JP6117837B2 - Plant glutamine phenylpyruvate transaminase gene and transgenic plant carrying it - Google Patents

Description

Detailed Description of the Invention

[Specification on rights to inventions based on federal-sponsored research or development]
The present invention relates to a contract number W-7405-ENG-36 given to the director of the University of California by the US Department of Energy, and a contract number DE-AC52- Made with support based on 06NA25396. The government has certain rights in this invention.

[Related Literature]
This specification claims priority to US Provisional Patent No. 61 / 190,581, filed Aug. 29, 2008.

[Background Technology]
The need for a more efficient and earth-friendly farming system is a top priority of interest to mankind as populations grow globally and available farmland continues to be lost or unusable It is. Improving yield, protein content, and plant growth rate has become a major goal in the development of agricultural systems that can be more effectively addressed by current challenges.

  In recent years, the production of many well-developed crops tends to be flat, and the importance of improved crop production techniques is only increasing. Many farms pay attention to time because costs and revenue depend on the rapid turnover of crops or time to market. Therefore, fast plant growth is an economically important goal for many agricultural businesses involving high value crops (such as cereals, vegetables, berries and other fruits).

  Genetic engineering is playing an increasingly important role in the development of sustainable agricultural technologies, but continues to play a role in the future. In recent years, many genetically modified plants and related technologies have been developed, and many of these plants and related technologies are widely used today (Factsheet: Genetically Modified Crops in the United States, Pew Initiative on Food and Biotechnology, August 2004, http://pewagbiotech.org/resources/factsheets). Currently, the variety of transgenic plants adopted is very large and increasing, and in 2006, about 250 million acres were cultivated.

  Although approval of transgenic plant technology may be increasing gradually (especially in the United States, Canada and Australia), many regions around the world (especially in Europe) will have genetically modified plants in agriculture. Implementation is still delayed. Therefore, genetically engineered plants that do not incorporate toxins or other potentially problematic substances into plants and / or the environment, consistent with the pursuit of a reliable and enduring agricultural policy There is a strong interest in the development of. There is also a strong interest in minimizing the cost of achieving the objectives (such as improving herbicide resistance, plague and disease resistance, and overall yield). Therefore, there remains a need for transgenic plants that can satisfy these objectives.

  The goal of fast plant growth has been pursued by numerous studies on various plant regulatory systems. Many of these regulation systems are not yet fully understood. In particular, the plant regulatory mechanisms that regulate carbon and nitrogen metabolism have not been fully elucidated. These regulatory mechanisms are presumed to have an indispensable influence on plant growth and development.

Carbon and nitrogen metabolism in photosynthetic organisms must be regulated in a coordinated manner that ensures the effective use of plant resources and energy. Conventional understanding of carbon and nitrogen metabolism includes details of metabolic pathways that are subsystems of the given steps and larger systems. In photosynthetic organisms, carbon metabolism begins with CO ₂ fixation and proceeds by two main processes (called C-3 metabolism and C-4 metabolism). In plants with C-3 metabolism, the enzyme ribulose bisphosphate carboxylase (RuBisCo) catalyzes the binding of CO ₂ and ribulose diphosphate to produce 3-phosphoglycerate. The said 3-phosphoglycerate is a 3 carbon compound (C-3) which a plant uses in order to synthesize | combine a carbon containing compound. In plants with C-4 metabolism, CO _2, in a reaction catalyzed by the enzyme phosphoenolpyruvate carboxylase to form acids containing 4 carbon coupled with phosphoenolpyruvate salt. This acid is transported to vascular sheath cells where it is decarboxylated to release CO ₂ and then combined with ribulose diphosphate in the same reaction used for C-3 plants.

