTGC GTGGTGCAATTGAGAGGATGAAGCAGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 19 Arabidopsis truncated GPT-30 construct amino acid sequence MAKIHRPIGATMTTVSTQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQG FPNFDGPDFVKEAAIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLVVDPEKEVT VTSGCTEAIAAAMLGLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLE ELKAAVTNKTRAILMNTPHNPTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEM DHISIASLPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATS TPAQWAAVAALKAPESYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFVVADH TPFGMENDVAFCEYLIEEVGVVAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIER MKQKLKRKV SEQ ID NO: 20: Arabidopsis truncated GPT-45 construct DNA sequence Arabidopsis GPT coding sequence with 45 residues in the targeting sequence removed ATGGCGACTCAGAACGAGTCTACTCAAAAACCCGTCCAGGTGGCGAAGAGATTA GAGAAGTTCAAGACTACTATTTTCACTCAAATGAGCATATTGGCAGTTAAACATG GAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAA AGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAACCAGTATGCTCGTGGATA CGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTTTCGTGAAGATACGGG TCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTACATCTGGTTGCACAGAAGCC ATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTG CACCGTTTTATGATTCCTATGAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAA AGGAATCACTTTACGTCCACCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGC TGCGGTAACTAACAAGACTCGAGCCATCCTTATGAACACTCCGCACAACCCGAC CGGGAAGATGTTCACTAGGGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGA AAACGATGTGCTTGTGTTCTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATG GATCACATTTCTATAGCTTCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGA ATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTG

CGCCGCCTCATCTGACTTGGGGAGTTCGACAAGCACACTCTTACCTCACATTCG CCACATCAACACCAGCACAATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAG TCTTACTTCAAAGAGCTGAAAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTA AGGGTTTGAAGGAAGTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTTTG TGGTTGCTGATCACACTCCATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTA TCTTATTGAAGAAGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAAT CCAGAAGAAGGGAAGAATTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACG TTGCGTGGTGCAATTGAGAGGATGAAGCAGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 21: Arabidopsis truncated GPT-45 construct amino acid sequence MATQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEA AIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAML GLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAIL MNTPHNPTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYER TVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKA PESYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCE YLIEEVGVVAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 22: Tomato Rubisco promoter TOMATO RuBisCo rbcS3C promoter sequence from KpnI to NcoI GGTACCGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTAC TTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGC TTCTGAAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCT TCCATTCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCT CCTCCTCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTG ATATATCCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTC TTTTGGGTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTG AAATGGATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAG GAGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTT AGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAG GGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAG CTCTAAAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCA TAGGTAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAAT ACACTAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGT AAGAATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGA AGTATAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAG AGACTGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAA TTATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTG TACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTT TCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATAT GGCGCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGG GGTTGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAA ATATTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGT GGGTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTA ATATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTC GTCAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGA AAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGA GGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAA TGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGG AAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATT TCAGCACCATGG SEQ ID NO: 23: Bamboo GPT DNA coding sequence ATGGCCTCCGCGGCCGTCTCCACCGTCGCCACCGCCGCCGACGGCGTCGCGA AGCCGACGGAGAAGCAGCCGGTACAGGTCGCAAAGCGTTTGGAAAAGTTTAAG ACAACAATTTTCACACAGATGAGCATGCTTGCCATCAAGCATGGAGCAATAAAC CTCGGCCAGGGCTTTCCGAATTTTGATGGCCCTGACTTTGTGAAAGAAGCTGCT ATTCAAGCTATCAATGCTGGGAAGAATCAGTATGCAAGAGGATATGGTGTGCCT GAACTGAACTCGGCTGTTGCTGAAAGGTTCCTGAAGGACAGTGGCTTGCAAGTC GATCCCGAGAAGGAAGTTACTGTCACATCTGGGTGCACGGAAGCGATAGCTGC AACGATATTGGGTCTTATCAACCCTGGCGATGAAGTGATCTTGTTTGCTCCATTC TATGATTCATACGAGGCTACGCTGTCGATGGCTGGTGCCAATGTAAAAGCCATT ACTCTCCGTCCTCCAGATTTTGCAGTCCCTCTTGAGGAGCTAAAGGCCACAGTC TCTAAGAACACCAGAGCGATAATGATAAACACACCACACAATCCTACTGGGAAA ATGTTTTCTAGGGAAGAACTTGAATTCATTGCTACTCTCTGCAAGAAAAATGATG TGTTGCTTTTTGCTGATGAGGTCTATGACAAGTTGGCATTTGAGGCAGATCATAT ATCAATGGCTTCTATTCCTGGCATGTATGAGAGGACTGTGACTATGAACTCTCTG GGGAAGACATTCTCTCTAACAGGATGGAAGATCGGTTGGGCAATAGCACCACCA CACCTGACATGGGGTGTAAGGCAGGCACACTCATTCCTCACATTTGCCACCTGC ACACCAATGCAATCGGCGGCGGCGGCGGCTCTTAGAGCACCAGATAGCTACTA TGGGGAGCTGAAGAGGGATTACGGTGCAAAGAAAGCGATACTAGTCGACGGAC TCAAGGCTGCAGGTTTTATTGTTTACCCTTCAAGTGGAACATACTTTGTCATGGT CGATCACACCCCGTTTGGTTTCGACAATGATATTGAGTTCTGCGAGTATTTGATC CGCGAAGTCGGTGTTGTCGCCATACCACCAAGCGTATTTTATCTCAACCCTGAG GATGGGAAGAACTTGGTGAGGTTCACCTTCTGCAAGGATGATGATACGCTGAGA GCCGCAGTTGAGAGGATGAAGACAAAGCTCAGGAAAAAATGA SEQ ID NO: 24: Bamboo GPT amino acid sequence MASAAVSTVATAADGVAKPTEKQPVQVAKRLEKFKTTIFTQMSMLAIKHGAINLGQG FPNFDGPDFVKEAAIQAINAGKNQYARGYGVPELNSAVAERFLKDSGLQVDPEKEV TVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPPDFAVPL EELKATVSKNTRAIMINTPHNPTGKMFSREELEFIATLCKKNDVLLFADEVYDKLAFE ADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFA TCTPMQSAAAAALRAPDSYYGELKRDYGAKKAILVDGLKAAGFIVYPSSGTYFVMV DHTPFGFDNDIEFCEYLIREVGVVAIPPSVFYLNPEDGKNLVRFTFCKDDDTLRAAVE RMKTKLRKK SEQ ID NO: 25: 1305.1 + rbcS3C promoter + catI intron with rice GPT gene. Cambia1305.1 with (3' end of) rbcS3C + rice GPT coding sequence. Underlined ATG is start site, parentheses are the catI intron and the underlined actagt is the speI cloning site used to splice in the rice gene. AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA ACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA ATGAATCTGGCCGGCTTTCTCGCCACGCCCGCGACCGCG ACCGCGACGCGGCATGAGATGCCGTTAAATCCCTCCTCCTCCGCCTCCTTCCTC CTCTCCTCGCTCCGCCGCTCGCTCGTCGCGTCGCTCCGGAAGGCCTCGCCGG CGGCGGCCGCGGCGCTCTCCCCCATGGCCTCCGCGTCCACCGTCGCCGCCGA GAACGGCGCCGCCAAGGCGGCGGCGGAGAAGCAGCAGCAGCAGCCTGTGCA GGTTGCAAAGCGGTTGGAAAAGTTTAAGACGACCATTTTCACACAGATGAGTAT GCTTGCCATCAAGCATGGAGCAATAAACCTTGGCCAGGGTTTTCCGAATTTCGA TGGCCCTGACTTTGTAAAAGAGGCTGCTATTCAAGCTATCAATGCTGGGAAGAA TCAGTACGCAAGAGGATATGGTGTGCCTGAACTGAACTCAGCTATTGCTGAAAG ATTCCTGAAGGACAGCGGACTGCAAGTCGATCCGGAGAAGGAAGTTACTGTCA CATCTGGATGCACAGAAGCTATAGCTGCAACAATTTTAGGTCTAATTAATCCAGG CGATGAAGTGATATTGTTTGCTCCATTCTATGATTCATATGAGGCTACCCTGTCA ATGGCTGGTGCCAACGTAAAAGCCATTACTCTCCGTCCTCCAGATTTTTCAGTC CCTCTTGAAGAGCTAAAGGCTGCAGTCTCGAAGAACACCAGAGCTATTATGATA AACACCCCGCACAATCCTACTGGGAAAATGTTTACAAGGGAAGAACTTGAGTTT ATTGCCACTCTCTGCAAGGAAAATGATGTGCTGCTTTTTGCTGATGAGGTCTAC GACAAGTTAGCTTTTGAGGCAGATCATATATCAATGGCTTCTATTCCTGGCATGT ATGAGAGGACCGTGACCATGAACTCTCTTGGGAAGACATTCTCTCTTACAGGAT GGAAGATCGGTTGGGCAATCGCACCGCCACACCTGACATGGGGTGTAAGGCAG GCACACTCATTCCTCACGTTTGCGACCTGCACACCAATGCAAGCAGCTGCAGCT GCAGCTCTGAGAGCACCAGATAGCTACTATGAGGAACTGAGGAGGGATTATGG AGCTAAGAAGGCATTGCTAGTCAACGGACTCAAGGATGCAGGTTTCATTGTCTA TCCTTCAAGTGGAACATACTTCGTCATGGTCGACCACACCCCATTTGGTTTCGA CAATGATATTGAGTTCTGCGAGTATTTGATTCGCGAAGTCGGTGTTGTCGCCATA CCACCTAGTGTATTTTATCTCAACCCTGAGGATGGGAAGAACTTGGTGAGGTTC ACCTTTTGCAAGGATGATGAGACGCTGAGAGCCGCGGTTGAGAGGATGAAGAC AAAGCTCAGGAAAAAATGA SEQ ID NO: 26: HORDEUM GPT SEQUENCE IN VECTOR Cambia1305.1 with (3' end of) rbcS3C + hordeum (IDI4) coding sequence. Underlined ATG is start site, parentheses are the catI intron and the underlined actagt is the speI cloning site used to splice in the hordeum gene. AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA ACCAATTATTTCAGCACC TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT

TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA ATGGCATCCGCCCCCGCCTCCGCCTCCGCGGCCCTCTCC ACCGCCGCCCCCGCCGACAACGGGGCCGCCAAGCCCACGGAGCAGCGGCCG GTACAGGTGGCTAAGCGATTGGAGAAGTTCAAAACAACAATTTTCACACAGATG AGCATGCTCGCAGTGAAGCATGGAGCAATAAACCTTGGACAGGGGTTTCCCAAT TTTGATGGCCCTGACTTTGTCAAAGATGCTGCTATTGAGGCTATCAAAGCTGGA AAGAATCAGTATGCAAGAGGATATGGTGTGCCTGAATTGAACTCAGCTGTTGCT GAGAGATTTCTCAAGGACAGTGGATTGCACATCGATCCTGATAAGGAAGTTACT GTTACATCTGGGTGCACAGAAGCAATAGCTGCAACGATATTGGGTCTGATCAAC CCTGGGGATGAAGTCATACTGTTTGCTCCATTCTATGATTCTTATGAGGCTACAC TGTCCATGGCTGGTGCGAATGTCAAAGCCATTACACTCCGCCCTCCGGACTTTG CAGTCCCTCTTGAAGAGCTAAAGGCTGCAGTCTCGAAGAATACCAGAGCAATAA TGATTAATACACCTCACAACCCTACCGGGAAAATGTTCACAAGGGAGGAACTTG AGTTCATTGCTGATCTCTGCAAGGAAAATGACGTGTTGCTCTTTGCCGATGAGG TCTACGACAAGCTGGCGTTTGAGGCGGATCACATATCAATGGCTTCTATTCCTG GCATGTATGAGAGGACCGTCACTATGAACTCCCTGGGGAAGACGTTCTCCTTGA CCGGATGGAAGATCGGCTGGGCGATAGCACCACCGCACCTGACATGGGGCGT AAGGCAGGCACACTCCTTCCTCACATTCGCCACCTCCACGCCGATGCAATCAGC AGCGGCGGCGGCCCTGAGAGCACCGGACAGCTACTTTGAGGAGCTGAAGAGG GACTACGGCGCAAAGAAAGCGCTGCTGGTGGACGGGCTCAAGGCGGCGGGCT TCATCGTCTACCCTTCGAGCGGAACCTACTTCATCATGGTCGACCACACCCCGT TCGGGTTCGACAACGACGTCGAGTTCTGCGAGTACTTGATCCGCGAGGTCGGC GTCGTGGCCATCCCGCCAAGCGTGTTCTACCTGAACCCGGAGGACGGGAAGAA CCTGGTGAGGTTCACCTTCTGCAAGGACGACGACACGCTAAGGGCGGCGGTG GACAGGATGAAGGCCAAGCTCAGGAAGAAATGATTGAGGGGCG SEQ ID NO: 27 Expression cassette, Arabidopsis GPT coding sequence (ATG underlined) under control of CMV 35S promoter (italics; promoter from Cambia 1201) CATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGG CGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTC AACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCT CAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACC TCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAA GGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGA AGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCA TCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTG ATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACC CTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTCTTGA CCATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTAGCTTCAAAGCCTC TCTTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAAAATCCATCGTCCT ATCGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTCTACTCAAAAACCC GTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATG AGCATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATT TCGACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAA AAACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGC GCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGT TACATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCT GGTGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCT CTATGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCA TCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTA TGAACACTCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAA ACCATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTAT ACGATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTAT GTATGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGA TGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACA AGCACACTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGT TGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAA TGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTT CCCATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGA GAACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGAT CCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTT TGCGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGC AGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 28 Cambia p1305.1 with (3' end of) rbcS3C + Arabidopsis GPT coding sequence. Underlined ATG is start site, parentheses are the catI intron and the underlined actagt is the speI cloning site used to splice in the Arabidopsis gene. AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA ACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA ATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTA GCTTCAAAGCCTCTCTTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAA AATCCATCGTCCTATCGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTC TACTCAAAAACCCGTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTAT TTTCACTCAAATGAGCATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAA GGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTA TTAAAGATGGTAAAAACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACT CTGCTATAGCTGCGCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGA AAGAAGTTACTGTTACATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGG GTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTAT GAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCA CCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACT CGAGCCATCCTTATGAACACTCCGCACAACCCGACCGGGAAGATGTTCACTAG GGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTT CTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCT TCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTT TCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTT GGGGAGTTCGACAAGCACACTCTTACCTCACATTCGCCACATCAACACCAGCAC AATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGA AAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCG GATTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTC CATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGG GGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAA TTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGA GAGGATGAAGCAGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 29 Arabidpsis GPT coding sequence (mature protein, no targeting sequence) GTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATGAGCATAT TGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACG GTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAACCA GTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTT TCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTACATCT GGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGGTGAT GAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCTCTATGG CTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCATCCCTTT GGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTATGAACAC TCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAAACCATTG CATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTATACGATAA GCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTATGTATGAA AGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAG ATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAAGCACA CTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGTTGCAGC TCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAATGTGAAA AAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTTCCCATCG AGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGAGAACGAT GTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGATCCCAACG AGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTTTGCGTTCT GTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGCAGAAGCTT AAGAGAAAAGTCTGA SEQ ID NO: 30 Arabidpsis GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEAAIQAIKDGKNQYAR

GYGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAMLGLINPGDEVILFAP FYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHNPTGKMFT REELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSL TGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDYN VKKETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCEYLIEEVGVVAIPTS VFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 31 Grape GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIKDGKNQYAR GYGVPDLNSAVADRFKKDTGLVVDPEKEVTVTSGCTEAIAATMLGLINPGDEVILFA PFYDSYEATLSMAGAQIKSITLRPPDFAVPMDELKSAISKNTRAILINTPHNPTGKMFT REELNVIASLCIENDVLVFTDEVYDKLAFEMDHISMASLPGMYERTVTMNSLGKTFS LTGWKIGWTVAPPHLTWGVRQAHSFLTFATCTPMQWAAATALRAPDSYYEELKRD YSAKKAILVEGLKAVGFRVYPSSGTYFVVVDHTPFGLKDDIAFCEYLIKEVGVVAIPT SVFYLHPEDGKNLVRFTFCKDEGTLRAAVERMKEKLKPKQ SEQ ID NO: 32 Rice GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAINAGKNQYAR GYGVPELNSAIAERFLKDSGLQVDPEKEVTVTSGCTEAIAATILGLINPGDEVILFAPF YDSYEATLSMAGANVKAITLRPPDFSVPLEELKAAVSKNTRAIMINTPHNPTGKMFT REELEFIATLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSL TGWKIGWAIAPPHLTWGVRQAHSFLTFATCTPMQAAAAAALRAPDSYYEELRRDY GAKKALLVNGLKDAGFIVYPSSGTYFVMVDHTPFGFDNDIEFCEYLIREVGVVAIPPS VFYLNPEDGKNLVRFTFCKDDETLRAAVERMKTKLRKK SEQ ID NO: 33 Soybean GPT amino acid sequence (-1 mature protein, no targeting sequence) AKRLEKFQTTIFTQMSLLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIRDGKNQYARG YGVPDLNIAIAERFKKDTGLVVDPEKEITVTSGCTEAIAATMIGLINPGDEVIMFAPFY DSYEATLSMAGAKVKGITLRPPDFAVPLEELKSTISKNTRAILINTPHNPTGKMFTRE ELNCIASLCIENDVLVFTDEVYDKLAFDMEHISMASLPGMFERTVTLNSLGKTFSLTG WKIGWAIAPPHLSWGVRQAHAFLTFATAHPFQCAAAAALRAPDSYYVELKRDYMAK RAILIEGLKAVGFKVFPSSGTYFVVVDHTPFGLENDVAFCEYLVKEVGVVAIPTSVFY LNPEEGKNLVRFTFCKDEETIRSAVERMKAKLRKVD SEQ ID NO: 34 Barley GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSMLAVKHGAINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYA RGYGVPELNSAVAERFLKDSGLHIDPDKEVTVTSGCTEAIAATILGLINPGDEVILFAP FYDSYEATLSMAGANVKAITLRPPDFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFT REELEFIADLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSL TGWKIGWAIAPPHLTWGVRQAHSFLTFATSTPMQSAAAAALRAPDSYFEELKRDYG AKKALLVDGLKAAGFIVYPSSGTYFIMVDHTPFGFDNDVEFCEYLIREVGVVAIPPSV FYLNPEDGKNLVRFTFCKDDDTLRAAVDRMKAKLRKK SEQ ID NO: 35 Zebra fish GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGNNQYA RGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGDEVIVFAP FYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPHNPTGKMFTP EELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVTMNSLGKTFSLT GWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAAAVALRAPDSYYTELKRDYM AKRSILVEGLKAVGFKVFPSSGTYFVVVDHTPFGHENDIAFCEYLVKEVGVVAIPTSV FYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRK SEQ ID NO: 36 Bamboo GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAINAGKNQYAR GYGVPELNSAVAERFLKDSGLQVDPEKEVTVTSGCTEAIAATILGLINPGDEVILFAP FYDSYEATLSMAGANVKAITLRPPDFAVPLEELKATVSKNTRAIMINTPHNPTGKMFS REELEFIATLCKKNDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSL TGWKIGWAIAPPHLTWGVRQAHSFLTFATCTPMQSAAAAALRAPDSYYGELKRDY GAKKAILVDGLKAAGFIVYPSSGTYFVMVDHTPFGFDNDIEFCEYLIREVGVVAIPPS VFYLNPEDGKNLVRFTFCKDDDTLRAAVERMKTKLRKK SEQ ID NO: 39 Rice rubisco promoter deposited in NCBI GenBank: AF143510.1 PstI cloning sites in bold; NcoI cloning site in italics, catI intron and part of Gus plus protein from Cambia 1305.1 vector in bold underline (sequence removed and not translated), 3' terminal SpeI cloning site in double underline. The construct also includes a PmlI 1305.1 cloning site CACGTG (also cuts in rice rbsc promoter), and a ZraI cloning site GACGTC, which can be added by PCR to clone into PmlI site of vector). CTGCAGCAAAGAAACGTTATTAGTTGGTGCTTTTGGTGGTAGGAATGTAGTTTTC TGACAAAGTCAATTACTGAATATAAAAAAAATCTGCACAGCTCTGCGTCAACAGT TGTCCAAGGGATGCCTCAAAAATCTGTGCAGATTATCAGTCGTCACGCAGAAGC AGAACATCATGGTGTGCTAGGTCAGCTTCTTGCATTGGGCCATGAATCCGGTTG GTTGTTAATCTCTCCTCTCTTATTCTCTTATATTAAGATGCATAACTCTTTTATGTA GTCTAAAAAAAAATCCAGTGGATCGGATAGTAGTACGTCATGGTGCCATTAGGT ACCGTTGAACCTAACAGATATTTATGCATGTGTATATATATAGCTATATAGACAAA ATTGATGCCGATTATAGACCCAAAAGCAATAGGTATATATAATATAATACAGACC ACACCACCAAACTAAGAATCGATCAAATAGACAAGGCATGTCTCCAAATTGTCTT AAACTATTTCCGTAGGTTCAGCCGTTCAGGAGTCGAATCAGCCTCTGCCGGCGT TTTCTTTGCACGTACGACGGACACACATGGGCATACCATATAGCTGGTCCATGA CATTAGGAGAGAGAACGTACGTGTTGACCTGTAGCTGAGATATAACAAGGTTGA TTATAATATCACCAAACATGAAATCATCCAAGGATGACCCATAACTATCACTACTA TAGTACTGCATCTGGTAAAAGAAATTGTATAGACTCTATTTCGAGCACTACCACA TAACGCCTGCAATGTGACACCCTACCTATTCACTAATGTGCCTCTTCCCACACG CTTTCCACCCGTACTGCTCACAGCTTTAAGAACCAGAACAAATGAGTAATATTAG TGTCGGTTCATGGCTAAAACCAGCACTGATGTACATGACCACATATGTCAAATG CTGCTTCTAGGCATGACCCGCTCTTACTAATACCTACTCATCGCTAGAAGAATTT TCGGCTGATAAATTTTCAATTTAAGCAAGAGTTATCTGCGTTGGTTCATAACTCA AACTGATGGCCCCAACCATATTAGTGCAAATTTCACATATGATCATAACCTTTTC ATATGAAATCGGATCGAGATGAACTTTATATAAACATTGTAGCTGTCGATGATAC CTACAATTTTATAGTTCACAACCTTTTTATTTCAAGTCATTTAAATGCCCAAATAG GTGTTTCAAATCTCAGATAGAAATGTTCAAAAGTAAAAAAGGTCCCTATCATAAC ATAATTGATATGTAAGTGAGTTGGAAAAAGATAAGTACGTGTGAGAGAGATCGG GGATCAAATTCTGGTGTAATAATGTATGTATTTCAGTCATAAAAATTGGTAGCAG TAGTTGGGGCTCTGTATATATACCGGTAAGGATGGGATGGTAGTAGAATAATTC TTTTTTTGTTTTTAGTTTTTTCTGGTCCAAAATTTCAAATTTGGATCCCTTACTTGT ACCAACTAATATTAATGAGTGTTGAGGGTAGTAGAGGTGCAACTTTACCATAATC CCTCTGTTTCAGGTTATAAGACGTTTTGACTTTAAATTTGACCAAGTTTATGCGCA AATATAGTAATATTTATAATACTATATTAGTTTCATTAAATAAATAATTGAATATATT TTCATAATAAATTTGTGTTGAGTTCAAAATATTATTAATTTTTTCTACAAACTTGGT CAAACTTGAAGCAGTTTGACTTTGACCAAAGTCAAAACGTCTTATAACTTGAAAC GGATGGATTACTTTTTTTGTGGGGACAAGTTTACAATGTTTAATAAAGCACAATC CATCTTAATGTTTTCAAGCTGAATATTGTAAAATTCATGGATAAACCAGCTTCTAA ATGTTTAACCGGGAAAATGTCGAACGACAAATTAATATTTTTAAGTGATGGGGAG TATTAATTAAGGAGTGACAACTCAACTTTCAATATCGTACTAAACTGTGGGATTTA TTTTCTAAAATTTTATACCCTGCCAATTCACGTGTTGTAGATCTTTTTTTTTCACTA ACCGACACCAGGTATATCAATTTTATTGAATATAGCAGCAAAAAGAATGTGTTGT ACTTGTAAACAAAAAGCAAACTGTACATAAAAAAAAATGCACTCCTATATAATTAA GCTCATAAAGATGCTTTGCTTCGTGAGGGCCCAAGTTTTGATGACCTTTTGCTTG ATCTCGAAATTAAAATTTAAGTACTGTTAAGGGAGTTCACACCACCATCAATTTTC AGCCTGAAGAAACAGTTAAACAACGACCCCGATGACCAGTCTACTGCTCTCCAC ATACTAGCTGCATTATTGATCACAAAACAAAACAAAACGAAATAAAAATCAGCAG CGAGAGTGTGCAGAGAGAGACAAAGGTGATCTGGCGTGGATATCTCCCCATCC ATCCTCACCCGCGCTGCCCATCACTCGCCGCCGCATACTCCATCATGTGGAGA GAGGAAGACGAGGACCACAGCCAGAGCCCGGGTCGAGATGCCACCACGGCCA CAACCCACGAGCCCGGCGCGACACCACCGCGCGCGCGTGAGCCAGCCACAAA CGCCCGCGGATAGGCGCGCGCACGCCGGCCAATCCTACCACATCCCCGGCCT CCGCGGCTCGCGAGCGCCGCTGCCATCCGATCCGCTGAGTTTTGGCTATTTAT ACGTACCGCGGGAGCCTGTGTGCAGAGCAGTGCATCTCAAGAAGTACTCGAGC AAAGAAGGAGAGAGCTTGGTGAGCTGCAGCCATGGTAGATCTGAGGGTAAATT TCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTG AGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAA ATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATG ATGATGATAGTTACAGAACCGACGAACTAGT SEQ ID NO: 40 Horeum GS1 coding sequence GCGCAGGCGGTTGTGCAGGCGATGCAGTGCCAGGTGGGGGTGAGGGGCAGG ACGGCCGTCCCGGCGAGGCAGCCCGCGGGCAGGGTGTGGGGCGTCAGGAGG GCCGCCCGCGCCACCTCCGGGTTCAAGGTGCTGGCGCTCGGCCCGGAGACCA CCGGGGTCATCCAGAGGATGCAGCAGCTGCTCGACATGGACACCACGCCCTTC ACCGACAAGATCATCGCCGAGTACATCTGGGTTGGAGGATCTGGAATTGACCTC AGAAGCAAATCAAGGACGATTTCGAAGCCAGTGGAGGACCCGTCAGAGCTGCC GAAATGGAACTACGACGGATCGAGCACGGGGCAGGCTCCTGGGGAAGACAGT GAAGTCATCCTATACCCACAGGCCATATTCAAGGACCCATTCCGAGGAGGCAAC

AACATACTGGTTATCTGTGACACCTACACACCACAGGGGGAACCCATCCCTACT AACAAACGCCACATGGCTGCACAAATCTTCAGTGACCCCAAGGTCACTTCACAA GTGCCATGGTTCGGAATCGAACAGGAGTACACTCTGATGCAGAGGGATGTGAA CTGGCCTCTTGGCTGGCCTGTTGGAGGGTACCCTGGCCCCCAGGGTCCATACT ACTGCGCCGTAGGATCAGACAAGTCATTTGGCCGTGACATATCAGATGCTCACT ACAAGGCGTGCCTTTACGCTGGAATTGAAATCAGTGGAACAAACGGGGAGGTC ATGCCTGGTCAGTGGGAGTACCAGGTTGGACCCAGCGTTGGTATTGATGCAGG AGACCACATATGGGCTTCCAGATACATTCTCGAGAGAATCACGGAGCAAGCTGG TGTGGTGCTCACCCTTGACCCAAAACCAATCCAGGGTGACTGGAACGGAGCTG GCTGCCACACAAACTACAGCACATTGAGCATGCGCGAGGATGGAGGTTTCGAC GTGATCAAGAAGGCAATCCTGAACCTTTCACTTCGCCATGACTTGCACATAGCC GCATATGGTGAAGGAAACGAGCGGAGGTTGACAGGGCTACACGAGACAGCTAG CATATCAGACTTCTCATGGGGTGTGGCGAACCGTGGCTGCTCTATTCGTGTGGG GCGAGACACCGAGGCGAAGGGCAAAGGATACCTGGAGGACCGTCGCCCGGCC TCCAACATGGACCCGTACACCGTGACGGCGCTGCTGGCCGAGACCACGATCCT GTGGGAGCCGACCCTCGAGGCGGAGGCCCTCGCTGCCAAGAAGCTGGCGCTG AAGGTATGA SEQ ID NO: 41 Horeum GS1 amino acid sequence AQAVVQAMQCQVGVRGRTAVPARQPAGRVWGVRRAARATSGFKVLALGPETTGV IQRMQQLLDMDTTPFTDKIIAEYIWVGGSGIDLRSKSRTISKPVEDPSELPKWNYDG SSTGQAPGEDSEVILYPQAIFKDPFRGGNNILVICDTYTPQGEPIPTNKRHMAAQIFS DPKVTSQVPWFGIEQEYTLMQRDVNWPLGWPVGGYPGPQGPYYCAVGSDKSFG RDISDAHYKACLYAGIEISGTNGEVMPGQWEYQVGPSVGIDAGDHIWASRYILERIT EQAGVVLTLDPKPIQGDWNGAGCHTNYSTLSMREDGGFDVIKKAILNLSLRHDLHIA AYGEGNERRLTGLHETASISDFSWGVANRGCSIRVGRDTEAKGKGYLEDRRPASN MDPYTVTALLAETTILWEPTLEAEALAAKKLALKV SEQ ID NO: 42: Expression cassette combining SEQ ID NO: 39 (5') and SEQ ID NO: 40 (3'), encoding the Rice rubisco promoter, catI intron and part of Gus plus protein, and hordeum GS1. Features shown as in SEQ ID NO: 39. Hordeum GS1 coding sequence begins after SpeI cloning site (double underline). CTGCAGCAAAGAAACGTTATTAGTTGGTGCTTTTGGTGGTAGGAATGTAGTTTTC TGACAAAGTCAATTACTGAATATAAAAAAAATCTGCACAGCTCTGCGTCAACAGT TGTCCAAGGGATGCCTCAAAAATCTGTGCAGATTATCAGTCGTCACGCAGAAGC AGAACATCATGGTGTGCTAGGTCAGCTTCTTGCATTGGGCCATGAATCCGGTTG GTTGTTAATCTCTCCTCTCTTATTCTCTTATATTAAGATGCATAACTCTTTTATGTA GTCTAAAAAAAAATCCAGTGGATCGGATAGTAGTACGTCATGGTGCCATTAGGT ACCGTTGAACCTAACAGATATTTATGCATGTGTATATATATAGCTATATAGACAAA ATTGATGCCGATTATAGACCCAAAAGCAATAGGTATATATAATATAATACAGACC ACACCACCAAACTAAGAATCGATCAAATAGACAAGGCATGTCTCCAAATTGTCTT AAACTATTTCCGTAGGTTCAGCCGTTCAGGAGTCGAATCAGCCTCTGCCGGCGT TTTCTTTGCACGTACGACGGACACACATGGGCATACCATATAGCTGGTCCATGA CATTAGGAGAGAGAACGTACGTGTTGACCTGTAGCTGAGATATAACAAGGTTGA TTATAATATCACCAAACATGAAATCATCCAAGGATGACCCATAACTATCACTACTA TAGTACTGCATCTGGTAAAAGAAATTGTATAGACTCTATTTCGAGCACTACCACA TAACGCCTGCAATGTGACACCCTACCTATTCACTAATGTGCCTCTTCCCACACG CTTTCCACCCGTACTGCTCACAGCTTTAAGAACCAGAACAAATGAGTAATATTAG TGTCGGTTCATGGCTAAAACCAGCACTGATGTACATGACCACATATGTCAAATG CTGCTTCTAGGCATGACCCGCTCTTACTAATACCTACTCATCGCTAGAAGAATTT TCGGCTGATAAATTTTCAATTTAAGCAAGAGTTATCTGCGTTGGTTCATAACTCA AACTGATGGCCCCAACCATATTAGTGCAAATTTCACATATGATCATAACCTTTTC ATATGAAATCGGATCGAGATGAACTTTATATAAACATTGTAGCTGTCGATGATAC CTACAATTTTATAGTTCACAACCTTTTTATTTCAAGTCATTTAAATGCCCAAATAG GTGTTTCAAATCTCAGATAGAAATGTTCAAAAGTAAAAAAGGTCCCTATCATAAC ATAATTGATATGTAAGTGAGTTGGAAAAAGATAAGTACGTGTGAGAGAGATCGG GGATCAAATTCTGGTGTAATAATGTATGTATTTCAGTCATAAAAATTGGTAGCAG TAGTTGGGGCTCTGTATATATACCGGTAAGGATGGGATGGTAGTAGAATAATTC TTTTTTTGTTTTTAGTTTTTTCTGGTCCAAAATTTCAAATTTGGATCCCTTACTTGT ACCAACTAATATTAATGAGTGTTGAGGGTAGTAGAGGTGCAACTTTACCATAATC CCTCTGTTTCAGGTTATAAGACGTTTTGACTTTAAATTTGACCAAGTTTATGCGCA AATATAGTAATATTTATAATACTATATTAGTTTCATTAAATAAATAATTGAATATATT TTCATAATAAATTTGTGTTGAGTTCAAAATATTATTAATTTTTTCTACAAACTTGGT CAAACTTGAAGCAGTTTGACTTTGACCAAAGTCAAAACGTCTTATAACTTGAAAC GGATGGATTACTTTTTTTGTGGGGACAAGTTTACAATGTTTAATAAAGCACAATC CATCTTAATGTTTTCAAGCTGAATATTGTAAAATTCATGGATAAACCAGCTTCTAA ATGTTTAACCGGGAAAATGTCGAACGACAAATTAATATTTTTAAAGTGATGGGGAG TATTAATTAAGGAGTGACAACTCAACTTTCAATATCGTACTAAACTGTGGGATTTA TTTTCTAAAATTTTATACCCTGCCAATTCACGTGTTGTAGATCTTTTTTTTTCACTA ACCGACACCAGGTATATCAATTTTATTGAATATAGCAGCAAAAAGAATGTGTTGT ACTTGTAAACAAAAAGCAAACTGTACATAAAAAAAAATGCACTCCTATATAATTAA GCTCATAAAGATGCTTTGCTTCGTGAGGGCCCAAGTTTTGATGACCTTTTGCTTG ATCTCGAAATTAAAATTTAAGTACTGTTAAGGGAGTTCACACCACCATCAATTTTC AGCCTGAAGAAACAGTTAAACAACGACCCCGATGACCAGTCTACTGCTCTCCAC ATACTAGCTGCATTATTGATCACAAAACAAAACAAAACGAAATAAAAATCAGCAG CGAGAGTGTGCAGAGAGAGACAAAGGTGATCTGGCGTGGATATCTCCCCATCC ATCCTCACCCGCGCTGCCCATCACTCGCCGCCGCATACTCCATCATGTGGAGA GAGGAAGACGAGGACCACAGCCAGAGCCCGGGTCGAGATGCCACCACGGCCA CAACCCACGAGCCCGGCGCGACACCACCGCGCGCGCGTGAGCCAGCCACAAA CGCCCGCGGATAGGCGCGCGCACGCCGGCCAATCCTACCACATCCCCGGCCT CCGCGGCTCGCGAGCGCCGCTGCCATCCGATCCGCTGAGTTTTGGCTATTTAT ACGTACCGCGGGAGCCTGTGTGCAGAGCAGTGCATCTCAAGAAGTACTCGAGC AAAGAAGGAGAGAGCTTGGTGAGCTGCAGCCATGGTAGATCTGAGGGTAAATTT CTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGA GCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATA TTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGA TGATAGTTACAGAACCGACGAACTAGTGCGCAGGCGGTTGTGCAGGCGATGCA GTGCCAGGTGGGGGTGAGGGGCAGGACGGCCGTCCCGGCGAGGCAGCCCGC GGGCAGGGTGTGGGGCGTCAGGAGGGCCGCCCGCGCCACCTCCGGGTTCAA GGTGCTGGCGCTCGGCCCGGAGACCACCGGGGTCATCCAGAGGATGCAGCAG CTGCTCGACATGGACACCACGCCCTTCACCGACAAGATCATCGCCGAGTACATC TGGGTTGGAGGATCTGGAATTGACCTCAGAAGCAAATCAAGGACGATTTCGAAG CCAGTGGAGGACCCGTCAGAGCTGCCGAAATGGAACTACGACGGATCGAGCAC GGGGCAGGCTCCTGGGGAAGACAGTGAAGTCATCCTATACCCACAGGCCATAT TCAAGGACCCATTCCGAGGAGGCAACAACATACTGGTTATCTGTGACACCTACA CACCACAGGGGGAACCCATCCCTACTAACAAACGCCACATGGCTGCACAAATCT TCAGTGACCCCAAGGTCACTTCACAAGTGCCATGGTTCGGAATCGAACAGGAGT ACACTCTGATGCAGAGGGATGTGAACTGGCCTCTTGGCTGGCCTGTTGGAGGG TACCCTGGCCCCCAGGGTCCATACTACTGCGCCGTAGGATCAGACAAGTCATTT GGCCGTGACATATCAGATGCTCACTACAAGGCGTGCCTTTACGCTGGAATTGAA ATCAGTGGAACAAACGGGGAGGTCATGCCTGGTCAGTGGGAGTACCAGGTTGG ACCCAGCGTTGGTATTGATGCAGGAGACCACATATGGGCTTCCAGATACATTCT CGAGAGAATCACGGAGCAAGCTGGTGTGGTGCTCACCCTTGACCCAAAACCAA TCCAGGGTGACTGGAACGGAGCTGGCTGCCACACAAACTACAGCACATTGAGC ATGCGCGAGGATGGAGGTTTCGACGTGATCAAGAAGGCAATCCTGAACCTTTCA CTTCGCCATGACTTGCACATAGCCGCATATGGTGAAGGAAACGAGCGGAGGTT GACAGGGCTACACGAGACAGCTAGCATATCAGACTTCTCATGGGGTGTGGCGA ACCGTGGCTGCTCTATTCGTGTGGGGCGAGACACCGAGGCGAAGGGCAAAGG ATACCTGGAGGACCGTCGCCCGGCCTCCAACATGGACCCGTACACCGTGACGG CGCTGCTGGCCGAGACCACGATCCTGTGGGAGCCGACCCTCGAGGCGGAGGC CCTCGCTGCCAAGAAGCTGGCGCTGAAGGTATGA SEQ ID NO: 43 Amino acid sequence of translation product of SEQ ID NO: 42. Amino-terminal bold residues from Gusplus and SpeI cloning site (intron removed) MVDLRNRRTSAQAVVQAMQCQVGVRGRTAVPARQPAGRVWGVRRAARATSGFK VLALGPETTGVIQRMQQLLDMDTTPFTDKIIAEYIWVGGSGIDLRSKSRTISKPVEDP SELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRGGNNILVICDTYTPQGEPIPT NKRHMAAQIFSDPKVTSQVPWFGIEQEYTLMQRDVNWPLGWPVGGYPGPQGPYY CAVGSDKSFGRDISDAHYKACLYAGIEISGTNGEVMPGQWEYQVGPSVGIDAGDHI WASRYILERITEQAGVVLTLDPKPIQGDWNGAGCHTNYSTLSMREDGGFDVIKKAIL NLSLRHDLHIAAYGEGNERRLTGLHETASISDFSWGVANRGCSIRVGRDTEAKGKG YLEDRRPASNMDPYTVTALLAETTILWEPTLEAEALAAKKLALKV SEQ ID NO: 44 Maize ubil promoter: 5'UTR intron shown in italics, TATA box at -30 is underlined, 5' and 3' PstI cloning sites in bold CTGCAGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGC ATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTG CAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTA TAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACAT GGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTA GTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCAT CCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAAT

TTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACT CTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGAC TAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTT GTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACA CCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACG GCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGG ACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAG CCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACGGCAGCTACGGGGG ATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGA CACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACA CACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACG CCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGGCGTTCCGGT CCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGT TTGTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAG ACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGG CTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCGTTGCAT AGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTC GGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGG GCGGTCGTTCTAGATCGGAGTAGAATTCTGTTTCAAACTACCTGGTGGATTTATT AATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATG ATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGA TGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGG GCGGTCGTTCATTCGTTCTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGT GTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGT TTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTT ACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTG AGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATA CTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCA TACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTG GTGTTACTTCTGCAG SEQ ID NO: 45 Hordeum GPT DNA coding sequence, including targeting sequence coding domain ATGGCATCCGCCCCCGCCTCCGCCTCCGCGGCCCTCTCCACCGCCGCCCCCG CCGACAACGGGGCCGCCAAGCCCACGGAGCAGCGGCCGGTACAGGTGGCTAA GCGATTGGAGAAGTTCAAAACAACAATTTTCACACAGATGAGCATGCTCGCAGT GAAGCATGGAGCAATAAACCTTGGACAGGGGTTTCCCAATTTTGATGGCCCTGA CTTTGTCAAAGATGCTGCTATTGAGGCTATCAAAGCTGGAAAGAATCAGTATGCA AGAGGATATGGTGTGCCTGAATTGAACTCAGCTGTTGCTGAGAGATTTCTCAAG GACAGTGGATTGCACATCGATCCTGATAAGGAAGTTACTGTTACATCTGGGTGC ACAGAAGCAATAGCTGCAACGATATTGGGTCTGATCAACCCTGGGGATGAAGTC ATACTGTTTGCTCCATTCTATGATTCTTATGAGGCTACACTGTCCATGGCTGGTG CGAATGTCAAAGCCATTACACTCCGCCCTCCGGACTTTGCAGTCCCTCTTGAAG AGCTAAAGGCTGCAGTCTCGAAGAATACCAGAGCAATAATGATTAATACACCTC ACAACCCTACCGGGAAAATGTTCACAAGGGAGGAACTTGAGTTCATTGCTGATC TCTGCAAGGAAAATGACGTGTTGCTCTTTGCCGATGAGGTCTACGACAAGCTGG CGTTTGAGGCGGATCACATATCAATGGCTTCTATTCCTGGCATGTATGAGAGGA CCGTCACTATGAACTCCCTGGGGAAGACGTTCTCCTTGACCGGATGGAAGATC GGCTGGGCGATAGCACCACCGCACCTGACATGGGGCGTAAGGCAGGCACACT CCTTCCTCACATTCGCCACCTCCACGCCGATGCAATCAGCAGCGGCGGCGGCC CTGAGAGCACCGGACAGCTACTTTGAGGAGCTGAAGAGGGACTACGGCGCAAA GAAAGCGCTGCTGGTGGACGGGCTCAAGGCGGCGGGCTTCATCGTCTACCCTT CGAGCGGAACCTACTTCATCATGGTCGACCACACCCCGTTCGGGTTCGACAAC GACGTCGAGTTCTGCGAGTACTTGATCCGCGAGGTCGGCGTCGTGGCCATCCC GCCAAGCGTGTTCTACCTGAACCCGGAGGACGGGAAGAACCTGGTGAGGTTCA CCTTCTGCAAGGACGACGACACGCTAAGGGCGGCGGTGGACAGGATGAAGGC CAAGCTCAGGAAGAAATGA SEQ ID NO: 46: Hordeum GPT amino acid sequence, including putative targeting sequence (in italics). MASAPASASAALSTAAPADNGAAKPTEQRPVQVAKRLEKFKTTIFTQMSMLAVKHG AINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYARGYGVPELNSAVAERFLKDSGLHI DPDKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPP DFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIADLCKENDVLLFADEVY DKLAFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAH SFLTFATSTPMQSAAAAALRAPDSYFEELKRDYGAKKALLVDGLKAAGFIVYPSSGT YFIMVDHTPFGFDNDVEFCEYLIREVGVVAIPPSVFYLNPEDGKNLVRFTFCKDDDT LRAAVDRMKAKLRKK SEQ ID NO: 47 Tomato rubisco small subunit (rbcS3C) promoter + Arabidopsis GS1 DNA coding sequence; NcoI/AflIII splice site shown in bold, ATG start of GS1 underlined. GTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTACTTTGTT GTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGCTTCTG AAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCTTCCAT TCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCTCCTCC TCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTGATATAT CCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGG GTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATG GATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGGAGTA ACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTTAGACA GATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAGGGTAT ACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAGCTCTA AAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCATAGG TAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAATACAC TAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGTAAGA ATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTA TAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGAC TGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATTAT CTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTGTACC ATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTTTCCC CAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATATGGC GCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGGGGT TGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAAATA TTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGTGG GTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAAT ATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGT CAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAAA AAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGAG GGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAAT GGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGGA AGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATTT CAGCACCATGTCTCTGCTCTCAGATCTCGTTAACCTCAACCTCACCGATGCCAC CGGGAAAATCATCGCCGAATACATATGGATCGGTGGATCTGGAATGGATATCAG AAGCAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCAAAGCTTCCCAA GTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGAAGACAGTGAAG TCATTCTATACCCTCAGGCAATATTCAAGGATCCCTTCAGGAAAGGCAACAACAT CCTGGTGATGTGTGATGCTTACACACCAGCTGGTGATCCTATTCCAACCAACAA GAGGCACAACGCTGCTAAGATCTTCAGCCACCCCGACGTTGCCAAGGAGGAGC CTTGGTATGGGATTGAGCAAGAATACACTTTGATGCAAAAGGATGTGAACTGGC CAATTGGTTGGCCTGTTGGTGGCTACCCTGGCCCTCAGGGACCTTACTACTGTG GTGTGGGAGCTGACAAAGCCATTGGTCGTGACATTGTGGATGCTCACTACAAG GCCTGTCTTTACGCCGGTATTGGTATTTCTGGTATCAATGGAGAAGTCATGCCA GGCCAGTGGGAGTTCCAAGTCGGCCCTGTTGAGGGTATTAGTTCTGGTGATCA AGTCTGGGTTGCTCGATACCTTCTCGAGAGGATCACTGAGATCTCTGGTGTAAT TGTCAGCTTCGACCCGAAACCAGTCCCGGGTGACTGGAATGGAGCTGGAGCTC ACTGCAACTACAGCACTAAGACAATGAGAAACGATGGAGGATTAGAAGTGATCA AGAAAGCGATAGGGAAGCTTCAGCTGAAACACAAAGAACACATTGCTGCTTACG GTGAAGGAAACGAGCGTCGTCTCACTGGAAAGCACGAAACCGCAGACATCAAC ACATTCTCTTGGGGAGTCGCGAACCGTGGAGCGTCAGTGAGAGTGGGACGTGA CACAGAGAAGGAAGGTAAAGGGTACTTCGAAGACAGAAGGCCAGCTTCTAACAT GGATCCTTACGTTGTCACCTCCATGATCGCTGAGACGACCATACTCGGTTGA SEQ ID NO: 48: Putative Clementine orange GPT coding sequence Derived from BioChain (Hayward, CA orange cDNA library, cat# C1634340; Derived from clementine PCR primers: 5'-ggccacatgtccgttgctaagtgcttggagaagttta-3' (AflIII oligo) [SEQ ID NO: ] 5'-cgggcacgtgtcattttctcctcagcttctccttcatcct-3' (PmlI oligo) [SEQ ID NO: ] ATG start site in bold, AflIII oligo binding site (start of putative mature coding sequence) is underlined; terminator sequence italicized. ATGCTTAAGCCGTCCGCCTTCGGGTCTTCTTTTTCTTCCTCAGCTCTGCTTTCGT TTTCGAAGCATTTGCATACAATAAGCATTACTGATTCTGTCAACACCAGAAGAAG AGGAATCAGTACCGCTTGCCCTAGGTACCCTTCTCTCATGGCGAGCTTGTCCAC

CGTTTCCACCAATCAAAGCGACACCATCCAGAAGACCAATCTTCAGCCTCAACA GGTTGCTAAGTGCTTGGAGAAGTTTAAAACTACAATCTTTACACAAATGAGTATG CTTGCCATCAAACATGGAGCTATAAATCTTGGTCAAGGCTTTCCCAACTTTGATG GCCCAGATTTTGTTAAAGATGCAGCGATTCAAGCCATAAGGGATGGGAAGAATC AATATGCTCGTGGACATGGGGTTCCAGAGTTCAACTCTGCCATTGCTTCCCGGT TTAAGAAAGATTCTGGGCTCGAGGTTGACCCTGAAAAGGAAGTTACTGTTACCT CTGGGTGCACCGAAGCCATTGCTGCAACCATCTTAGGTTTGATTAATCCTGGAG ATGAGGTGATCCTTTTTGCACCTTTCTATGATTCCTATGAAGCTACTCTCTCCAT GGCTGGTGCTAAAATTAAATGCATCACATTGCGCCCTCCAGAATTTGCCATCCC CATTGAAGAGCTCAAGTCTACAATCTCAAAAAATACTCGTGCAATTCTTATGAAC ACTCCACATAACCCCACTGGAAAGATGTTCACTAGGGAGGAACTTAATGTTATTG CATCTCTTTGCATTGAGAATGATGTGTTGGTTTTTAGTGATGAGGTCTATGATAA GTTGGCTTTTGAAATGGATCACATTTCCATAGCCTCTCTTCCTGGAATGTATGAG CGTACTGTAACCATGAATTCCTTAGGGAAGACATTCTCTTTAACAGGGTGGAAG ATCGGGTGGGCAATAGCTCCACCGCACCTTACATGGGGGGTGCGGCAGGCAC ACTCTTTTCTCACGTTTGCCACATCCACTCCAATGCAGTGGGCAGCTACAGCAG CCCTTAGAGCTCCGGAGACGTACTATGAGGAGCTAAAGAGAGATTACTCGGCAA AGAAGGCAATTTTGGTGGAGGGATTGAATGCTGTTGGTTTCAAGGTATTCCCAT CTAGTGGGACATACTTTGTGGTTGTAGATCACACCCCATTTGGGCACGAAACTG ATATTGCATTTTGTGAATATCTGATCAAGGAAGTTGGGGTTGTGGCAATTCCGAC CAGCGTATTTTACTTGAATCCAGAGGATGGAAAGAATTTGGTGAGATTTACCTTC TGCAAAGATGAAGGAACTTTGAGGTCTGCAGTTGACAGGATGAAGGAGAAGCT GAGGAGAAAATGA SEQ ID NO: 49: Putative Clementine orange GPT amino acid sequence; putative mature protein sequence begins at VAK shown in bold underline. MLKPSAFGSSFSSSALLSFSKHLHTISITDSVNTRRRGISTACPRYPSLMASLSTVST NQSDTIQKTNLQPQQVAKCLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVK DAAIQAIRDGKNQYARGHGVPEFNSAIASRFKKDSGLEVDPEKEVTVTSGCTEAIAA TILGLINPGDEVILFAPFYDSYEATLSMAGAKIKCITLRPPEFAIPIEELKSTISKNTRAIL MNTPHNPTGKMFTREELNVIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYE RTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFATSTPMQWAATAALR APETYYEELKRDYSAKKAILVEGLNAVGFKVFPSSGTYFVVVDHTPFGHETDIAFCE YLIKEVGVVAIPTSVFYLNPEDGKNLVRFTFCKDEGTLRSAVDRMKEKLRRK

Sequence CWU 1

5111323DNAAradopsis thaliana 1atgtacctgg acataaatgg tgtgatgatc aaacagttta gcttcaaagc ctctcttctc 60ccattctctt ctaatttccg acaaagctcc gccaaaatcc atcgtcctat cggagccacc 120atgaccacag tttcgactca gaacgagtct actcaaaaac ccgtccaggt ggcgaagaga 180ttagagaagt tcaagactac tattttcact caaatgagca tattggcagt taaacatgga 240gcgatcaatt taggccaagg ctttcccaat ttcgacggtc ctgattttgt taaagaagct 300gcgatccaag ctattaaaga tggtaaaaac cagtatgctc gtggatacgg cattcctcag 360ctcaactctg ctatagctgc gcggtttcgt gaagatacgg gtcttgttgt tgatcctgag 420aaagaagtta ctgttacatc tggttgcaca gaagccatag ctgcagctat gttgggttta 480ataaaccctg gtgatgaagt cattctcttt gcaccgtttt atgattccta tgaagcaaca 540ctctctatgg ctggtgctaa agtaaaagga atcactttac gtccaccgga cttctccatc 600cctttggaag agcttaaagc tgcggtaact aacaagactc gagccatcct tatgaacact 660ccgcacaacc cgaccgggaa gatgttcact agggaggagc ttgaaaccat tgcatctctc 720tgcattgaaa acgatgtgct tgtgttctcg gatgaagtat acgataagct tgcgtttgaa 780atggatcaca tttctatagc ttctcttccc ggtatgtatg aaagaactgt gaccatgaat 840tccctgggaa agactttctc tttaaccgga tggaagatcg gctgggcgat tgcgccgcct 900catctgactt ggggagttcg acaagcacac tcttacctca cattcgccac atcaacacca 960gcacaatggg cagccgttgc agctctcaag gcaccagagt cttacttcaa agagctgaaa 1020agagattaca atgtgaaaaa ggagactctg gttaagggtt tgaaggaagt cggatttaca 1080gtgttcccat cgagcgggac ttactttgtg gttgctgatc acactccatt tggaatggag 1140aacgatgttg ctttctgtga gtatcttatt gaagaagttg gggtcgttgc gatcccaacg 1200agcgtctttt atctgaatcc agaagaaggg aagaatttgg ttaggtttgc gttctgtaaa 1260gacgaagaga cgttgcgtgg tgcaattgag aggatgaagc agaagcttaa gagaaaagtc 1320tga 13232440PRTAradopsis thaliana 2Met Tyr Leu Asp Ile Asn Gly Val Met Ile Lys Gln Phe Ser Phe Lys1 5 10 15Ala Ser Leu Leu Pro Phe Ser Ser Asn Phe Arg Gln Ser Ser Ala Lys 20 25 30Ile His Arg Pro Ile Gly Ala Thr Met Thr Thr Val Ser Thr Gln Asn 35 40 45Glu Ser Thr Gln Lys Pro Val Gln Val Ala Lys Arg Leu Glu Lys Phe 50 55 60Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu Ala Val Lys His Gly65 70 75 80Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro Asp Phe 85 90 95Val Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp Gly Lys Asn Gln Tyr 100 105 110Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn Ser Ala Ile Ala Ala Arg 115 120 125Phe Arg Glu Asp Thr Gly Leu Val Val Asp Pro Glu Lys Glu Val Thr 130 135 140Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Ala Met Leu Gly Leu145 150 155 160Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp Ser 165 170 175Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Lys Val Lys Gly Ile Thr 180 185 190Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu Glu Leu Lys Ala Ala 195 200 205Val Thr Asn Lys Thr Arg Ala Ile Leu Met Asn Thr Pro His Asn Pro 210 215 220Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu Thr Ile Ala Ser Leu225 230 235 240Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp Glu Val Tyr Asp Lys 245 250 255Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu Pro Gly Met 260 265 270Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser Leu 275 280 285Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr Trp 290 295 300Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe Ala Thr Ser Thr Pro305 310 315 320Ala Gln Trp Ala Ala Val Ala Ala Leu Lys Ala Pro Glu Ser Tyr Phe 325 330 335Lys Glu Leu Lys Arg Asp Tyr Asn Val Lys Lys Glu Thr Leu Val Lys 340 345 350Gly Leu Lys Glu Val Gly Phe Thr Val Phe Pro Ser Ser Gly Thr Tyr 355 360 365Phe Val Val Ala Asp His Thr Pro Phe Gly Met Glu Asn Asp Val Ala 370 375 380Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val Val Ala Ile Pro Thr385 390 395 400Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys Asn Leu Val Arg Phe 405 410 415Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg Gly Ala Ile Glu Arg Met 420 425 430Lys Gln Lys Leu Lys Arg Lys Val 435 44031374DNAMedicago sativa 3atttccgttt tcgttttcat ttgattcatt gaatcaaatc gaatcgaatc tttaggattc 60aatacagatt ccttagattt tactaagttt gaaaccaaaa ccaaaacatg tctctccttt 120cagatcttat caaccttgac ctctccgaaa ccaccgagaa aatcatcgcc gaatacatat 180ggattggtgg atctggtttg gacttgagga gcaaagcaag gactctacca ggaccagtta 240ctgacccttc acagcttccc aagtggaact atgatggttc cagcacaggt caagctcctg 300gagaagatag tgaagttatt atctacccac aagccatttt caaggaccca tttagaaggg 360gtaacaatat cttggttatg tgtgatgcat acactccagc tggagagccc attcccacca 420acaagagaca tgcagctgcc aagattttca gccatcctga tgttgttgct gaagtaccat 480ggtatggtat tgagcaagaa tacaccttgt tgcagaaaga catcaattgg cctcttggtt 540ggccagttgg tggttttcct ggacctcagg gaccatacta ttgtggagct ggtgctgaca 600aggcatttgg ccgtgacatt gttgactcac attacaaagc ctgtctttat gccggcatca 660acatcagtgg aatcaatggt gaagtgatgc ctggtcaatg ggaattccaa gttggtccct 720cagttggtat ctctgctggt gatgagatat gggttgctcg ttacattttg gagaggatca 780ctgaggttgc tggtgtggtg ctttcctttg acccaaaacc aattaagggt gattggaatg 840gtgctggtgc tcacacaaat tacagcacca agtctatgag agaagatggt ggctatgaag 900tcatcttgaa agcaattgag aagcttggga agaagcacaa ggagcacatt gctgcttatg 960gagaaggcaa cgagcgtaga ttgacagggc gacatgagac agctgacatt aacaccttct 1020tatggggtgt tgcaaaccgt ggtgcgtcga ttagagttgg aagggacaca gagaaagcag 1080ggaaaggtta tttcgaggat aggaggccat catctaacat ggatccatat gttgttactt 1140ccatgattgc agacaccacc attctctgga aaccataagc caccacacac acatgcattg 1200aagtatttga aagtcattgt tgattccgca ttagaatttg gtcattgttt tttctaggat 1260ttggatttgt gttattgtta tggttcacac tttgtttgtt tgaatttgag gccttgttat 1320aggtttcata tttctttctc ttgttctaag taaatgtcag aataataatg taat 13744356PRTMedicago sativa 4Met Ser Leu Leu Ser Asp Leu Ile Asn Leu Asp Leu Ser Glu Thr Thr1 5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Leu Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Thr Asp Pro Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Ile Tyr Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Ala Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val Val Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly 130 135 140Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155 160Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Ile Val Asp Ser His Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Val Ala Gly Val Val Leu Ser Phe Asp Pro Lys Pro Ile Lys225 230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu Asp Gly Gly Tyr Glu Val Ile Leu Lys Ala Ile Glu Lys 260 265 270Leu Gly Lys Lys His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys Ala Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ser Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Asp Thr Thr Ile 340 345 350Leu Trp Lys Pro 35551419DNAMedicago sativa 5atcgatgaat tcgagctcgg tacccatttc cgttttcgtt ttcatttgat tcattgaatc 60aaatcgaatc gaatctttag gattcaatac agattcctta gattttacta agtttgaaac 120caaaaccaaa acatgtctct cctttcagat cttatcaacc ttgacctctc cgaaaccacc 180gagaaaatca tcgccgaata catatggatt ggtggatctg gtttggactt gaggagcaaa 240gcaaggactc taccaggacc agttactgac ccttcacagc ttcccaagtg gaactatgat 300ggttccagca caggtcaagc tcctggagaa gatagtgaag ttattatcta cccacaagcc 360attttcaagg acccatttag aaggggtaac aatatcttgg ttatgtgtga tgcatacact 420ccagctggag agcccattcc caccaacaag agacatgcag ctgccaagat tttcagccat 480cctgatgttg ttgctgaagt accatggtat ggtattgagc aagaatacac cttgttgcag 540aaagacatca attggcctct tggttggcca gttggtggtt ttcctggacc tcagggacca 600tactattgtg gagctggtgc tgacaaggca tttggccgtg acattgttga ctcacattac 660aaagcctgtc tttatgccgg catcaacatc agtggaatca atggtgaagt gatgcctggt 720caatgggaat tccaagttgg tccctcagtt ggtatctctg ctggtgatga gatatgggtt 780gctcgttaca ttttggagag gatcactgag gttgctggtg tggtgctttc ctttgaccca 840aaaccaatta agggtgattg gaatggtgct ggtgctcaca caaattacag caccaagtct 900atgagagaag atggtggcta tgaagtcatc ttgaaagcaa ttgagaagct tgggaagaag 960cacaaggagc acattgctgc ttatggagaa ggcaacgagc gtagattgac agggcgacat 1020gagacagctg acattaacac cttcttatgg ggtgttgcaa accgtggtgc gtcgattaga 1080gttggaaggg acacagagaa agcagggaaa ggttatttcg aggataggag gccatcatct 1140aacatggatc catatgttgt tacttccatg attgcagaca ccaccattct ctggaaacca 1200taagccacca cacacacatg cattgaagta tttgaaagtc attgttgatt ccgcattaga 1260atttggtcat tgttttttct aggatttgga tttgtgttat tgttatggtt cacactttgt 1320ttgtttgaat ttgaggcctt gttataggtt tcatatttct ttctcttgtt ctaagtaaat 1380gtcagaataa taatgtaatg gggatcctct agagtcgag 141961302DNAArtificial SequenceSynthetic plasmid vector sequence 6aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240tctctgctct cagatctcgt taacctcaac ctcaccgatg ccaccgggaa aatcatcgcc 300gaatacatat ggatcggtgg atctggaatg gatatcagaa gcaaagccag gacactacca 360ggaccagtga ctgatccatc aaagcttccc aagtggaact acgacggatc cagcaccggt 420caggctgctg gagaagacag tgaagtcatt ctataccctc aggcaatatt caaggatccc 480ttcaggaaag gcaacaacat cctggtgatg tgtgatgctt acacaccagc tggtgatcct 540attccaacca acaagaggca caacgctgct aagatcttca gccaccccga cgttgccaag 600gaggagcctt ggtatgggat tgagcaagaa tacactttga tgcaaaagga tgtgaactgg 660ccaattggtt ggcctgttgg tggctaccct ggccctcagg gaccttacta ctgtggtgtg 720ggagctgaca aagccattgg tcgtgacatt gtggatgctc actacaaggc ctgtctttac 780gccggtattg gtatttctgg tatcaatgga gaagtcatgc caggccagtg ggagttccaa 840gtcggccctg ttgagggtat tagttctggt gatcaagtct gggttgctcg ataccttctc 900gagaggatca ctgagatctc tggtgtaatt gtcagcttcg acccgaaacc agtcccgggt 960gactggaatg gagctggagc tcactgcaac tacagcacta agacaatgag aaacgatgga 1020ggattagaag tgatcaagaa agcgataggg aagcttcagc tgaaacacaa agaacacatt 1080gctgcttacg gtgaaggaaa cgagcgtcgt ctcactggaa agcacgaaac cgcagacatc 1140aacacattct cttggggagt cgcgaaccgt ggagcgtcag tgagagtggg acgtgacaca 1200gagaaggaag gtaaagggta cttcgaagac agaaggccag cttctaacat ggatccttac 1260gttgtcacct ccatgatcgc tgagacgacc atactcggtt ga 13027364PRTArabidopsis thalianaSITE(1)..(9)amino terminal 9 amino acids vector encoded sequence 7Met Val Asp Leu Arg Asn Arg Arg Thr Ser Met Ser Leu Leu Ser Asp1 5 10 15Leu Val Asn Leu Asn Leu Thr Asp Ala Thr Gly Lys Ile Ile Ala Glu 20 25 30Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp Ile Arg Ser Lys Ala Arg 35 40 45Thr Leu Pro Gly Pro Val Thr Asp Pro Ser Lys Leu Pro Lys Trp Asn 50 55 60Tyr Asp Gly Ser Ser Thr Gly Gln Ala Ala Gly Glu Asp Ser Glu Val65 70 75 80Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp Pro Phe Arg Lys Gly Asn 85 90 95Asn Ile Leu Val Met Cys Asp Ala Tyr Thr Pro Ala Gly Asp Pro Ile 100 105 110Pro Thr Asn Lys Arg His Asn Ala Ala Lys Ile Phe Ser His Pro Asp 115 120 125Val Ala Lys Glu Glu Pro Trp Tyr Gly Ile Glu Gln Glu Tyr Thr Leu 130 135 140Met Gln Lys Asp Val Asn Trp Pro Ile Gly Trp Pro Val Gly Gly Tyr145 150 155 160Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala 165 170 175Ile Gly Arg Asp Ile Val Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala 180 185 190Gly Ile Gly Ile Ser Gly Ile Asn Gly Glu Val Met Pro Gly Gln Trp 195 200 205Glu Phe Gln Val Gly Pro Val Glu Gly Ile Ser Ser Gly Asp Gln Val 210 215 220Trp Val Ala Arg Tyr Leu Leu Glu Arg Ile Thr Glu Ile Ser Gly Val225 230 235 240Ile Val Ser Phe Asp Pro Lys Pro Val Pro Gly Asp Trp Asn Gly Ala 245 250 255Gly Ala His Cys Asn Tyr Ser Thr Lys Thr Met Arg Asn Asp Gly Gly 260 265 270Leu Glu Val Ile Lys Lys Ala Ile Gly Lys Leu Gln Leu Lys His Lys 275 280 285Glu His Ile Ala Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly 290 295 300Lys His Glu Thr Ala Asp Ile Asn Thr Phe Ser Trp Gly Val Ala Asn305 310 315 320Arg Gly Ala Ser Val Arg Val Gly Arg Asp Thr Glu Lys Glu Gly Lys 325 330 335Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser Asn Met Asp Pro Tyr Val 340 345 350Val Thr Ser Met Ile Ala Glu Thr Thr Ile Leu Gly 355 36081817DNAArtificial SequenceSynthetic plasmid vector sequence including vitis vinifera GPT coding sequence 8aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac agaaccgacg aactagtatg cagctctctc aatgtacctg 480gacattccca gagttgctta aaagaccagc ctttttaagg aggagtattg atagtatttc 540gagtagaagt aggtccagct ccaagtatcc atctttcatg gcgtccgcat caacggtctc 600cgctccaaat acggaggctg agcagaccca taacccccct caacctctac aggttgcaaa 660gcgcttggag aaattcaaaa caacaatctt tactcaaatg agcatgcttg ccatcaaaca 720tggagcaata aaccttggcc aagggtttcc caactttgat ggtcctgagt ttgtcaaaga 780agcagcaatt caagccatta aggatgggaa aaaccaatat gctcgtggat atggagttcc 840tgatctcaac tctgctgttg ctgatagatt caagaaggat acaggactcg tggtggaccc 900cgagaaggaa gttactgtta cttctggatg tacagaagca attgctgcta ctatgctagg 960cttgataaat cctggtgatg aggtgatcct ctttgctcca ttttatgatt cctatgaagc 1020cactctatcc atggctggtg cccaaataaa atccatcact ttacgtcctc cggattttgc 1080tgtgcccatg gatgagctca agtctgcaat ctcaaagaat acccgtgcaa tccttataaa 1140cactccccat aaccccacag gaaagatgtt cacaagggag gaactgaatg tgattgcatc 1200cctctgcatt gagaatgatg tgttggtgtt tactgatgaa gtttacgaca agttggcttt 1260cgaaatggat cacatttcca tggcttctct tcctgggatg tacgagagga ccgtgactat 1320gaattcctta gggaaaactt tctccctgac tggatggaag attggttgga cagtagctcc 1380cccacacctg acatggggag tgaggcaagc ccactcattc ctcacgtttg ctacctgcac 1440cccaatgcaa tgggcagctg caacagccct ccgggcccca gactcttact atgaagagct 1500aaagagagat tacagtgcaa agaaggcaat cctggtggag ggattgaagg ctgtcggttt 1560cagggtatac ccatcaagtg ggacctattt tgtggtggtg gatcacaccc catttgggtt 1620gaaagacgat attgcgtttt gtgagtatct gatcaaggaa gttggggtgg tagcaattcc 1680gacaagcgtt ttctacttac acccagaaga tggaaagaac cttgtgaggt ttaccttctg 1740taaagacgag ggaactctga gagctgcagt tgaaaggatg aaggagaaac tgaagcctaa 1800acaatagggg cacgtga 18179459PRTVitis vinifera 9Met Val Asp Leu Arg Asn Arg Arg Thr Ser Met Gln Leu Ser Gln Cys1 5 10 15Thr Trp Thr Phe Pro Glu Leu Leu Lys Arg Pro Ala Phe Leu Arg Arg 20 25 30Ser Ile Asp Ser Ile Ser Ser Arg Ser Arg Ser Ser Ser Lys Tyr Pro 35 40