  Numerous studies have shown that various metabolites are important for plant regulation of nitrogen metabolism. These compounds include malic acid, which is an organic acid, and glutamic acid and glutamine, which are amino acids. Nitrogen is absorbed by photosynthetic organisms by the action of the enzyme glutamine synthetase (GS). Glutamine synthetase catalyzes the binding of ammonia and glutamic acid to produce glutamine. GS plays an important role in nitrogen absorption in plants and produces glutamine in an ATP-dependent reaction by catalyzing the addition of ammonia to glutamic acid (Miflin and Habash, 2002, Journal of Experimental Botany, Vol. 53). , No. 370, pp. 979-987). GS also reabsorbs ammonia released as a result of light absorption and cessation of protein and nitrogen transport compound function. GS enzymes can be divided into two general categories. One represents the cytoplasmic form (GS1) and the other represents the plastid (ie, chloroplast) form (GS2).

  Previous studies have demonstrated that increased expression of GS1 increases the level of GS activity and plant growth, but the reports are inconsistent. For example, Fuentes et al. Show that overexpression of alfalfa GS1 (cytoplasmic form) by the CaMV S35 promoter in tobacco increases the level of GS expression and GS activity in leaf tissue and increases growth in nitrogen deficient conditions. However, it has been reported that it does not affect growth in optimal nitrogen fertile conditions (Fuentes et al., 2001, J. Exp. Botany 52: 1071-81). Temple et al. Reported on transgenic tobacco plants overexpressing sequences encoding full-length alfalfa GS1 containing very high levels of GS transcripts, and GS polypeptides incorporated into active enzymes. No phenotypic effects have been reported in (Temple et al., 1993, Molecular and General Genetics 236: 315-325). Corruzi et al. Have reported that transgenic tobacco overexpressing the pea cytosolic GS1 transgene under the control of the CaMV S35 promoter has increased GS activity, increased cytosolic GS protein, and improved growth characteristics. (US Patent Application No. 6,107,547). More recently, Unkefer et al. Have reported that transgenic tobacco plants overexpressing alfalfa GS1 in foliar tissues (after screening for increased GS activity in leaves to roots, an increased S1 transgene copy number is obtained. , Plants genetically isolated by self-pollination) have been found to produce increased amounts of 2-hydroxy-5-oxoproline in their foliate sites. The 2-hydroxy-5-oxoproline has been found to lead to significantly increased growth rates throughout wild-type tobacco plants (US Pat. No. 6,555,500, US Pat. (See application 6,593,275, US patent application 6,831,040).

  In addition, Unkefer et al. Describe the use of 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) to improve plant development (US No. 6,555,500, U.S. Pat. No. 6,593,275, U.S. Pat. No. 6,831,040). In particular, Unkefer et al. Show that increased concentrations of 2-hydroxy-5-oxoproline in leafy tissue (related to root tissue) trigger a cascade of events that will enhance plant growth characteristics. It is disclosed. Unkefer et al. Show that the leaf concentration of 2-hydroxy-5-oxoproline can be increased to trigger plant growth properties (specifically, 2-hydroxy-5-oxo directly on the plant leaf. A method has been described (by administering a solution of proline and overexpressing glutamine synthetase specifically in leaf tissue).

  A number of transaminases and hydrolyase enzymes known to be related to the synthesis of animal 2-hydroxy-5-oxoproline have been identified in animal liver and pancreatic tissues (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197: 304). Although the biochemical synthesis of 2-hydroxy-5-oxoproline is known in plants, it has not been fully characterized. Furthermore, the importance of the function of 2-hydroxy-5-oxoproline in plants and the size of the pool (tissue concentration) is not known. Ultimately, in the art what transaminase or hydrolase (s) may be present and / or what transaminase or hydrolase (s) is present in the plant Provides no clear guidance on what can function when catalyzing the synthesis of 2-hydroxy-5-oxoproline, and the plant transaminase has been reported, isolated or characterized Absent.

[Summary of the Invention]
The present invention relates to transgenic plants that exhibit enhanced growth rates, seed and fruit yields, and total biomass yields, and methods for producing transgenic plants with enhanced growth. In one embodiment, a transgenic plant designed to overexpress glutamine phenylpyruvate transaminase (GPT) is provided.

  Applicants identify the enzyme glutamine phenylpyruvate transaminase (GPT) as a catalyst for 2-hydroxy-5-oxoproline (2-oxoglutamate) synthetase in plants. 2-Oxoglutaramate is a powerful signal metabolite that regulates the function of many genes involved in photosynthesis applications, carbon fixation and nitrogen metabolism.

  By specifically increasing the concentration of the signal metabolite 2-oxoglutaramate (ie, in leaf tissue), the transgene plants of the present invention can produce higher total yields in a short period of time. Can provide agriculture with enhanced productivity across a wide range of crops. Importantly, unlike many of the transgenic plants described to date, the present invention utilizes natural plant genes that encode natural plant enzymes. The enhanced growth characteristics of the transgenic plants of the present invention are essentially achieved by incorporating additional GPT capabilities into the plant. Thus, the transgenic plants of the present invention did not express any toxic substances, growth hormones, viral or bacterial genetic products and were therefore delayed in some parts of the world so far Many concerns about the adoption of transgenic plants disappear.

  In one embodiment, the present invention provides a transgenic plant comprising a GPT transgene, wherein the GPT transgene is operably linked to a plant promoter. In certain embodiments, the GPT transgene comprises (a) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and (b) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 25, selected from the group consisting of amino acid sequences having at least 75% identity with any one of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and having GPT activity A polypeptide having the amino acid sequence of

  In some embodiments, the GPT transgene is integrated into the genome of the plant. The transgenic plants of the present invention can be monocotyledonous or dicotyledonous.

  The present invention also provides seeds of any generation of the transgenic plant of the present invention, wherein the progeny include a GPT transgene. Similarly, the present invention provides seeds of any generation of the transgenic plant of the present invention, wherein said seed comprises a GPT transgene. The transgenic plants of the present invention may exhibit one or more enhanced growth characteristics when compared to similar wild type plants or non-transformed plants. The growth characteristics are not limited to enhanced growth rate, increased biomass yield, increased seed yield, increased flower or flower bud yield, increased fruit or pod yield, large leaves, and increased Examples include levels of GPT activity and / or increased levels of 2-oxoglutaramate. In some embodiments, the transgenic plants of the present invention exhibit improved nitrogen utilization efficiency.

  Also provided are methods for producing the transgenic plants of the present invention and seeds thereof, enhanced growth characteristics, improved nitrogen utilization efficiency, and salts compared to similar wild-type or non-transformed plants. Or a method relating to the production of plants having increased resistance to germination or growth in a salty environment.

[Brief description of the drawings]
FIG. Nitrogen absorption and 2-oxoglutaramate chemical synthesis: a schematic metabolic pathway.

  FIG. It is a photograph which shows the comparison of the transgenic tobacco plant which overexpresses GPT with respect to a wild-type tobacco plant. From left to right, wild type plant, alfalfa GS1 transgene, Arabidopsis GPT transgene. See Example 3 below.

  FIG. It is a photograph which shows the comparison of the transgenic microtome tomato plant which overexpresses GPT with respect to a wild-type tomato plant. (A) Wild type plant, (B) Arabidopsis GPT transgene. See Example 4 below.

  FIG. It is a photograph showing a comparison of leaf size between a wild-type tobacco plant (upper leaf) and a GPT transgenic tobacco plant (lower leaf).