45Ser Phe Met Ala Ser Ala Ser Thr Val Ser Ala Pro Asn Thr Glu Ala 50 55 60Glu Gln Thr His Asn Pro Pro Gln Pro Leu Gln Val Ala Lys Arg Leu65 70 75 80Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile 85 90 95Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly 100 105 110Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp Gly Lys 115 120 125Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp Leu Asn Ser Ala Val 130 135 140Ala Asp Arg Phe Lys Lys Asp Thr Gly Leu Val Val Asp Pro Glu Lys145 150 155 160Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Met 165 170 175Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe 180 185 190Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Gln Ile Lys 195 200 205Ser Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Met Asp Glu Leu 210 215 220Lys Ser Ala Ile Ser Lys Asn Thr Arg Ala Ile Leu Ile Asn Thr Pro225 230 235 240His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Asn Val Ile 245 250 255Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe Thr Asp Glu Val 260 265 270Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile Ser Met Ala Ser Leu 275 280 285Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr 290 295 300Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Thr Val Ala Pro Pro His305 310 315 320Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr 325 330 335Cys Thr Pro Met Gln Trp Ala Ala Ala Thr Ala Leu Arg Ala Pro Asp 340 345 350Ser Tyr Tyr Glu Glu Leu Lys Arg Asp Tyr Ser Ala Lys Lys Ala Ile 355 360 365Leu Val Glu Gly Leu Lys Ala Val Gly Phe Arg Val Tyr Pro Ser Ser 370 375 380Gly Thr Tyr Phe Val Val Val Asp His Thr Pro Phe Gly Leu Lys Asp385 390 395 400Asp Ile Ala Phe Cys Glu Tyr Leu Ile Lys Glu Val Gly Val Val Ala 405 410 415Ile Pro Thr Ser Val Phe Tyr Leu His Pro Glu Asp Gly Lys Asn Leu 420 425 430Val Arg Phe Thr Phe Cys Lys Asp Glu Gly Thr Leu Arg Ala Ala Val 435 440 445Glu Arg Met Lys Glu Lys Leu Lys Pro Lys Gln 450 455101446DNAArtificial SequenceSynthetic DNA encoding Oryza sativa GPT protein, codons optimized for expression in E. coli 10atgtggatga acctggcagg ctttctggca accccggcaa ccgcaaccgc aacccgtcat 60gaaatgccgc tgaacccgag cagcagcgcg agctttctgc tgagcagcct gcgtcgtagc 120ctggtggcga gcctgcgtaa agcgagcccg gcagcagcag cagcactgag cccgatggca 180agcgcaagca ccgtggcagc agaaaacggt gcagcaaaag cagcagcaga aaaacagcag 240cagcagccgg tgcaggtggc gaaacgtctg gaaaaattta aaaccaccat ttttacccag 300atgagcatgc tggcgattaa acatggcgcg attaacctgg gccagggctt tccgaacttt 360gatggcccgg attttgtgaa agaagcggcg attcaggcga ttaacgcggg caaaaaccag 420tatgcgcgtg gctatggcgt gccggaactg aacagcgcga ttgcggaacg ttttctgaaa 480gatagcggcc tgcaggtgga tccggaaaaa gaagtgaccg tgaccagcgg ctgcaccgaa 540gcgattgcgg cgaccattct gggcctgatt aacccgggcg atgaagtgat tctgtttgcg 600ccgttttatg atagctatga agcgaccctg agcatggcgg gcgcgaacgt gaaagcgatt 660accctgcgtc cgccggattt tagcgtgccg ctggaagaac tgaaagcggc cgtgagcaaa 720aacacccgtg cgattatgat taacaccccg cataacccga ccggcaaaat gtttacccgt 780gaagaactgg aatttattgc gaccctgtgc aaagaaaacg atgtgctgct gtttgcggat 840gaagtgtatg ataaactggc gtttgaagcg gatcatatta gcatggcgag cattccgggc 900atgtatgaac gtaccgtgac catgaacagc ctgggcaaaa cctttagcct gaccggctgg 960aaaattggct gggcgattgc gccgccgcat ctgacctggg gcgtgcgtca ggcacatagc 1020tttctgacct ttgcaacctg caccccgatg caggcagccg ccgcagcagc actgcgtgca 1080ccggatagct attatgaaga actgcgtcgt gattatggcg cgaaaaaagc gctgctggtg 1140aacggcctga aagatgcggg ctttattgtg tatccgagca gcggcaccta ttttgtgatg 1200gtggatcata ccccgtttgg ctttgataac gatattgaat tttgcgaata tctgattcgt 1260gaagtgggcg tggtggcgat tccgccgagc gtgttttatc tgaacccgga agatggcaaa 1320aacctggtgc gttttacctt ttgcaaagat gatgaaaccc tgcgtgcggc ggtggaacgt 1380atgaaaacca aactgcgtaa aaaaaagctt gcggccgcac tcgagcacca ccaccaccac 1440cactga 144611481PRTArtificial SequenceOryza sativa GPT protein sequence with amino- and carboxyl-terminal vector sequences 11Met Trp Met Asn Leu Ala Gly Phe Leu Ala Thr Pro Ala Thr Ala Thr1 5 10 15Ala Thr Arg His Glu Met Pro Leu Asn Pro Ser Ser Ser Ala Ser Phe 20 25 30Leu Leu Ser Ser Leu Arg Arg Ser Leu Val Ala Ser Leu Arg Lys Ala 35 40 45Ser Pro Ala Ala Ala Ala Ala Leu Ser Pro Met Ala Ser Ala Ser Thr 50 55 60Val Ala Ala Glu Asn Gly Ala Ala Lys Ala Ala Ala Glu Lys Gln Gln65 70 75 80Gln Gln Pro Val Gln Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr 85 90 95Ile Phe Thr Gln Met Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn 100 105 110Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu 115 120 125Ala Ala Ile Gln Ala Ile Asn Ala Gly Lys Asn Gln Tyr Ala Arg Gly 130 135 140Tyr Gly Val Pro Glu Leu Asn Ser Ala Ile Ala Glu Arg Phe Leu Lys145 150 155 160Asp Ser Gly Leu Gln Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser 165 170 175Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro 180 185 190Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala 195 200 205Thr Leu Ser Met Ala Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro 210 215 220Pro Asp Phe Ser Val Pro Leu Glu Glu Leu Lys Ala Ala Val Ser Lys225 230 235 240Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys 245 250 255Met Phe Thr Arg Glu Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys Glu 260 265 270Asn Asp Val Leu Leu Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe 275 280 285Glu Ala Asp His Ile Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg 290 295 300Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp305 310 315 320Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg 325 330 335Gln Ala His Ser Phe Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ala 340 345 350Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser Tyr Tyr Glu Glu Leu 355 360 365Arg Arg Asp Tyr Gly Ala Lys Lys Ala Leu Leu Val Asn Gly Leu Lys 370 375 380Asp Ala Gly Phe Ile Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met385 390 395 400Val Asp His Thr Pro Phe Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu 405 410 415Tyr Leu Ile Arg Glu Val Gly Val Val Ala Ile Pro Pro Ser Val Phe 420 425 430Tyr Leu Asn Pro Glu Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys 435 440 445Lys Asp Asp Glu Thr Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys 450 455 460Leu Arg Lys Lys Lys Leu Ala Ala Ala Leu Glu His His His His His465 470 475 480His121251DNAArtificial SequenceSynthetic DNA encoding Glycine max GPT protein, codons optimized for expression in E. coli 12atgcatcatc accatcacca tggtaagcct atccctaacc ctctcctcgg tctcgattct 60acggaaaacc tgtattttca gggaattgat cccttcaccg cgaaacgtct ggaaaaattt 120cagaccacca tttttaccca gatgagcctg ctggcgatta aacatggcgc gattaacctg 180ggccagggct ttccgaactt tgatggcccg gaatttgtga aagaagcggc gattcaggcg 240attcgtgatg gcaaaaacca gtatgcgcgt ggctatggcg tgccggatct gaacattgcg 300attgcggaac gttttaaaaa agataccggc ctggtggtgg atccggaaaa agaaattacc 360gtgaccagcg gctgcaccga agcgattgcg gcgaccatga ttggcctgat taacccgggc 420gatgaagtga ttatgtttgc gccgttttat gatagctatg aagcgaccct gagcatggcg 480ggcgcgaaag tgaaaggcat taccctgcgt ccgccggatt ttgcggtgcc gctggaagaa 540ctgaaaagca ccattagcaa aaacacccgt gcgattctga ttaacacccc gcataacccg 600accggcaaaa tgtttacccg tgaagaactg aactgcattg cgagcctgtg cattgaaaac 660gatgtgctgg tgtttaccga tgaagtgtat gataaactgg cgtttgatat ggaacatatt 720agcatggcga gcctgccggg catgtttgaa cgtaccgtga ccctgaacag cctgggcaaa 780acctttagcc tgaccggctg gaaaattggc tgggcgattg cgccgccgca tctgagctgg 840ggcgtgcgtc aggcgcatgc gtttctgacc tttgcaaccg cacatccgtt tcagtgcgca 900gcagcagcag cactgcgtgc accggatagc tattatgtgg aactgaaacg tgattatatg 960gcgaaacgtg cgattctgat tgaaggcctg aaagcggtgg gctttaaagt gtttccgagc 1020agcggcacct attttgtggt ggtggatcat accccgtttg gcctggaaaa cgatgtggcg 1080ttttgcgaat atctggtgaa agaagtgggc gtggtggcga ttccgaccag cgtgttttat 1140ctgaacccgg aagaaggcaa aaacctggtg cgttttacct tttgcaaaga tgaagaaacc 1200attcgtagcg cggtggaacg tatgaaagcg aaactgcgta aagtcgacta a 125113416PRTArtificial SequenceGlycine max GPT amino acid sequence and amino-terminal vector sequence 13Met His His His His His His Gly Lys Pro Ile Pro Asn Pro Leu Leu1 5 10 15Gly Leu Asp Ser Thr Glu Asn Leu Tyr Phe Gln Gly Ile Asp Pro Phe 20 25 30Thr Ala Lys Arg Leu Glu Lys Phe Gln Thr Thr Ile Phe Thr Gln Met 35 40 45Ser Leu Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 50 55 60Pro Asn Phe Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala65 70 75 80Ile Arg Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp 85 90 95Leu Asn Ile Ala Ile Ala Glu Arg Phe Lys Lys Asp Thr Gly Leu Val 100 105 110Val Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr Glu Ala 115 120 125Ile Ala Ala Thr Met Ile Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 130 135 140Met Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala145 150 155 160Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 165 170 175Pro Leu Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg Ala Ile 180 185 190Leu Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 195 200 205Glu Leu Asn Cys Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 210 215 220Phe Thr Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu His Ile225 230 235 240Ser Met Ala Ser Leu Pro Gly Met Phe Glu Arg Thr Val Thr Leu Asn 245 250 255Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala 260 265 270Ile Ala Pro Pro His Leu Ser Trp Gly Val Arg Gln Ala His Ala Phe 275 280 285Leu Thr Phe Ala Thr Ala His Pro Phe Gln Cys Ala Ala Ala Ala Ala 290 295 300Leu Arg Ala Pro Asp Ser Tyr Tyr Val Glu Leu Lys Arg Asp Tyr Met305 310 315 320Ala Lys Arg Ala Ile Leu Ile Glu Gly Leu Lys Ala Val Gly Phe Lys 325 330 335Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro 340 345 350Phe Gly Leu Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu Val Lys Glu 355 360 365Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu 370 375 380Glu Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Glu Thr385 390 395 400Ile Arg Ser Ala Val Glu Arg Met Lys Ala Lys Leu Arg Lys Val Asp 405 410 415141278DNAHordeum vulgare 14atggtagatc tgaggaaccg acgaactagt atggcatccg cccccgcctc cgcctccgcg 60gccctctcca ccgccgcccc cgccgacaac ggggccgcca agcccacgga gcagcggccg 120gtacaggtgg ctaagcgatt ggagaagttc aaaacaacaa ttttcacaca gatgagcatg 180ctcgcagtga agcatggagc aataaacctt ggacaggggt ttcccaattt tgatggccct 240gactttgtca aagatgctgc tattgaggct atcaaagctg gaaagaatca gtatgcaaga 300ggatatggtg tgcctgaatt gaactcagct gttgctgaga gatttctcaa ggacagtgga 360ttgcacatcg atcctgataa ggaagttact gttacatctg ggtgcacaga agcaatagct 420gcaacgatat tgggtctgat caaccctggg gatgaagtca tactgtttgc tccattctat 480gattcttatg aggctacact gtccatggct ggtgcgaatg tcaaagccat tacactccgc 540cctccggact ttgcagtccc tcttgaagag ctaaaggctg cagtctcgaa gaataccaga 600gcaataatga ttaatacacc tcacaaccct accgggaaaa tgttcacaag ggaggaactt 660gagttcattg ctgatctctg caaggaaaat gacgtgttgc tctttgccga tgaggtctac 720gacaagctgg cgtttgaggc ggatcacata tcaatggctt ctattcctgg catgtatgag 780aggaccgtca ctatgaactc cctggggaag acgttctcct tgaccggatg gaagatcggc 840tgggcgatag caccaccgca cctgacatgg ggcgtaaggc aggcacactc cttcctcaca 900ttcgccacct ccacgccgat gcaatcagca gcggcggcgg ccctgagagc accggacagc 960tactttgagg agctgaagag ggactacggc gcaaagaaag cgctgctggt ggacgggctc 1020aaggcggcgg gcttcatcgt ctacccttcg agcggaacct acttcatcat ggtcgaccac 1080accccgttcg ggttcgacaa cgacgtcgag ttctgcgagt acttgatccg cgaggtcggc 1140gtcgtggcca tcccgccaag cgtgttctac ctgaacccgg aggacgggaa gaacctggtg 1200aggttcacct tctgcaagga cgacgacacg ctaagggcgg cggtggacag gatgaaggcc 1260aagctcagga agaaatga 127815425PRTHordeum vulgare 15Met Val Asp Leu Arg Asn Arg Arg Thr Ser Met Ala Ser Ala Pro Ala1 5 10 15Ser Ala Ser Ala Ala Leu Ser Thr Ala Ala Pro Ala Asp Asn Gly Ala 20 25 30Ala Lys Pro Thr Glu Gln Arg Pro Val Gln Val Ala Lys Arg Leu Glu 35 40 45Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Val Lys 50 55 60His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro65 70 75 80Asp Phe Val Lys Asp Ala Ala Ile Glu Ala Ile Lys Ala Gly Lys Asn 85 90 95Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu Leu Asn Ser Ala Val Ala 100 105 110Glu Arg Phe Leu Lys Asp Ser Gly Leu His Ile Asp Pro Asp Lys Glu 115 120 125Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile Leu 130 135 140Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr145 150 155 160Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Asn Val Lys Ala 165 170 175Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Leu Glu Glu Leu Lys 180 185 190Ala Ala Val Ser Lys Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His 195 200 205Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu Phe Ile Ala 210 215 220Asp Leu Cys Lys Glu Asn Asp Val Leu Leu Phe Ala Asp Glu Val Tyr225 230 235 240Asp Lys Leu Ala Phe Glu Ala Asp His Ile Ser Met Ala Ser Ile Pro 245 250 255Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe 260 265 270Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu 275 280 285Thr Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr Ser 290 295 300Thr Pro Met Gln Ser Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser305 310 315 320Tyr Phe Glu Glu Leu Lys Arg Asp Tyr Gly Ala Lys Lys Ala Leu Leu 325 330 335Val Asp Gly Leu Lys Ala Ala Gly Phe Ile Val Tyr Pro Ser Ser Gly 340 345 350Thr Tyr Phe Ile Met Val Asp His Thr Pro Phe Gly Phe Asp Asn Asp 355 360 365Val Glu Phe Cys Glu Tyr Leu Ile Arg Glu Val Gly Val Val Ala Ile 370 375 380Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu Asp Gly Lys Asn Leu Val385 390 395 400Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr Leu Arg Ala Ala Val Asp 405 410 415Arg Met Lys Ala Lys