Detailed Description of the Invention
(Definition)
Unless defined otherwise, all technical terms, annotations, and other scientific terms used herein are intended to have the meanings that are commonly understood by those of ordinary skill in the areas relevant to the present invention. In some cases, terms having a generally understood meaning are defined herein to clarify and / or arrange relationships. Also, what is included in such definitions herein should not necessarily be described in order to represent substantial differences from what is commonly understood in the art. The techniques and methods described or referenced herein are generally well understood and routinely used by those skilled in the art using conventional theory. Such techniques and methods include, for example, molecular cloning techniques widely used in Sambrook et al., Molecular cloning: A Laboratory Manual 3rd. Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Molecular Biology Conventional protocols (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 um protocol (Wan, ed., Humana Press , 2 nd edition, 2006) can be mentioned. Where appropriate, methods involving the use of commercially available kits and reagents are generally performed according to industrially defined protocols and / or parameters unless otherwise stated.

  The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof (“polynucleotides”) in either single-stranded or double-stranded form. Unless otherwise limited, the term “polynucleotide” encompasses nucleic acids containing known analogues of natural nucleotides. Such analogs have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Also, unless otherwise indicated, certain nucleic acid sequences implicitly include conservatively modified variants (eg, altered codon substitutes) and complementary sequences, as well as explicitly indicated sequences. Specifically, an altered codon substitute is such that the sequence generated at position 3 of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (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 mRAN encoded by a gene.

  The term “promoter” refers to a nucleic acid regulatory sequence or sequence that directs the transcription of an operably linked nucleic acid. As used herein, a “plant promoter” is a promoter that functions in plants. A promoter necessarily includes a nucleic acid sequence near the initiation region of transcription (such as a TATA factor in the case of a polymerase type II promoter). The promoter also optionally includes a terminal enhancer factor or receptor factor. The enhancer factor or receptor factor can be located approximately several thousand base pairs from the start region 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 (eg, an array of promoters or transcription factor binding sites) and a second nucleic acid sequence. Here, the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. The term applies to naturally occurring amino acids to which one or more amino acid residues correspond, as well as amino acid polymers that are artificial chemical mimics of naturally occurring and non-naturally occurring amino acid polymers.

  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 and those that are post-modified. Examples of such amino acids include hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bonded to a hydrogen, carboxyl group, amino group, and R group). Examples of the amino acid analog include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. 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.

  As used herein, amino acids are represented by the three letter symbols commonly known, or by one symbol recommended by the International Pure Chemical Association-International Biochemical Association (IUPAC-IUB) Biochemical Nomenclature. obtain. Similarly, nucleotides can be represented by the generally accepted single letter code.

  The term “plant” includes whole plants, plant tissues (such as leaves, stems, flowers, roots, embryos and parts thereof), seedlings, seeds and plant cells, and their progeny. The classification of plants that can be used in the method of the present invention is usually similar to the classification of higher plants that are susceptible to transformation techniques. Such higher plants include angiosperms (monocotyledons and dicotyledons) and gymnosperms. These include plants at various ploidy stages, including multiploid, diploid, haploid and hemizygous.

  The terms “GPT polynucleotide” and “GPT nucleic acid” are used interchangeably herein and refer to the full-length gene encoding a polynucleotide associated with catalyzing the synthesis of 2-oxoglutaramate, or Refers to a length of polynucleotide sequence. The “GPT polynucleotide” and “GPT nucleic acid” include polynucleotides that include both translated (coding) and untranslated sequences, and their complements. The term “GPT coding sequence” refers to the portion of the gene that is transcribed and encodes the GPT protein. The term “targeting sequence” refers to the amino terminus of a protein that directs the protein to the intracellular compartment of the cell (eg, the chloroplast in a plant cell). In addition, GPT polynucleotides are defined by their ability to hybridize to GPT polynucleotides or PCR products derived from GPT polynucleotides under defined conditions.

  A “GPT transgene” has a nucleic acid molecule and is exogenous to a transgenic plant or plant embryo, tissue or seed, or a transgenic plant progenitor plant or plant embryo, tissue or plant having a GPT polynucleotide. A nucleic acid molecule comprising a GPT polynucleotide that is exogenous to the seed.

  Examples of GPT polynucleotides of the present invention are provided herein and include GPT polynucleotides of Arabidopsis, rice, barley, bamboo, soybeans, grapes, and zebrafish.