Leu Arg Lys Lys 420 425161200DNAArtificial SequenceSynthetic DNA encoding Danio rerio GPT protein, codons optimized for expression in E. coli, including 5' and 3' vector sequences 16atgtccgtgg cgaaacgtct ggaaaaattt aaaaccacca tttttaccca gatgagcatg 60ctggcgatta aacatggcgc gattaacctg ggccagggct ttccgaactt tgatggcccg 120gattttgtga aagaagcggc gattcaggcg attcgtgatg gcaacaacca gtatgcgcgt 180ggctatggcg tgccggatct gaacattgcg attagcgaac gttataaaaa agataccggc 240ctggcggtgg atccggaaaa agaaattacc gtgaccagcg gctgcaccga agcgattgcg 300gcgaccgtgc tgggcctgat taacccgggc gatgaagtga ttgtgtttgc gccgttttat 360gatagctatg aagcgaccct gagcatggcg ggcgcgaaag tgaaaggcat taccctgcgt 420ccgccggatt ttgcgctgcc gattgaagaa ctgaaaagca ccattagcaa aaacacccgt 480gcgattctgc tgaacacccc gcataacccg accggcaaaa tgtttacccc ggaagaactg 540aacaccattg cgagcctgtg cattgaaaac gatgtgctgg tgtttagcga tgaagtgtat 600gataaactgg cgtttgatat ggaacatatt agcattgcga gcctgccggg catgtttgaa 660cgtaccgtga ccatgaacag cctgggcaaa acctttagcc tgaccggctg gaaaattggc 720tgggcgattg cgccgccgca tctgacctgg ggcgtgcgtc aggcgcatgc gtttctgacc 780tttgcaacca gcaacccgat gcagtgggca gcagcagtgg cactgcgtgc accggatagc 840tattataccg aactgaaacg tgattatatg gcgaaacgta gcattctggt ggaaggcctg 900aaagcggtgg gctttaaagt gtttccgagc agcggcacct attttgtggt ggtggatcat 960accccgtttg gccatgaaaa cgatattgcg ttttgcgaat atctggtgaa agaagtgggc 1020gtggtggcga ttccgaccag cgtgttttat ctgaacccgg aagaaggcaa aaacctggtg 1080cgttttacct tttgcaaaga tgaaggcacc ctgcgtgcgg cggtggatcg tatgaaagaa 1140aaactgcgta aagtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga 120017399PRTDanio rerioMISC_FEATURE(1)..(399)Amino- and carboxy-terminal amino acids shown 17Met Ser Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr1 5 10 15Gln Met Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln 20 25 30Gly Phe Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile 35 40 45Gln Ala Ile Arg Asp Gly Asn Asn Gln Tyr Ala Arg Gly Tyr Gly Val 50 55 60Pro Asp Leu Asn Ile Ala Ile Ser Glu Arg Tyr Lys Lys Asp Thr Gly65 70 75 80Leu Ala Val Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr 85 90 95Glu Ala Ile Ala Ala Thr Val Leu Gly Leu Ile Asn Pro Gly Asp Glu 100 105 110Val Ile Val Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser 115 120 125Met Ala Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe 130 135 140Ala Leu Pro Ile Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg145 150 155 160Ala Ile Leu Leu Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr 165 170 175Pro Glu Glu Leu Asn Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val 180 185 190Leu Val Phe Ser Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu 195 200 205His Ile Ser Ile Ala Ser Leu Pro Gly Met Phe Glu Arg Thr Val Thr 210 215 220Met Asn Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly225 230 235 240Trp Ala Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His 245 250 255Ala Phe Leu Thr Phe Ala Thr Ser Asn Pro Met Gln Trp Ala Ala Ala 260 265 270Val Ala Leu Arg Ala Pro Asp Ser Tyr Tyr Thr Glu Leu Lys Arg Asp 275 280 285Tyr Met Ala Lys Arg Ser Ile Leu Val Glu Gly Leu Lys Ala Val Gly 290 295 300Phe Lys Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His305 310 315 320Thr Pro Phe Gly His Glu Asn Asp Ile Ala Phe Cys Glu Tyr Leu Val 325 330 335Lys Glu Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn 340 345 350Pro Glu Glu Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu 355 360 365Gly Thr Leu Arg Ala Ala Val Asp Arg Met Lys Glu Lys Leu Arg Lys 370 375 380Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His385 390 395181236DNAArabidopsis thaliana 18atggccaaaa tccatcgtcc tatcggagcc accatgacca cagtttcgac tcagaacgag 60tctactcaaa aacccgtcca ggtggcgaag agattagaga agttcaagac tactattttc 120actcaaatga gcatattggc agttaaacat ggagcgatca atttaggcca aggctttccc 180aatttcgacg gtcctgattt tgttaaagaa gctgcgatcc aagctattaa agatggtaaa 240aaccagtatg ctcgtggata cggcattcct cagctcaact ctgctatagc tgcgcggttt 300cgtgaagata cgggtcttgt tgttgatcct gagaaagaag ttactgttac atctggttgc 360acagaagcca tagctgcagc tatgttgggt ttaataaacc ctggtgatga agtcattctc 420tttgcaccgt tttatgattc ctatgaagca acactctcta tggctggtgc taaagtaaaa 480ggaatcactt tacgtccacc ggacttctcc atccctttgg aagagcttaa agctgcggta 540actaacaaga ctcgagccat ccttatgaac actccgcaca acccgaccgg gaagatgttc 600actagggagg agcttgaaac cattgcatct ctctgcattg aaaacgatgt gcttgtgttc 660tcggatgaag tatacgataa gcttgcgttt gaaatggatc acatttctat agcttctctt 720cccggtatgt atgaaagaac tgtgaccatg aattccctgg gaaagacttt ctctttaacc 780ggatggaaga tcggctgggc gattgcgccg cctcatctga cttggggagt tcgacaagca 840cactcttacc tcacattcgc cacatcaaca ccagcacaat gggcagccgt tgcagctctc 900aaggcaccag agtcttactt caaagagctg aaaagagatt acaatgtgaa aaaggagact 960ctggttaagg gtttgaagga agtcggattt acagtgttcc catcgagcgg gacttacttt 1020gtggttgctg atcacactcc atttggaatg gagaacgatg ttgctttctg tgagtatctt 1080attgaagaag ttggggtcgt tgcgatccca acgagcgtct tttatctgaa tccagaagaa 1140gggaagaatt tggttaggtt tgcgttctgt aaagacgaag agacgttgcg tggtgcaatt 1200gagaggatga agcagaagct taagagaaaa gtctga 123619411PRTArabidopsis thaliana 19Met Ala Lys Ile His Arg Pro Ile Gly Ala Thr Met Thr Thr Val Ser1 5 10 15Thr Gln Asn Glu Ser Thr Gln Lys Pro Val Gln Val Ala Lys Arg Leu 20 25 30Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu Ala Val 35 40 45Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly 50 55 60Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp Gly Lys65 70 75 80Asn Gln Tyr Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn Ser Ala Ile 85 90 95Ala Ala Arg Phe Arg Glu Asp Thr Gly Leu Val Val Asp Pro Glu Lys 100 105 110Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Ala Met 115 120 125Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe 130 135 140Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Lys Val Lys145 150 155 160Gly Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu Glu Leu 165 170 175Lys Ala Ala Val Thr Asn Lys Thr Arg Ala Ile Leu Met Asn Thr Pro 180 185 190His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu Thr Ile 195 200 205Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp Glu Val 210 215 220Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu225 230 235 240Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr 245 250 255Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His 260 265 270Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe Ala Thr 275 280 285Ser Thr Pro Ala Gln Trp Ala Ala Val Ala Ala Leu Lys Ala Pro Glu 290 295 300Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr Asn Val Lys Lys Glu Thr305 310 315 320Leu Val Lys Gly Leu Lys Glu Val Gly Phe Thr Val Phe Pro Ser Ser 325 330 335Gly Thr Tyr Phe Val Val Ala Asp His Thr Pro Phe Gly Met Glu Asn 340 345 350Asp Val Ala Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val Val Ala 355 360 365Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys Asn Leu 370 375 380Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg Gly Ala Ile385 390 395 400Glu Arg Met Lys Gln Lys Leu Lys Arg Lys Val 405 410201194DNAArabidopsis thaliana 20atggcgactc agaacgagtc tactcaaaaa cccgtccagg tggcgaagag attagagaag 60ttcaagacta ctattttcac tcaaatgagc atattggcag ttaaacatgg agcgatcaat 120ttaggccaag gctttcccaa tttcgacggt cctgattttg ttaaagaagc tgcgatccaa 180gctattaaag atggtaaaaa ccagtatgct cgtggatacg gcattcctca gctcaactct 240gctatagctg cgcggtttcg tgaagatacg ggtcttgttg ttgatcctga gaaagaagtt 300actgttacat ctggttgcac agaagccata gctgcagcta tgttgggttt aataaaccct 360ggtgatgaag tcattctctt tgcaccgttt tatgattcct atgaagcaac actctctatg 420gctggtgcta aagtaaaagg aatcacttta cgtccaccgg acttctccat ccctttggaa 480gagcttaaag ctgcggtaac taacaagact cgagccatcc ttatgaacac tccgcacaac 540ccgaccggga agatgttcac tagggaggag cttgaaacca ttgcatctct ctgcattgaa 600aacgatgtgc ttgtgttctc ggatgaagta tacgataagc ttgcgtttga aatggatcac 660atttctatag cttctcttcc cggtatgtat gaaagaactg tgaccatgaa ttccctggga 720aagactttct ctttaaccgg atggaagatc ggctgggcga ttgcgccgcc tcatctgact 780tggggagttc gacaagcaca ctcttacctc acattcgcca catcaacacc agcacaatgg 840gcagccgttg cagctctcaa ggcaccagag tcttacttca aagagctgaa aagagattac 900aatgtgaaaa aggagactct ggttaagggt ttgaaggaag tcggatttac agtgttccca 960tcgagcggga cttactttgt ggttgctgat cacactccat ttggaatgga gaacgatgtt 1020gctttctgtg agtatcttat tgaagaagtt ggggtcgttg cgatcccaac gagcgtcttt 1080tatctgaatc cagaagaagg gaagaatttg gttaggtttg cgttctgtaa agacgaagag 1140acgttgcgtg gtgcaattga gaggatgaag cagaagctta agagaaaagt ctga 119421397PRTArabidopsis thaliana 21Met Ala Thr Gln Asn Glu Ser Thr Gln Lys Pro Val Gln Val Ala Lys1 5 10 15Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu 20 25 30Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe 35 40 45Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp 50 55 60Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn Ser65 70 75 80Ala Ile Ala Ala Arg Phe Arg Glu Asp Thr Gly Leu Val Val Asp Pro 85 90 95Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala 100 105 110Ala Met Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala 115 120 125Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Lys 130 135 140Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu145 150 155 160Glu Leu Lys Ala Ala Val Thr Asn Lys Thr Arg Ala Ile Leu Met Asn 165 170 175Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu 180 185 190Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp 195 200 205Glu Val Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala 210 215 220Ser Leu Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly225 230 235 240Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro 245 250 255Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe 260 265 270Ala Thr Ser Thr Pro Ala Gln Trp Ala Ala Val Ala Ala Leu Lys Ala 275 280 285Pro Glu Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr Asn Val Lys Lys 290 295 300Glu Thr Leu Val Lys Gly Leu Lys Glu Val Gly Phe Thr Val Phe Pro305 310 315 320Ser Ser Gly Thr Tyr Phe Val Val Ala Asp His Thr Pro Phe Gly Met 325 330 335Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val 340 345 350Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys 355 360 365Asn Leu Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg Gly 370 375 380Ala Ile Glu Arg Met Lys Gln Lys Leu Lys Arg Lys Val385 390 395221680DNALycopersicon esculentum 22ggtaccgttt gaatcctcct taaagttttt ctctggagaa actgtagtaa ttttactttg 60ttgtgttccc ttcatctttt gaattaatgg catttgtttt aatactaatc tgcttctgaa 120acttgtaatg tatgtatatc agtttcttat aatttatcca agtaatatct tccattctct 180atgcaattgc ctgcataagc tcgacaaaag agtacatcaa cccctcctcc tctggactac 240tctagctaaa cttgaatttc cccttaagat tatgaaattg atatatcctt aacaaacgac 300tccttctgtt ggaaaatgta gtacttgtct ttcttctttt gggtatatat agtttatata 360caccatacta tgtacaacat ccaagtagag tgaaatggat acatgtacaa gacttatttg 420attgattgat gacttgagtt gccttaggag taacaaattc ttaggtcaat aaatcgttga 480tttgaaatta atctctctgt cttagacaga taggaattat gacttccaat ggtccagaaa 540gcaaagttcg cactgagggt atacttggaa ttgagacttg cacaggtcca gaaaccaaag 600ttcccatcga gctctaaaat cacatctttg gaatgaaatt caattagaga taagttgctt 660catagcatag gtaaaatgga agatgtgaag taacctgcaa taatcagtga aatgacatta 720atacactaaa tacttcatat gtaattatcc tttccaggtt aacaatactc tataaagtaa 780gaattatcag aaatgggctc atcaaacttt tgtactatgt atttcatata aggaagtata 840actatacata agtgtataca caactttatt cctattttgt aaaggtggag agactgtttt 900cgatggatct aaagcaatat gtctataaaa tgcattgata taataattat ctgagaaaat 960ccagaattgg cgttggatta tttcagccaa atagaagttt gtaccatact tgttgattcc 1020ttctaagtta aggtgaagta tcattcataa acagttttcc ccaaagtact actcaccaag 1080tttccctttg tagaattaac agttcaaata tatggcgcag aaattactct atgcccaaaa 1140ccaaacgaga aagaaacaaa atacaggggt tgcagacttt attttcgtgt tagggtgtgt 1200tttttcatgt aattaatcaa aaaatattat gacaaaaaca tttatacata tttttactca 1260acactctggg tatcagggtg ggttgtgttc gacaatcaat atggaaagga agtattttcc 1320ttattttttt agttaatatt ttcagttata ccaaacatac cttgtgatat tatttttaaa 1380aatgaaaaac tcgtcagaaa gaaaaagcaa aagcaacaaa aaaattgcaa gtatttttta 1440aaaaagaaaa aaaaaacata tcttgtttgt cagtatggga agtttgagat aaggacgagt 1500gaggggttaa aattcagtgg ccattgattt tgtaatgcca agaaccacaa aatccaatgg 1560ttaccattcc tgtaagatga ggtttgctaa ctctttttgt ccgttagata ggaagcctta 1620tcactatata tacaaggcgt cctaataacc tcttagtaac caattatttc agcaccatgg 1680231230DNAPhyllostachys bambusoides 23atggcctccg cggccgtctc caccgtcgcc accgccgccg acggcgtcgc gaagccgacg 60gagaagcagc cggtacaggt cgcaaagcgt ttggaaaagt ttaagacaac aattttcaca 120cagatgagca tgcttgccat caagcatgga gcaataaacc tcggccaggg ctttccgaat 180tttgatggcc ctgactttgt gaaagaagct gctattcaag ctatcaatgc tgggaagaat 240cagtatgcaa gaggatatgg tgtgcctgaa ctgaactcgg ctgttgctga aaggttcctg 300aaggacagtg gcttgcaagt cgatcccgag aaggaagtta ctgtcacatc tgggtgcacg 360gaagcgatag ctgcaacgat attgggtctt atcaaccctg gcgatgaagt gatcttgttt 420gctccattct atgattcata cgaggctacg ctgtcgatgg ctggtgccaa tgtaaaagcc 480attactctcc gtcctccaga ttttgcagtc cctcttgagg agctaaaggc cacagtctct 540aagaacacca gagcgataat gataaacaca ccacacaatc ctactgggaa aatgttttct 600agggaagaac ttgaattcat tgctactctc tgcaagaaaa atgatgtgtt gctttttgct 660gatgaggtct atgacaagtt ggcatttgag gcagatcata tatcaatggc ttctattcct 720ggcatgtatg agaggactgt gactatgaac tctctgggga agacattctc tctaacagga 780tggaagatcg gttgggcaat agcaccacca cacctgacat ggggtgtaag gcaggcacac 840tcattcctca catttgccac ctgcacacca atgcaatcgg cggcggcggc ggctcttaga 900gcaccagata gctactatgg ggagctgaag agggattacg gtgcaaagaa agcgatacta 960gtcgacggac tcaaggctgc aggttttatt gtttaccctt caagtggaac atactttgtc 1020atggtcgatc acaccccgtt tggtttcgac aatgatattg agttctgcga gtatttgatc 1080cgcgaagtcg gtgttgtcgc cataccacca agcgtatttt atctcaaccc tgaggatggg 1140aagaacttgg tgaggttcac cttctgcaag gatgatgata cgctgagagc cgcagttgag 1200aggatgaaga caaagctcag gaaaaaatga 123024409PRTPhyllostachys bambusoides 24Met Ala Ser Ala Ala Val Ser Thr Val Ala Thr Ala Ala Asp Gly Val1 5 10 15Ala Lys Pro Thr Glu Lys Gln Pro Val Gln Val Ala Lys Arg Leu Glu 20 25 30Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile Lys 35 40 45His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro 50 55 60Asp Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Asn Ala Gly Lys Asn65 70 75 80Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu Leu Asn Ser Ala Val Ala 85 90 95Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln Val Asp Pro