  Partial length GPT polynucleotides include polynucleotide sequences encoding NPT or C-terminal truncated GPT, polynucleotide sequences encoding mature GPT (not including targeting sequences), and sequences encoding GPT domains. It is done. Examples of GPT polynucleotides encoding GPT truncated at the N-terminus include Arabidopsis thaliana −30, −45, and −56 constructs. These are coding sequences in which the first 30, 45, and 56 amino acids have been removed from the amino acids of the full-length GPT structure of SEQ ID NO: 2, respectively.

  When using the GPT polynucleotides of the present invention in the production of transformed cells and transgenic plants, the inserted polynucleotide sequences need not be identical, but as described further below, Those skilled in the art will appreciate that they need only be “substantially identical” to the gene sequence of a GPT polynucleotide. The term “GPT polynucleotide”, strictly speaking, encompasses such substantially identical variants. Similarly, one skilled in the art will understand that due to codon degeneracy, many polynucleotide sequences will encode the same polypeptide and all such polynucleotide sequences are intended to be included within the term GPT polynucleotide. . More specifically, the term is substantially identical to a GPT polynucleotide sequence disclosed herein and is due to a mutation in the wild-type GPT polypeptide or the function of the GPT polypeptide (eg, , Resulting from a conservative substitution of amino acids in the GPT polypeptide) (which is determined as described below). Thus, the term “GPT polynucleotide” also includes such substantially identical variants.

  The term “conservatively modified variant” applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or substantially the same amino acid sequence, or a nucleic acid that does not encode an amino acid sequence relative to a substantially identical sequence. Point to. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, 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 converted to any corresponding codon without altering the encoded protein. The nucleic acid variant is a “silent variant” and is a type of variant that is conservatively modified. Also, as used herein, all nucleic acid sequences that encode a polypeptide represent all possible silent variants of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually only the codon for methionine, and TGG, which is usually only the codon for tryptophan), can be modified to produce a functionally identical molecule. to understand. Thus, silent variations of nucleic acid sequences encoding polypeptides are each associated with each described sequence.

  With respect to amino acid sequences, one skilled in the art understands that individual substitutions, deletions or additions to the sequence of a nucleic acid, peptide, polypeptide or protein are “conservatively modified variants”. Such substitutions, deletions or additions alter, add or delete one amino acid or a few percent of the encoded sequence, and in “conservatively modified variants” the changes are chemically similar Result in the substitution of an amino acid with the amino acid being Conservative substitution tables showing amino acids that are functionally similar are known in the art. Such conservatively modified variants are added to polymorphic variants, interspecies homologs and alleles of the present invention and are not removed.

  The following eight groups each contain amino acids that are conservative substitutions: 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, eg, Creighton, Proteins (1984)).

Macromolecular structures (eg, polypeptide structures) can be described in terms of various stages of the tissue. For a general discussion of this organization, see, for example, 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). You can refer to it. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to a three-dimensional structure within a locally arranged polypeptide. These structures are commonly known as domains. A domain is a portion of a polypeptide that forms a small unit of the polypeptide, typically 25 to about 500 amino acids in length. A standard domain consists of smaller units of organization (eg, a zone of β-sheets and α-helices). “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to a three-dimensional structure formed by a non-covalent relationship of independent tertiary units.

  The term “isolated” refers to material that is substantially or essentially free from components that normally occur simultaneously with the material, either in the wild-type or as found in nature. However, the term “isolated” is not intended to refer to components present in the electrophoresis gel or in another separation medium. The isolated components are either away from this separation medium and are ready for use in another application or are already in use in a new application / environment. An “isolated” antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment.