Glu Lys Glu 100 105 110Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile Leu 115 120 125Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr 130 135 140Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Asn Val Lys Ala145 150 155 160Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Leu Glu Glu Leu Lys 165 170 175Ala Thr Val Ser Lys Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His 180 185 190Asn Pro Thr Gly Lys Met Phe Ser Arg Glu Glu Leu Glu Phe Ile Ala 195 200 205Thr Leu Cys Lys Lys Asn Asp Val Leu Leu Phe Ala Asp Glu Val Tyr 210 215 220Asp Lys Leu Ala Phe Glu Ala Asp His Ile Ser Met Ala Ser Ile Pro225 230 235 240Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe 245 250 255Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu 260 265 270Thr Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr Cys 275 280 285Thr Pro Met Gln Ser Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser 290 295 300Tyr Tyr Gly Glu Leu Lys Arg Asp Tyr Gly Ala Lys Lys Ala Ile Leu305 310 315 320Val Asp Gly Leu Lys Ala Ala Gly Phe Ile Val Tyr Pro Ser Ser Gly 325 330 335Thr Tyr Phe Val Met Val Asp His Thr Pro Phe Gly Phe Asp Asn Asp 340 345 350Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu Val Gly Val Val Ala Ile 355 360 365Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu Asp Gly Lys Asn Leu Val 370 375 380Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr Leu Arg Ala Ala Val Glu385 390 395 400Arg Met Lys Thr Lys Leu Arg Lys Lys 405251858DNAOryza sativa 25aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac agaaccgacg aactagtatg aatctggccg gctttctcgc 480cacgcccgcg accgcgaccg cgacgcggca tgagatgccg ttaaatccct cctcctccgc 540ctccttcctc ctctcctcgc tccgccgctc gctcgtcgcg tcgctccgga aggcctcgcc 600ggcggcggcc gcggcgctct cccccatggc ctccgcgtcc accgtcgccg ccgagaacgg 660cgccgccaag gcggcggcgg agaagcagca gcagcagcct gtgcaggttg caaagcggtt 720ggaaaagttt aagacgacca ttttcacaca gatgagtatg cttgccatca agcatggagc 780aataaacctt ggccagggtt ttccgaattt cgatggccct gactttgtaa aagaggctgc 840tattcaagct atcaatgctg ggaagaatca gtacgcaaga ggatatggtg tgcctgaact 900gaactcagct attgctgaaa gattcctgaa ggacagcgga ctgcaagtcg atccggagaa 960ggaagttact gtcacatctg gatgcacaga agctatagct gcaacaattt taggtctaat 1020taatccaggc gatgaagtga tattgtttgc tccattctat gattcatatg aggctaccct 1080gtcaatggct ggtgccaacg taaaagccat tactctccgt cctccagatt tttcagtccc 1140tcttgaagag ctaaaggctg cagtctcgaa gaacaccaga gctattatga taaacacccc 1200gcacaatcct actgggaaaa tgtttacaag ggaagaactt gagtttattg ccactctctg 1260caaggaaaat gatgtgctgc tttttgctga tgaggtctac gacaagttag cttttgaggc 1320agatcatata tcaatggctt ctattcctgg catgtatgag aggaccgtga ccatgaactc 1380tcttgggaag acattctctc ttacaggatg gaagatcggt tgggcaatcg caccgccaca 1440cctgacatgg ggtgtaaggc aggcacactc attcctcacg tttgcgacct gcacaccaat 1500gcaagcagct gcagctgcag ctctgagagc accagatagc tactatgagg aactgaggag 1560ggattatgga gctaagaagg cattgctagt caacggactc aaggatgcag gtttcattgt 1620ctatccttca agtggaacat acttcgtcat ggtcgaccac accccatttg gtttcgacaa 1680tgatattgag ttctgcgagt atttgattcg cgaagtcggt gttgtcgcca taccacctag 1740tgtattttat ctcaaccctg aggatgggaa gaacttggtg aggttcacct tttgcaagga 1800tgatgagacg ctgagagccg cggttgagag gatgaagaca aagctcagga aaaaatga 1858261724DNAArtificial SequenceSynthetic DNA encoding Hordeum vulgare GPT protein 26aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac agaaccgacg aactagtatg gcatccgccc ccgcctccgc 480ctccgcggcc ctctccaccg ccgcccccgc cgacaacggg gccgccaagc ccacggagca 540gcggccggta caggtggcta agcgattgga gaagttcaaa acaacaattt tcacacagat 600gagcatgctc gcagtgaagc atggagcaat aaaccttgga caggggtttc ccaattttga 660tggccctgac tttgtcaaag atgctgctat tgaggctatc aaagctggaa agaatcagta 720tgcaagagga tatggtgtgc ctgaattgaa ctcagctgtt gctgagagat ttctcaagga 780cagtggattg cacatcgatc ctgataagga agttactgtt acatctgggt gcacagaagc 840aatagctgca acgatattgg gtctgatcaa ccctggggat gaagtcatac tgtttgctcc 900attctatgat tcttatgagg ctacactgtc catggctggt gcgaatgtca aagccattac 960actccgccct ccggactttg cagtccctct tgaagagcta aaggctgcag tctcgaagaa 1020taccagagca ataatgatta atacacctca caaccctacc gggaaaatgt tcacaaggga 1080ggaacttgag ttcattgctg atctctgcaa ggaaaatgac gtgttgctct ttgccgatga 1140ggtctacgac aagctggcgt ttgaggcgga tcacatatca atggcttcta ttcctggcat 1200gtatgagagg accgtcacta tgaactccct ggggaagacg ttctccttga ccggatggaa 1260gatcggctgg gcgatagcac caccgcacct gacatggggc gtaaggcagg cacactcctt 1320cctcacattc gccacctcca cgccgatgca atcagcagcg gcggcggccc tgagagcacc 1380ggacagctac tttgaggagc tgaagaggga ctacggcgca aagaaagcgc tgctggtgga 1440cgggctcaag gcggcgggct tcatcgtcta cccttcgagc ggaacctact tcatcatggt 1500cgaccacacc ccgttcgggt tcgacaacga cgtcgagttc tgcgagtact tgatccgcga 1560ggtcggcgtc gtggccatcc cgccaagcgt gttctacctg aacccggagg acgggaagaa 1620cctggtgagg ttcaccttct gcaaggacga cgacacgcta agggcggcgg tggacaggat 1680gaaggccaag ctcaggaaga aatgattgag gggcgcacgt gtga 1724271868DNAArtificial SequenceSynthetic DNA encoding Arabidopsis thaliana GPT protein 27catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 60gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 120gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 300ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 360acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 480gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggactct 540tgaccatgta cctggacata aatggtgtga tgatcaaaca gtttagcttc aaagcctctc 600ttctcccatt ctcttctaat ttccgacaaa gctccgccaa aatccatcgt cctatcggag 660ccaccatgac cacagtttcg actcagaacg agtctactca aaaacccgtc caggtggcga 720agagattaga gaagttcaag actactattt tcactcaaat gagcatattg gcagttaaac 780atggagcgat caatttaggc caaggctttc ccaatttcga cggtcctgat tttgttaaag 840aagctgcgat ccaagctatt aaagatggta aaaaccagta tgctcgtgga tacggcattc 900ctcagctcaa ctctgctata gctgcgcggt ttcgtgaaga tacgggtctt gttgttgatc 960ctgagaaaga agttactgtt acatctggtt gcacagaagc catagctgca gctatgttgg 1020gtttaataaa ccctggtgat gaagtcattc tctttgcacc gttttatgat tcctatgaag 1080caacactctc tatggctggt gctaaagtaa aaggaatcac tttacgtcca ccggacttct 1140ccatcccttt ggaagagctt aaagctgcgg taactaacaa gactcgagcc atccttatga 1200acactccgca caacccgacc gggaagatgt tcactaggga ggagcttgaa accattgcat 1260ctctctgcat tgaaaacgat gtgcttgtgt tctcggatga agtatacgat aagcttgcgt 1320ttgaaatgga tcacatttct atagcttctc ttcccggtat gtatgaaaga actgtgacca 1380tgaattccct gggaaagact ttctctttaa ccggatggaa gatcggctgg gcgattgcgc 1440cgcctcatct gacttgggga gttcgacaag cacactctta cctcacattc gccacatcaa 1500caccagcaca atgggcagcc gttgcagctc tcaaggcacc agagtcttac ttcaaagagc 1560tgaaaagaga ttacaatgtg aaaaaggaga ctctggttaa gggtttgaag gaagtcggat 1620ttacagtgtt cccatcgagc gggacttact ttgtggttgc tgatcacact ccatttggaa 1680tggagaacga tgttgctttc tgtgagtatc ttattgaaga agttggggtc gttgcgatcc 1740caacgagcgt cttttatctg aatccagaag aagggaagaa tttggttagg tttgcgttct 1800gtaaagacga agagacgttg cgtggtgcaa ttgagaggat gaagcagaag cttaagagaa 1860aagtctga 1868281780DNAArtificial SequenceSynthetic DNA encoding Arabidopsis thaliana GPT protein 28aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac agaaccgacg aactagtatg tacctggaca taaatggtgt 480gatgatcaaa cagtttagct tcaaagcctc tcttctccca ttctcttcta atttccgaca 540aagctccgcc aaaatccatc gtcctatcgg agccaccatg accacagttt cgactcagaa 600cgagtctact caaaaacccg tccaggtggc gaagagatta gagaagttca agactactat 660tttcactcaa atgagcatat tggcagttaa acatggagcg atcaatttag gccaaggctt 720tcccaatttc gacggtcctg attttgttaa agaagctgcg atccaagcta ttaaagatgg 780taaaaaccag tatgctcgtg gatacggcat tcctcagctc aactctgcta tagctgcgcg 840gtttcgtgaa gatacgggtc ttgttgttga tcctgagaaa gaagttactg ttacatctgg 900ttgcacagaa gccatagctg cagctatgtt gggtttaata aaccctggtg atgaagtcat 960tctctttgca ccgttttatg attcctatga agcaacactc tctatggctg gtgctaaagt 1020aaaaggaatc actttacgtc caccggactt ctccatccct ttggaagagc ttaaagctgc 1080ggtaactaac aagactcgag ccatccttat gaacactccg cacaacccga ccgggaagat 1140gttcactagg gaggagcttg aaaccattgc atctctctgc attgaaaacg atgtgcttgt 1200gttctcggat gaagtatacg ataagcttgc gtttgaaatg gatcacattt ctatagcttc 1260tcttcccggt atgtatgaaa gaactgtgac catgaattcc ctgggaaaga ctttctcttt 1320aaccggatgg aagatcggct gggcgattgc gccgcctcat ctgacttggg gagttcgaca 1380agcacactct tacctcacat tcgccacatc aacaccagca caatgggcag ccgttgcagc 1440tctcaaggca ccagagtctt acttcaaaga gctgaaaaga gattacaatg tgaaaaagga 1500gactctggtt aagggtttga aggaagtcgg atttacagtg ttcccatcga gcgggactta 1560ctttgtggtt gctgatcaca ctccatttgg aatggagaac gatgttgctt tctgtgagta 1620tcttattgaa gaagttgggg tcgttgcgat cccaacgagc gtcttttatc tgaatccaga 1680agaagggaag aatttggtta ggtttgcgtt ctgtaaagac gaagagacgt tgcgtggtgc 1740aattgagagg atgaagcaga agcttaagag aaaagtctga 1780291155DNAArabidopsis thaliana 29gtggcgaaga gattagagaa gttcaagact actattttca ctcaaatgag catattggca 60gttaaacatg gagcgatcaa tttaggccaa ggctttccca atttcgacgg tcctgatttt 120gttaaagaag ctgcgatcca agctattaaa gatggtaaaa accagtatgc tcgtggatac 180ggcattcctc agctcaactc tgctatagct gcgcggtttc gtgaagatac gggtcttgtt 240gttgatcctg agaaagaagt tactgttaca tctggttgca cagaagccat agctgcagct 300atgttgggtt taataaaccc tggtgatgaa gtcattctct ttgcaccgtt ttatgattcc 360tatgaagcaa cactctctat ggctggtgct aaagtaaaag gaatcacttt acgtccaccg 420gacttctcca tccctttgga agagcttaaa gctgcggtaa ctaacaagac tcgagccatc 480cttatgaaca ctccgcacaa cccgaccggg aagatgttca ctagggagga gcttgaaacc 540attgcatctc tctgcattga aaacgatgtg cttgtgttct cggatgaagt atacgataag 600cttgcgtttg aaatggatca catttctata gcttctcttc ccggtatgta tgaaagaact 660gtgaccatga attccctggg aaagactttc tctttaaccg gatggaagat cggctgggcg 720attgcgccgc ctcatctgac ttggggagtt cgacaagcac actcttacct cacattcgcc 780acatcaacac cagcacaatg ggcagccgtt gcagctctca aggcaccaga gtcttacttc 840aaagagctga aaagagatta caatgtgaaa aaggagactc tggttaaggg tttgaaggaa 900gtcggattta cagtgttccc atcgagcggg acttactttg tggttgctga tcacactcca 960tttggaatgg agaacgatgt tgctttctgt gagtatctta ttgaagaagt tggggtcgtt 1020gcgatcccaa cgagcgtctt ttatctgaat ccagaagaag ggaagaattt ggttaggttt 1080gcgttctgta aagacgaaga gacgttgcgt ggtgcaattg agaggatgaa gcagaagctt 1140aagagaaaag tctga 115530384PRTArabidopsis thaliana 30Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Ile Leu Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Lys Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Ile Pro Gln 50 55 60Leu Asn Ser Ala Ile Ala Ala Arg Phe Arg Glu Asp Thr Gly Leu Val65 70 75 80Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Ala Met Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile 130 135 140Pro Leu Glu Glu Leu Lys Ala Ala Val Thr Asn Lys Thr Arg Ala Ile145 150 155 160Leu Met Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 165 170 175Glu Leu Glu Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 180 185 190Phe Ser Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile 195 200 205Ser Ile Ala Ser Leu Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr 245 250 255Leu Thr Phe Ala Thr Ser Thr Pro Ala Gln Trp Ala Ala Val Ala Ala 260 265 270Leu Lys Ala Pro Glu Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr Asn 275 280 285Val Lys Lys Glu Thr Leu Val Lys Gly Leu Lys Glu Val Gly Phe Thr 290 295 300Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Ala Asp His Thr Pro305 310 315 320Phe Gly Met Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu Ile Glu Glu 325 330 335Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Glu Gly Lys Asn Leu Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr 355 360 365Leu Arg Gly Ala Ile Glu Arg Met Lys Gln Lys Leu Lys Arg Lys Val 370 375 38031384PRTVitis vinifera 31Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Lys Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp 50 55 60Leu Asn Ser Ala Val Ala Asp Arg Phe Lys Lys Asp Thr Gly Leu Val65 70 75 80Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Met Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Gln Ile Lys Ser Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130 135 140Pro Met Asp Glu Leu Lys Ser Ala Ile Ser Lys Asn Thr Arg Ala Ile145 150 155 160Leu Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 165 170 175Glu Leu Asn Val Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 180 185 190Phe Thr Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Met Asp His Ile 195 200 205Ser Met Ala Ser Leu Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Thr225 230 235 240Val Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe 245 250 255Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Trp Ala Ala Ala Thr Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr Glu Glu Leu Lys Arg Asp Tyr Ser 275 280 285Ala Lys Lys Ala Ile Leu Val Glu Gly Leu Lys Ala Val Gly Phe Arg 290 295 300Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro305 310 315 320Phe Gly Leu Lys Asp Asp Ile Ala Phe Cys Glu Tyr Leu Ile Lys Glu

325 330 335Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu His Pro Glu 340 345 350Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Gly Thr 355 360 365Leu Arg Ala Ala Val Glu Arg Met Lys Glu Lys Leu Lys Pro Lys Gln 370 375 38032383PRTOryza sativa 32Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Asn Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu 50 55 60Leu Asn Ser Ala Ile Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln65 70 75 80Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ser Val 130 135 140Pro Leu Glu Glu Leu Lys Ala Ala Val Ser Lys Asn Thr Arg Ala Ile145 150 155 160Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 165 170 175Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys Glu Asn Asp Val Leu Leu 180 185 190Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile 195 200 205Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe 245 250 255Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ala Ala Ala Ala Ala Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr Glu Glu Leu Arg Arg Asp Tyr Gly 275 280 285Ala Lys Lys Ala Leu Leu Val Asn Gly Leu Lys Asp Ala Gly Phe Ile 290 295 300Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met Val Asp His Thr Pro305 310 315 320Phe Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325 330 335Val Gly Val Val Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Glu Thr 355 360 365Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys Leu Arg Lys Lys 370 375 38033383PRTGlycine max 33Ala Lys Arg Leu Glu Lys Phe Gln Thr Thr Ile Phe Thr Gln Met Ser1 5 10 15Leu Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro 20 25 30Asn Phe Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala Ile 35 40 45Arg Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp Leu 50 55 60Asn Ile Ala Ile Ala Glu Arg Phe Lys Lys Asp Thr Gly Leu Val Val65 70 75 80Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr Glu Ala Ile 85 90 95Ala Ala Thr Met Ile Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Met 100 105 110Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly 115 120 125Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro 130 135 140Leu Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg Ala Ile Leu145 150 155 160Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu 165 170 175Leu Asn Cys Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe 180 185 190Thr Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu His Ile Ser 195 200 205Met Ala Ser Leu Pro Gly Met Phe Glu Arg Thr Val Thr Leu Asn Ser 210 215 220Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile225 230 235 240Ala Pro Pro His Leu Ser Trp Gly Val Arg Gln Ala His Ala Phe Leu 245 250 255Thr Phe Ala Thr Ala His Pro Phe Gln Cys Ala Ala Ala Ala Ala Leu 260 265 270Arg Ala Pro Asp Ser Tyr Tyr Val Glu Leu Lys Arg Asp Tyr Met Ala 275 280 285Lys Arg Ala Ile Leu Ile Glu Gly Leu Lys Ala Val Gly Phe Lys Val 290 295 300Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro Phe305 310 315 320Gly Leu Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu Val Lys Glu Val 325 330 335Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu Glu 340 345 350Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Glu Thr Ile 355 360 365Arg Ser Ala Val Glu Arg Met Lys Ala Lys Leu Arg Lys Val Asp 370 375 38034383PRTHordeum vulgare 34Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Asp Ala Ala Ile Glu Ala 35 40 45Ile Lys Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu 50 55 60 Leu Asn Ser Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu His65 70 75 80Ile Asp Pro Asp Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130 135 140Pro Leu Glu Glu Leu Lys Ala Ala Val Ser Lys Asn Thr Arg Ala Ile145 150 155 160Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 165 170 175Glu Leu Glu Phe Ile Ala Asp Leu Cys Lys Glu Asn Asp Val Leu Leu 180 185 190Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile 195 200 205Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe 245 250 255Leu Thr Phe Ala Thr Ser Thr Pro Met Gln Ser Ala Ala Ala Ala Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Phe Glu Glu Leu Lys Arg Asp Tyr Gly 275 280 285Ala Lys Lys Ala Leu Leu Val Asp Gly Leu Lys Ala Ala Gly Phe Ile 290 295 300Val Tyr Pro Ser Ser Gly Thr Tyr Phe Ile Met Val Asp His Thr Pro305 310 315 320Phe Gly Phe Asp Asn Asp Val Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325 330 335Val Gly Val Val Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr 355 360 365Leu Arg Ala Ala Val Asp Arg Met Lys Ala Lys Leu Arg Lys Lys 370 375 38035382PRTDanio rerio 35Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Arg Asp Gly Asn Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp 50 55 60Leu Asn Ile Ala Ile Ser Glu Arg Tyr Lys Lys Asp Thr Gly Leu Ala65 70 75 80Val Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Val Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Val Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ala Leu 130 135 140Pro Ile Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg Ala Ile145 150 155 160Leu Leu Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Pro Glu 165 170 175Glu Leu Asn Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 180 185 190Phe Ser Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu His Ile 195 200 205Ser Ile Ala Ser Leu Pro Gly Met Phe Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ala Phe 245 250 255Leu Thr Phe Ala Thr Ser Asn Pro Met Gln Trp Ala Ala Ala Val Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr Thr Glu Leu Lys Arg Asp Tyr Met 275 280 285Ala Lys Arg Ser Ile Leu Val Glu Gly Leu Lys Ala Val Gly Phe Lys 290 295 300Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro305 310 315 320Phe Gly His Glu Asn Asp Ile Ala Phe Cys Glu Tyr Leu Val Lys Glu 325 330 335Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Glu Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Gly Thr 355 360 365Leu Arg Ala Ala Val Asp Arg Met Lys Glu Lys Leu Arg Lys 370 375 38036383PRTPhyllostachys bambusoides 36Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Asn Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu 50 55 60Leu Asn Ser Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln65 70 75 80Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130 135 140Pro Leu Glu Glu Leu Lys Ala Thr Val Ser Lys Asn Thr Arg Ala Ile145 150 155 160Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Ser Arg Glu 165 170 175Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys Lys Asn Asp Val Leu Leu 180 185 190Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile 195 200 205Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe 245 250 255Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ser Ala Ala Ala Ala Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr Gly Glu Leu Lys Arg Asp Tyr Gly 275 280 285Ala Lys Lys Ala Ile Leu Val Asp Gly Leu Lys Ala Ala Gly Phe Ile 290 295 300Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met Val Asp His Thr Pro305 310 315 320Phe Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325 330 335Val Gly Val Val Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr 355 360 365Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys Leu Arg Lys Lys 370 375 3803734DNAArtificial SequenceSynthetic primer sequence 37cccatcgatg tacctggaca taaatggtgt gatg 343837DNAArtificial SequenceSynthetic primer sequence 38gatggtacct cagacttttc tcttaagctt ctgcttc 37392992DNAArtificial SequenceSynthetic expression cassette 39ctgcagcaaa gaaacgttat tagttggtgc ttttggtggt aggaatgtag ttttctgaca 60aagtcaatta ctgaatataa aaaaaatctg cacagctctg cgtcaacagt tgtccaaggg 120atgcctcaaa aatctgtgca gattatcagt cgtcacgcag aagcagaaca tcatggtgtg 180ctaggtcagc ttcttgcatt gggccatgaa tccggttggt tgttaatctc tcctctctta 240ttctcttata ttaagatgca taactctttt atgtagtcta aaaaaaaatc cagtggatcg 300gatagtagta cgtcatggtg ccattaggta ccgttgaacc taacagatat ttatgcatgt 360gtatatatat agctatatag acaaaattga tgccgattat agacccaaaa gcaataggta 420tatataatat aatacagacc acaccaccaa actaagaatc gatcaaatag acaaggcatg 480tctccaaatt gtcttaaact atttccgtag gttcagccgt tcaggagtcg aatcagcctc 540tgccggcgtt ttctttgcac gtacgacgga cacacatggg cataccatat agctggtcca 600tgacattagg agagagaacg tacgtgttga cctgtagctg agatataaca aggttgatta 660taatatcacc aaacatgaaa tcatccaagg atgacccata actatcacta ctatagtact 720gcatctggta aaagaaattg tatagactct atttcgagca ctaccacata acgcctgcaa 780tgtgacaccc tacctattca ctaatgtgcc tcttcccaca cgctttccac ccgtactgct 840cacagcttta agaaccagaa caaatgagta atattagtgt cggttcatgg ctaaaaccag 900cactgatgta catgaccaca tatgtcaaat gctgcttcta ggcatgaccc gctcttacta 960atacctactc atcgctagaa gaattttcgg ctgataaatt ttcaatttaa gcaagagtta 1020tctgcgttgg ttcataactc aaactgatgg ccccaaccat attagtgcaa atttcacata 1080tgatcataac cttttcatat gaaatcggat cgagatgaac tttatataaa cattgtagct 1140gtcgatgata cctacaattt tatagttcac aaccttttta tttcaagtca tttaaatgcc 1200caaataggtg tttcaaatct cagatagaaa tgttcaaaag taaaaaaggt ccctatcata 1260acataattga tatgtaagtg agttggaaaa agataagtac gtgtgagaga gatcggggat 1320caaattctgg tgtaataatg tatgtatttc agtcataaaa attggtagca gtagttgggg 1380ctctgtatat ataccggtaa ggatgggatg gtagtagaat aattcttttt ttgtttttag 1440ttttttctgg tccaaaattt caaatttgga tcccttactt gtaccaacta atattaatga 1500gtgttgaggg tagtagaggt gcaactttac cataatccct ctgtttcagg ttataagacg 1560ttttgacttt aaatttgacc aagtttatgc gcaaatatag taatatttat aatactatat 1620tagtttcatt aaataaataa ttgaatatat tttcataata aatttgtgtt gagttcaaaa 1680tattattaat tttttctaca aacttggtca aacttgaagc agtttgactt tgaccaaagt 1740caaaacgtct tataacttga aacggatgga ttactttttt tgtggggaca agtttacaat 1800gtttaataaa gcacaatcca tcttaatgtt ttcaagctga atattgtaaa attcatggat 1860aaaccagctt ctaaatgttt aaccgggaaa atgtcgaacg acaaattaat atttttaagt 1920gatggggagt attaattaag gagtgacaac tcaactttca atatcgtact aaactgtggg 1980atttattttc taaaatttta taccctgcca attcacgtgt tgtagatctt tttttttcac 2040taaccgacac caggtatatc aattttattg aatatagcag caaaaagaat gtgttgtact 2100tgtaaacaaa aagcaaactg tacataaaaa aaaatgcact cctatataat taagctcata 2160aagatgcttt gcttcgtgag ggcccaagtt ttgatgacct tttgcttgat ctcgaaatta 2220aaatttaagt actgttaagg gagttcacac caccatcaat tttcagcctg aagaaacagt 2280taaacaacga ccccgatgac cagtctactg ctctccacat actagctgca ttattgatca 2340caaaacaaaa caaaacgaaa taaaaatcag cagcgagagt gtgcagagag agacaaaggt 2400gatctggcgt ggatatctcc ccatccatcc tcacccgcgc tgcccatcac tcgccgccgc 2460atactccatc atgtggagag aggaagacga ggaccacagc cagagcccgg gtcgagatgc 2520caccacggcc acaacccacg agcccggcgc gacaccaccg cgcgcgcgtg agccagccac 2580aaacgcccgc ggataggcgc gcgcacgccg gccaatccta ccacatcccc ggcctccgcg 2640gctcgcgagc gccgctgcca tccgatccgc tgagttttgg ctatttatac gtaccgcggg 2700agcctgtgtg cagagcagtg catctcaaga agtactcgag caaagaagga gagagcttgg 2760tgagctgcag

ccatggtaga tctgagggta aatttctagt ttttctcctt cattttcttg 2820gttaggaccc ttttctcttt ttattttttt gagctttgat ctttctttaa actgatctat 2880tttttaattg attggttatg gtgtaaatat tacatagctt taactgataa tctgattact 2940ttatttcgtg tgtctatgat gatgatgata gttacagaac cgacgaacta gt 2992401281DNAHordeum vulgare 40gcgcaggcgg ttgtgcaggc gatgcagtgc caggtggggg tgaggggcag gacggccgtc 60ccggcgaggc agcccgcggg cagggtgtgg ggcgtcagga gggccgcccg cgccacctcc 120gggttcaagg tgctggcgct cggcccggag accaccgggg tcatccagag gatgcagcag 180ctgctcgaca tggacaccac gcccttcacc gacaagatca tcgccgagta catctgggtt 240ggaggatctg gaattgacct cagaagcaaa tcaaggacga tttcgaagcc agtggaggac 300ccgtcagagc tgccgaaatg gaactacgac ggatcgagca cggggcaggc tcctggggaa 360gacagtgaag tcatcctata cccacaggcc atattcaagg acccattccg aggaggcaac 420aacatactgg ttatctgtga cacctacaca ccacaggggg aacccatccc tactaacaaa 480cgccacatgg ctgcacaaat cttcagtgac cccaaggtca cttcacaagt gccatggttc 540ggaatcgaac aggagtacac tctgatgcag agggatgtga actggcctct tggctggcct 600gttggagggt accctggccc ccagggtcca tactactgcg ccgtaggatc agacaagtca 660tttggccgtg acatatcaga tgctcactac aaggcgtgcc tttacgctgg aattgaaatc 720agtggaacaa acggggaggt catgcctggt cagtgggagt accaggttgg acccagcgtt 780ggtattgatg caggagacca catatgggct tccagataca ttctcgagag aatcacggag 840caagctggtg tggtgctcac ccttgaccca aaaccaatcc agggtgactg gaacggagct 900ggctgccaca caaactacag cacattgagc atgcgcgagg atggaggttt cgacgtgatc 960aagaaggcaa tcctgaacct ttcacttcgc catgacttgc acatagccgc atatggtgaa 1020ggaaacgagc ggaggttgac agggctacac gagacagcta gcatatcaga cttctcatgg 1080ggtgtggcga accgtggctg ctctattcgt gtggggcgag acaccgaggc gaagggcaaa 1140ggatacctgg aggaccgtcg cccggcctcc aacatggacc cgtacaccgt gacggcgctg 1200ctggccgaga ccacgatcct gtgggagccg accctcgagg cggaggccct cgctgccaag 1260aagctggcgc tgaaggtatg a 128141426PRTHordeum vulgare 41Ala Gln Ala Val Val Gln Ala Met Gln Cys Gln Val Gly Val Arg Gly1 5 10 15Arg Thr Ala Val Pro Ala Arg Gln Pro Ala Gly Arg Val Trp Gly Val 20 25 30Arg Arg Ala Ala Arg Ala Thr Ser Gly Phe Lys Val Leu Ala Leu Gly 35 40 45Pro Glu Thr Thr Gly Val Ile Gln Arg Met Gln Gln Leu Leu Asp Met 50 55 60Asp Thr Thr Pro Phe Thr Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val65 70 75 80Gly Gly Ser Gly Ile Asp Leu Arg Ser Lys Ser Arg Thr Ile Ser Lys 85 90 95Pro Val Glu Asp Pro Ser Glu Leu Pro Lys Trp Asn Tyr Asp Gly Ser 100 105 110Ser Thr Gly Gln Ala Pro Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro 115 120 125Gln Ala Ile Phe Lys Asp Pro Phe Arg Gly Gly Asn Asn Ile Leu Val 130 135 140Ile Cys Asp Thr Tyr Thr Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys145 150 155 160Arg His Met Ala Ala Gln Ile Phe Ser Asp Pro Lys Val Thr Ser Gln 165 170 175Val Pro Trp Phe Gly Ile Glu Gln Glu Tyr Thr Leu Met Gln Arg Asp 180 185 190Val Asn Trp Pro Leu Gly Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln 195 200 205Gly Pro Tyr Tyr Cys Ala Val Gly Ser Asp Lys Ser Phe Gly Arg Asp 210 215 220Ile Ser Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala Gly Ile Glu Ile225 230 235 240Ser Gly Thr Asn Gly Glu Val Met Pro Gly Gln Trp Glu Tyr Gln Val 245 250 255Gly Pro Ser Val Gly Ile Asp Ala Gly Asp His Ile Trp Ala Ser Arg 260 265 270Tyr Ile Leu Glu Arg Ile Thr Glu Gln Ala Gly Val Val Leu Thr Leu 275 280 285Asp Pro Lys Pro Ile Gln Gly Asp Trp Asn Gly Ala Gly Cys His Thr 290 295 300Asn Tyr Ser Thr Leu Ser Met Arg Glu Asp Gly Gly Phe Asp Val Ile305 310 315 320Lys Lys Ala Ile Leu Asn Leu Ser Leu Arg His Asp Leu His Ile Ala 325 330 335Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly Leu His Glu Thr 340 345 350Ala Ser Ile Ser Asp Phe Ser Trp Gly Val Ala Asn Arg Gly Cys Ser 355 360 365Ile Arg Val Gly Arg Asp Thr Glu Ala Lys Gly Lys Gly Tyr Leu Glu 370 375 380Asp Arg Arg Pro Ala Ser Asn Met Asp Pro Tyr Thr Val Thr Ala Leu385 390 395 400Leu Ala Glu Thr Thr Ile Leu Trp Glu Pro Thr Leu Glu Ala Glu Ala 405 410 415Leu Ala Ala Lys Lys Leu Ala Leu Lys Val 420 425424273DNAArtificial SequenceSynthetic expression cassette 42ctgcagcaaa gaaacgttat tagttggtgc ttttggtggt aggaatgtag ttttctgaca 60aagtcaatta ctgaatataa aaaaaatctg cacagctctg cgtcaacagt tgtccaaggg 120atgcctcaaa aatctgtgca gattatcagt cgtcacgcag aagcagaaca tcatggtgtg 180ctaggtcagc ttcttgcatt gggccatgaa tccggttggt tgttaatctc tcctctctta 240ttctcttata ttaagatgca taactctttt atgtagtcta aaaaaaaatc cagtggatcg 300gatagtagta cgtcatggtg ccattaggta ccgttgaacc taacagatat ttatgcatgt 360gtatatatat agctatatag acaaaattga tgccgattat agacccaaaa gcaataggta 420tatataatat aatacagacc acaccaccaa actaagaatc gatcaaatag acaaggcatg 480tctccaaatt gtcttaaact atttccgtag gttcagccgt tcaggagtcg aatcagcctc 540tgccggcgtt ttctttgcac gtacgacgga cacacatggg cataccatat agctggtcca 600tgacattagg agagagaacg tacgtgttga cctgtagctg agatataaca aggttgatta 660taatatcacc aaacatgaaa tcatccaagg atgacccata actatcacta ctatagtact 720gcatctggta aaagaaattg tatagactct atttcgagca ctaccacata acgcctgcaa 780tgtgacaccc tacctattca ctaatgtgcc tcttcccaca cgctttccac ccgtactgct 840cacagcttta agaaccagaa caaatgagta atattagtgt cggttcatgg ctaaaaccag 900cactgatgta catgaccaca tatgtcaaat gctgcttcta ggcatgaccc gctcttacta 960atacctactc atcgctagaa gaattttcgg ctgataaatt ttcaatttaa gcaagagtta 1020tctgcgttgg ttcataactc aaactgatgg ccccaaccat attagtgcaa atttcacata 1080tgatcataac cttttcatat gaaatcggat cgagatgaac tttatataaa cattgtagct 1140gtcgatgata cctacaattt tatagttcac aaccttttta tttcaagtca tttaaatgcc 1200caaataggtg tttcaaatct cagatagaaa tgttcaaaag taaaaaaggt ccctatcata 1260acataattga tatgtaagtg agttggaaaa agataagtac gtgtgagaga gatcggggat 1320caaattctgg tgtaataatg tatgtatttc agtcataaaa attggtagca gtagttgggg 1380ctctgtatat ataccggtaa ggatgggatg gtagtagaat aattcttttt ttgtttttag 1440ttttttctgg tccaaaattt caaatttgga tcccttactt gtaccaacta atattaatga 1500gtgttgaggg tagtagaggt gcaactttac cataatccct ctgtttcagg ttataagacg 1560ttttgacttt aaatttgacc aagtttatgc gcaaatatag taatatttat aatactatat 1620tagtttcatt aaataaataa ttgaatatat tttcataata aatttgtgtt gagttcaaaa 1680tattattaat tttttctaca aacttggtca aacttgaagc agtttgactt tgaccaaagt 1740caaaacgtct tataacttga aacggatgga ttactttttt tgtggggaca agtttacaat 1800gtttaataaa gcacaatcca tcttaatgtt ttcaagctga atattgtaaa attcatggat 1860aaaccagctt ctaaatgttt aaccgggaaa atgtcgaacg acaaattaat atttttaagt 1920gatggggagt attaattaag gagtgacaac tcaactttca atatcgtact aaactgtggg 1980atttattttc taaaatttta taccctgcca attcacgtgt tgtagatctt tttttttcac 2040taaccgacac caggtatatc aattttattg aatatagcag caaaaagaat gtgttgtact 2100tgtaaacaaa aagcaaactg tacataaaaa aaaatgcact cctatataat taagctcata 2160aagatgcttt gcttcgtgag ggcccaagtt ttgatgacct tttgcttgat ctcgaaatta 2220aaatttaagt actgttaagg gagttcacac caccatcaat tttcagcctg aagaaacagt 2280taaacaacga ccccgatgac cagtctactg ctctccacat actagctgca ttattgatca 2340caaaacaaaa caaaacgaaa taaaaatcag cagcgagagt gtgcagagag agacaaaggt 2400gatctggcgt ggatatctcc ccatccatcc tcacccgcgc tgcccatcac tcgccgccgc 2460atactccatc atgtggagag aggaagacga ggaccacagc cagagcccgg gtcgagatgc 2520caccacggcc acaacccacg agcccggcgc gacaccaccg cgcgcgcgtg agccagccac 2580aaacgcccgc ggataggcgc gcgcacgccg gccaatccta ccacatcccc ggcctccgcg 2640gctcgcgagc gccgctgcca tccgatccgc tgagttttgg ctatttatac gtaccgcggg 2700agcctgtgtg cagagcagtg catctcaaga agtactcgag caaagaagga gagagcttgg 2760tgagctgcag ccatggtaga tctgagggta aatttctagt ttttctcctt cattttcttg 2820gttaggaccc ttttctcttt ttattttttt gagctttgat ctttctttaa actgatctat 2880tttttaattg attggttatg gtgtaaatat tacatagctt taactgataa tctgattact 2940ttatttcgtg tgtctatgat gatgatgata gttacagaac cgacgaacta gtgcgcaggc 3000ggttgtgcag gcgatgcagt gccaggtggg ggtgaggggc aggacggccg tcccggcgag 3060gcagcccgcg ggcagggtgt ggggcgtcag gagggccgcc cgcgccacct ccgggttcaa 3120ggtgctggcg ctcggcccgg agaccaccgg ggtcatccag aggatgcagc agctgctcga 3180catggacacc acgcccttca ccgacaagat catcgccgag tacatctggg ttggaggatc 3240tggaattgac ctcagaagca aatcaaggac gatttcgaag ccagtggagg acccgtcaga 3300gctgccgaaa tggaactacg acggatcgag cacggggcag gctcctgggg aagacagtga 3360agtcatccta tacccacagg ccatattcaa ggacccattc cgaggaggca acaacatact 3420ggttatctgt gacacctaca caccacaggg ggaacccatc cctactaaca aacgccacat 3480ggctgcacaa atcttcagtg accccaaggt cacttcacaa gtgccatggt tcggaatcga 3540acaggagtac actctgatgc agagggatgt gaactggcct cttggctggc ctgttggagg 3600gtaccctggc ccccagggtc catactactg cgccgtagga tcagacaagt catttggccg 3660tgacatatca gatgctcact acaaggcgtg cctttacgct ggaattgaaa tcagtggaac 3720aaacggggag gtcatgcctg gtcagtggga gtaccaggtt ggacccagcg ttggtattga 3780tgcaggagac cacatatggg cttccagata cattctcgag agaatcacgg agcaagctgg 3840tgtggtgctc acccttgacc caaaaccaat ccagggtgac tggaacggag ctggctgcca 3900cacaaactac agcacattga gcatgcgcga ggatggaggt ttcgacgtga tcaagaaggc 3960aatcctgaac ctttcacttc gccatgactt gcacatagcc gcatatggtg aaggaaacga 4020gcggaggttg acagggctac acgagacagc tagcatatca gacttctcat ggggtgtggc 4080gaaccgtggc tgctctattc gtgtggggcg agacaccgag gcgaagggca aaggatacct 4140ggaggaccgt cgcccggcct ccaacatgga cccgtacacc gtgacggcgc tgctggccga 4200gaccacgatc ctgtgggagc cgaccctcga ggcggaggcc ctcgctgcca agaagctggc 4260gctgaaggta tga 427343436PRTArtificial SequenceTranslation product of SEQ ID NO 42 DNA 43Met Val Asp Leu Arg Asn Arg Arg Thr Ser Ala Gln Ala Val Val Gln1 5 10 15Ala Met Gln Cys Gln Val Gly Val Arg Gly Arg Thr Ala Val Pro Ala 20 25 30Arg Gln Pro Ala Gly Arg Val Trp Gly Val Arg Arg Ala Ala Arg Ala 35 40 45Thr Ser Gly Phe Lys Val Leu Ala Leu Gly Pro Glu Thr Thr Gly Val 50 55 60Ile Gln Arg Met Gln Gln Leu Leu Asp Met Asp Thr Thr Pro Phe Thr65 70 75 80Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Ile Asp 85 90 95Leu Arg Ser Lys Ser Arg Thr Ile Ser Lys Pro Val Glu Asp Pro Ser 100 105 110Glu Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 115 120 125Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp 130 135 140Pro Phe Arg Gly Gly Asn Asn Ile Leu Val Ile Cys Asp Thr Tyr Thr145 150 155 160Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys Arg His Met Ala Ala Gln 165 170 175Ile Phe Ser Asp Pro Lys Val Thr Ser Gln Val Pro Trp Phe Gly Ile 180 185 190Glu Gln Glu Tyr Thr Leu Met Gln Arg Asp Val Asn Trp Pro Leu Gly 195 200 205Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala 210 215 220Val Gly Ser Asp Lys Ser Phe Gly Arg Asp Ile Ser Asp Ala His Tyr225 230 235 240Lys Ala Cys Leu Tyr Ala Gly Ile Glu Ile Ser Gly Thr Asn Gly Glu 245 250 255Val Met Pro Gly Gln Trp Glu Tyr Gln Val Gly Pro Ser Val Gly Ile 260 265 270Asp Ala Gly Asp His Ile Trp Ala Ser Arg Tyr Ile Leu Glu Arg Ile 275 280 285Thr Glu Gln Ala Gly Val Val Leu Thr Leu Asp Pro Lys Pro Ile Gln 290 295 300Gly Asp Trp Asn Gly Ala Gly Cys His Thr Asn Tyr Ser Thr Leu Ser305 310 315 320Met Arg Glu Asp Gly Gly Phe Asp Val Ile Lys Lys Ala Ile Leu Asn 325 330 335Leu Ser Leu Arg His Asp Leu His Ile Ala Ala Tyr Gly Glu Gly Asn 340 345 350Glu Arg Arg Leu Thr Gly Leu His Glu Thr Ala Ser Ile Ser Asp Phe 355 360 365Ser Trp Gly Val Ala Asn Arg Gly Cys Ser Ile Arg Val Gly Arg Asp 370 375 380Thr Glu Ala Lys Gly Lys Gly Tyr Leu Glu Asp Arg Arg Pro Ala Ser385 390 395 400Asn Met Asp Pro Tyr Thr Val Thr Ala Leu Leu Ala Glu Thr Thr Ile 405 410 415Leu Trp Glu Pro Thr Leu Glu Ala Glu Ala Leu Ala Ala Lys Lys Leu 420 425 430Ala Leu Lys Val 435441992DNAZea mays 44ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980ttacttctgc ag 1992451248DNAHordeum vulgare 45atggcatccg cccccgcctc cgcctccgcg gccctctcca ccgccgcccc cgccgacaac 60ggggccgcca agcccacgga gcagcggccg gtacaggtgg ctaagcgatt ggagaagttc 120aaaacaacaa ttttcacaca gatgagcatg ctcgcagtga agcatggagc aataaacctt 180ggacaggggt ttcccaattt tgatggccct gactttgtca aagatgctgc tattgaggct 240atcaaagctg gaaagaatca gtatgcaaga ggatatggtg tgcctgaatt gaactcagct 300gttgctgaga gatttctcaa ggacagtgga ttgcacatcg atcctgataa ggaagttact 360gttacatctg ggtgcacaga agcaatagct gcaacgatat tgggtctgat caaccctggg 420gatgaagtca tactgtttgc tccattctat gattcttatg aggctacact gtccatggct 480ggtgcgaatg tcaaagccat tacactccgc cctccggact ttgcagtccc tcttgaagag 540ctaaaggctg cagtctcgaa gaataccaga gcaataatga ttaatacacc tcacaaccct 600accgggaaaa tgttcacaag ggaggaactt gagttcattg ctgatctctg caaggaaaat 660gacgtgttgc tctttgccga tgaggtctac gacaagctgg cgtttgaggc ggatcacata 720tcaatggctt ctattcctgg catgtatgag aggaccgtca ctatgaactc cctggggaag 780acgttctcct tgaccggatg gaagatcggc tgggcgatag caccaccgca cctgacatgg 840ggcgtaaggc aggcacactc cttcctcaca ttcgccacct ccacgccgat gcaatcagca 900gcggcggcgg ccctgagagc accggacagc tactttgagg agctgaagag ggactacggc 960gcaaagaaag cgctgctggt ggacgggctc aaggcggcgg gcttcatcgt ctacccttcg 1020agcggaacct acttcatcat ggtcgaccac accccgttcg ggttcgacaa cgacgtcgag 1080ttctgcgagt acttgatccg cgaggtcggc gtcgtggcca tcccgccaag cgtgttctac 1140ctgaacccgg aggacgggaa gaacctggtg aggttcacct tctgcaagga cgacgacacg 1200ctaagggcgg cggtggacag gatgaaggcc aagctcagga agaaatga 124846415PRTHordeum vulgare 46Met Ala Ser Ala Pro Ala Ser Ala Ser Ala Ala Leu Ser Thr Ala Ala1 5 10 15Pro Ala Asp Asn Gly Ala Ala Lys Pro Thr Glu Gln Arg Pro Val Gln 20 25 30Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met 35 40 45Ser Met Leu Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 50 55 60Pro Asn Phe Asp Gly Pro

Asp Phe Val Lys Asp Ala Ala Ile Glu Ala65 70 75 80Ile Lys Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu 85 90 95Leu Asn Ser Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu His 100 105 110Ile Asp Pro Asp Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 115 120 125Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 130 135 140Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala145 150 155 160Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 165 170 175Pro Leu Glu Glu Leu Lys Ala Ala Val Ser Lys Asn Thr Arg Ala Ile 180 185 190Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 195 200 205Glu Leu Glu Phe Ile Ala Asp Leu Cys Lys Glu Asn Asp Val Leu Leu 210 215 220Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile225 230 235 240Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 245 250 255Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala 260 265 270Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe 275 280 285Leu Thr Phe Ala Thr Ser Thr Pro Met Gln Ser Ala Ala Ala Ala Ala 290 295 300Leu Arg Ala Pro Asp Ser Tyr Phe Glu Glu Leu Lys Arg Asp Tyr Gly305 310 315 320Ala Lys Lys Ala Leu Leu Val Asp Gly Leu Lys Ala Ala Gly Phe Ile 325 330 335Val Tyr Pro Ser Ser Gly Thr Tyr Phe Ile Met Val Asp His Thr Pro 340 345 350Phe Gly Phe Asp Asn Asp Val Glu Phe Cys Glu Tyr Leu Ile Arg Glu 355 360 365Val Gly Val Val Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu 370 375 380Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr385 390 395 400Leu Arg Ala Ala Val Asp Arg Met Lys Ala Lys Leu Arg Lys Lys 405 410 415472735DNAArtificial SequenceSynthetic expression cassette 47gtttgaatcc tccttaaagt ttttctctgg agaaactgta gtaattttac tttgttgtgt 60tcccttcatc ttttgaatta atggcatttg ttttaatact aatctgcttc tgaaacttgt 120aatgtatgta tatcagtttc ttataattta tccaagtaat atcttccatt ctctatgcaa 180ttgcctgcat aagctcgaca aaagagtaca tcaacccctc ctcctctgga ctactctagc 240taaacttgaa tttcccctta agattatgaa attgatatat ccttaacaaa cgactccttc 300tgttggaaaa tgtagtactt gtctttcttc ttttgggtat atatagttta tatacaccat 360actatgtaca acatccaagt agagtgaaat ggatacatgt acaagactta tttgattgat 420tgatgacttg agttgcctta ggagtaacaa attcttaggt caataaatcg ttgatttgaa 480attaatctct ctgtcttaga cagataggaa ttatgacttc caatggtcca gaaagcaaag 540ttcgcactga gggtatactt ggaattgaga cttgcacagg tccagaaacc aaagttccca 600tcgagctcta aaatcacatc tttggaatga aattcaatta gagataagtt gcttcatagc 660ataggtaaaa tggaagatgt gaagtaacct gcaataatca gtgaaatgac attaatacac 720taaatacttc atatgtaatt atcctttcca ggttaacaat actctataaa gtaagaatta 780tcagaaatgg gctcatcaaa cttttgtact atgtatttca tataaggaag tataactata 840cataagtgta tacacaactt tattcctatt ttgtaaaggt ggagagactg ttttcgatgg 900atctaaagca atatgtctat aaaatgcatt gatataataa ttatctgaga aaatccagaa 960ttggcgttgg attatttcag ccaaatagaa gtttgtacca tacttgttga ttccttctaa 1020gttaaggtga agtatcattc ataaacagtt ttccccaaag tactactcac caagtttccc 1080tttgtagaat taacagttca aatatatggc gcagaaatta ctctatgccc aaaaccaaac 1140gagaaagaaa caaaatacag gggttgcaga ctttattttc gtgttagggt gtgttttttc 1200atgtaattaa tcaaaaaata ttatgacaaa aacatttata catattttta ctcaacactc 1260tgggtatcag ggtgggttgt gttcgacaat caatatggaa aggaagtatt ttccttattt 1320ttttagttaa tattttcagt tataccaaac ataccttgtg atattatttt taaaaatgaa 1380aaactcgtca gaaagaaaaa gcaaaagcaa caaaaaaatt gcaagtattt tttaaaaaag 1440aaaaaaaaaa catatcttgt ttgtcagtat gggaagtttg agataaggac gagtgagggg 1500ttaaaattca gtggccattg attttgtaat gccaagaacc acaaaatcca atggttacca 1560ttcctgtaag atgaggtttg ctaactcttt ttgtccgtta gataggaagc cttatcacta 1620tatatacaag gcgtcctaat aacctcttag taaccaatta tttcagcacc atgtctctgc 1680tctcagatct cgttaacctc aacctcaccg atgccaccgg gaaaatcatc gccgaataca 1740tatggatcgg tggatctgga atggatatca gaagcaaagc caggacacta ccaggaccag 1800tgactgatcc atcaaagctt cccaagtgga actacgacgg atccagcacc ggtcaggctg 1860ctggagaaga cagtgaagtc attctatacc ctcaggcaat attcaaggat cccttcagga 1920aaggcaacaa catcctggtg atgtgtgatg cttacacacc agctggtgat cctattccaa 1980ccaacaagag gcacaacgct gctaagatct tcagccaccc cgacgttgcc aaggaggagc 2040cttggtatgg gattgagcaa gaatacactt tgatgcaaaa ggatgtgaac tggccaattg 2100gttggcctgt tggtggctac cctggccctc agggacctta ctactgtggt gtgggagctg 2160acaaagccat tggtcgtgac attgtggatg ctcactacaa ggcctgtctt tacgccggta 2220ttggtatttc tggtatcaat ggagaagtca tgccaggcca gtgggagttc caagtcggcc 2280ctgttgaggg tattagttct ggtgatcaag tctgggttgc tcgatacctt ctcgagagga 2340tcactgagat ctctggtgta attgtcagct tcgacccgaa accagtcccg ggtgactgga 2400atggagctgg agctcactgc aactacagca ctaagacaat gagaaacgat ggaggattag 2460aagtgatcaa gaaagcgata gggaagcttc agctgaaaca caaagaacac attgctgctt 2520acggtgaagg aaacgagcgt cgtctcactg gaaagcacga aaccgcagac atcaacacat 2580tctcttgggg agtcgcgaac cgtggagcgt cagtgagagt gggacgtgac acagagaagg 2640aaggtaaagg gtacttcgaa gacagaaggc cagcttctaa catggatcct tacgttgtca 2700cctccatgat cgctgagacg accatactcg gttga 2735481371DNACitrus reticulata 48atgcttaagc cgtccgcctt cgggtcttct ttttcttcct cagctctgct ttcgttttcg 60aagcatttgc atacaataag cattactgat tctgtcaaca ccagaagaag aggaatcagt 120accgcttgcc ctaggtaccc ttctctcatg gcgagcttgt ccaccgtttc caccaatcaa 180agcgacacca tccagaagac caatcttcag cctcaacagg ttgctaagtg cttggagaag 240tttaaaacta caatctttac acaaatgagt atgcttgcca tcaaacatgg agctataaat 300cttggtcaag gctttcccaa ctttgatggc ccagattttg ttaaagatgc agcgattcaa 360gccataaggg atgggaagaa tcaatatgct cgtggacatg gggttccaga gttcaactct 420gccattgctt cccggtttaa gaaagattct gggctcgagg ttgaccctga aaaggaagtt 480actgttacct ctgggtgcac cgaagccatt gctgcaacca tcttaggttt gattaatcct 540ggagatgagg tgatcctttt tgcacctttc tatgattcct atgaagctac tctctccatg 600gctggtgcta aaattaaatg catcacattg cgccctccag aatttgccat ccccattgaa 660gagctcaagt ctacaatctc aaaaaatact cgtgcaattc ttatgaacac tccacataac 720cccactggaa agatgttcac tagggaggaa cttaatgtta ttgcatctct ttgcattgag 780aatgatgtgt tggtttttag tgatgaggtc tatgataagt tggcttttga aatggatcac 840atttccatag cctctcttcc tggaatgtat gagcgtactg taaccatgaa ttccttaggg 900aagacattct ctttaacagg gtggaagatc gggtgggcaa tagctccacc gcaccttaca 960tggggggtgc ggcaggcaca ctcttttctc acgtttgcca catccactcc aatgcagtgg 1020gcagctacag cagcccttag agctccggag acgtactatg aggagctaaa gagagattac 1080tcggcaaaga aggcaatttt ggtggaggga ttgaatgctg ttggtttcaa ggtattccca 1140tctagtggga catactttgt ggttgtagat cacaccccat ttgggcacga aactgatatt 1200gcattttgtg aatatctgat caaggaagtt ggggttgtgg caattccgac cagcgtattt 1260tacttgaatc cagaggatgg aaagaatttg gtgagattta ccttctgcaa agatgaagga 1320actttgaggt ctgcagttga caggatgaag gagaagctga ggagaaaatg a 137149456PRTCitrus reticulata 49Met Leu Lys Pro Ser Ala Phe Gly Ser Ser Phe Ser Ser Ser Ala Leu1 5 10 15Leu Ser Phe Ser Lys His Leu His Thr Ile Ser Ile Thr Asp Ser Val 20 25 30Asn Thr Arg Arg Arg Gly Ile Ser Thr Ala Cys Pro Arg Tyr Pro Ser 35 40 45Leu Met Ala Ser Leu Ser Thr Val Ser Thr Asn Gln Ser Asp Thr Ile 50 55 60Gln Lys Thr Asn Leu Gln Pro Gln Gln Val Ala Lys Cys Leu Glu Lys65 70 75 80Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile Lys His 85 90 95Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro Asp 100 105 110Phe Val Lys Asp Ala Ala Ile Gln Ala Ile Arg Asp Gly Lys Asn Gln 115 120 125Tyr Ala Arg Gly His Gly Val Pro Glu Phe Asn Ser Ala Ile Ala Ser 130 135 140Arg Phe Lys Lys Asp Ser Gly Leu Glu Val Asp Pro Glu Lys Glu Val145 150 155 160Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Ile Leu Gly 165 170 175Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp 180 185 190Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Lys Ile Lys Cys Ile 195 200 205Thr Leu Arg Pro Pro Glu Phe Ala Ile Pro Ile Glu Glu Leu Lys Ser 210 215 220Thr Ile Ser Lys Asn Thr Arg Ala Ile Leu Met Asn Thr Pro His Asn225 230 235 240Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Asn Val Ile Ala Ser 245 250 255Leu Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp Glu Val Tyr Asp 260 265 270Lys Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu Pro Gly 275 280 285Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser 290 295 300Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr305 310 315 320Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr Ser Thr 325 330 335Pro Met Gln Trp Ala Ala Thr Ala Ala Leu Arg Ala Pro Glu Thr Tyr 340 345 350Tyr Glu Glu Leu Lys Arg Asp Tyr Ser Ala Lys Lys Ala Ile Leu Val 355 360 365Glu Gly Leu Asn Ala Val Gly Phe Lys Val Phe Pro Ser Ser Gly Thr 370 375 380Tyr Phe Val Val Val Asp His Thr Pro Phe Gly His Glu Thr Asp Ile385 390 395 400Ala Phe Cys Glu Tyr Leu Ile Lys Glu Val Gly Val Val Ala Ile Pro 405 410 415Thr Ser Val Phe Tyr Leu Asn Pro Glu Asp Gly Lys Asn Leu Val Arg 420 425 430Phe Thr Phe Cys Lys Asp Glu Gly Thr Leu Arg Ser Ala Val Asp Arg 435 440 445Met Lys Glu Lys Leu Arg Arg Lys 450 4555037DNAArtificial SequenceSynthetic primer sequence 50ggccacatgt ccgttgctaa gtgcttggag aagttta 375140DNAArtificial SequenceSynthetic primer sequence 51cgggcacgtg tcattttctc ctcagcttct ccttcatcct 40

Claims

1. A transgenic plant comprising a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a plant promoter.

2. (canceled)

3. The transgenic plant of claim 1, wherein each plant promoter is heterologous to the plant.

4. The transgenic plant of claim 1, wherein the plant promoter operably linked to the GPT transgene is heterologous to the species from which the GPT transgene is derived, and wherein the plant promoter operably linked to the GS transgene is heterologous to the species from which the GS transgene is derived.

5. The transgenic plant of claim 4, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49 and has GPT activity.

6. The transgenic plant of claim 4, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected form the group consisting of (a) SEQ ID NO: 4, SEQ ID NO: 7 from residue 11, and SEQ ID NO: 41, and (b) an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 41.

7. The transgenic plant according to claim 1, wherein the GPT and GS transgenes are incorporated into the genome of the plant.

8. The transgenic plant according to claim 1, wherein the GPT and/or the GS transgenes are incorporated into the plastome of the plant.

9-15. (canceled)

16. The transgenic plant according to claim 1, which is a plant of the phylum Tracheophyta.

17. The transgenic plant of claim 16, wherein the transgenic plant is selected from the group consisting of fruits including apples, avocado, banana, blackberry, blueberry, cherries, cranberries, cantaloupes, grapefruit, lemons, limes, nectarines, oranges, peaches, pineapples, pears, plums, tangelos, tangerines, papaya, mango, strawberry, raspberry, grape, kiwi fruit, olives, and coconuts; vegetables including brussel sprouts, radish, mung beans, carrots, sugarbeet, yams, horseradish, flax, sesame, turnip, sweet potato, cabbage, canola, cucumbers, eggplant, lettuce, onion, okra, parsnips, pumpkins and spinach; flowering plants including lily, carnation, chrysanthemum, petunia, geranium, violet, gladioli, orchid, and lilac; trees including oil palm; ornamental plants; canola; lupins; and cotton.

18. The transgenic plant of claim 16, wherein the transgenic plant is selected from the group consisting of plants of the families Poaceae, Gossypium, Fabaceae, Brassicaceae, Rutaceae, Rubiaceae, Cucurbitaceae, Rosaceae, Asteraceae, Amaranthaceae and Brassicaceae.

19. The transgenic plant of claim 16, wherein the transgenic plant is selected from the group consisting of plants of the genera Avena, Hordeum, Oryza, Panicum, Phleum, Saccharum, Secale, Sorghum, Triticum, Zea, Pennisetum, Lycopersicon, Capiscum, Fagopyrum, Triticosecale, Chenopodium, Digitaria, Manihot, Ipomoea, Olea, Daucus, Pastinaca, Raphanus, Dioscorea, Armoracia, Elaeis, Linum, Carthamus, Sesamum, Vitis, and Solanum.

20-22. (canceled)

23. A method of producing a plant having increased nitrogen use efficiency relative to an analogous wild type or untransformed plant, comprising:(a) introducing a GPT transgene into the plant;(b) introducing a GS transgene into the plant or a progeny of the plant;(c) expressing the GPT transgene and the GS transgene in the plant or the progeny of the plant; and,(d) selecting a plant having an increased nitrogen use efficiency relative to a plant of the same species that does not comprise a GPT transgene or a GS transgene.

24-29. (canceled)

30. A transplastomic plant or cell line carrying a GPT transgene and a GS transgene expression cassette, wherein said expression cassette is flanked by sequences from the plant or plant cell's plastome.

31-35. (canceled)

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