  Contaminants in the natural environment are substances that can interfere with the use of diagnostic antibodies, or the use of pharmaceutical antibodies, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) purified to 95% (antibody weight) or higher, most preferably 99% (weight) or higher as measured by the Raleigh method, or (2) rotational cap sequencing. Purified to an amount sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by the device, or (3) in reducing or non-reducing conditions by Coomassie Blue, or preferably silver staining Purified to homogeneity by SDS-PAGE. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody in the natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

  The term “heterologous” when used with respect to a portion of a nucleic acid indicates that the nucleic acid contains two or more subsequences that are not found in the same relationship to each other in nature. For example, encoding two or more sequences from unrelated genes arranged to create a new functional nucleic acid (eg, encoding a nucleic acid encoding a protein from one source and a peptide sequence from another source) The nucleic acid having a nucleic acid is typically produced recombinantly. 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 (eg, a fusion protein).

  With respect to two or more nucleic acid or polypeptide sequences, the term “identical” or percentage of “identity” refers to when the entire comparison region is compared and aligned, or to a sequence comparison algorithm or manual sequence test and When specifying regions as measured by eye, they are the same or the same for maximum matching (ie, about 70% identity across a particular region, preferably about 75%, about 80% , About 85%, about 90%, or about 95% identity) refers to two or more sequences or subsequences that contain a certain percentage of amino acid residues or nucleotides. This definition also refers to the complement of a test sequence that has substantial sequence complementarity or subsequence complementarity when the test sequence is substantially identical to a reference sequence. This definition also refers to the complement of a test sequence that has substantial sequence complementarity or subsequence complementarity when the test sequence is substantially identical to a reference sequence.

  When percentage sequence identity is used in reference to a polypeptide, it is understood that portions of residues that are not identical often differ by conservative substitutions of amino acids. Here, since the amino acid residue is replaced with another amino acid residue having similar chemical properties (eg, charge or hydrophobicity), the functional properties of the polypeptide do not change. If the sequence is not a conservative substitution, the percent sequence identity can be modified upstream to correct for conservative natural substitutions.

  For sequence comparison, typically one sequence acts as a reference sequence, and a test sequence is compared to that reference sequence. 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 are used, or alternative parameters are specified. The sequence comparison algorithm then calculates the ratio of sequence identity of the test sequence to the reference sequence based on the program parameters.

  As used herein, a “comparison window” is 20 to 600 of sequences that can be compared to a reference sequence at the same number of consecutive positions after optimal alignment of the two sequences. Relates to any one segment at a plurality of consecutive positions selected from the group consisting of about 50 to about 200, more usually about 100 to about 150. Methods for arranging sequences for comparison are well known in the art. The optimal arrangement of the sequences for comparison is described, for example, in the Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443 by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2: 482. By homologous placement algorithms, Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85: 2444, research on similar methods, these algorithms (the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., By performing computer control of Madison, WI's GAP, BESTFIT, FASTA, and TFASTA) or by manual alignment and visually (eg, Current Protocols in Molecular Biology (Ausubel et al., Eds. 1995 supplement) ).

  Preferred examples of algorithms suitable for measuring percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms (Altschul et al., 1977, Nuc. Acids Res. 25: 3389-3402 and Altschul et al., Respectively). al., 1990, J. Mol. Biol. 215: 403-410). BLAST and BLAST 2.0 are typically used with the initial parameters described herein to determine the percent sequence identity of the nucleic acids and proteins of the invention. Software for performing BLAST analyzes is publicly available by the National Center for Biotechnology Information. The algorithm first involves identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. Here, the query sequence matches or satisfies a certain positive number of reference scores T when arranged with words of the same length in the database sequence. T is considered the word close to the reference point (Altschul et al. Above). These hits near the beginning of the word serve as the root for initiating searches to find long HSPs containing that word. The word hits are extended in both directions along each sequence as long as the cumulative sequence score can be increased. For nucleotide sequences, the cumulative score is calculated by parameter M (reward score for matching residue pairs; always> 0) and parameter N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate a cumulative score. The expansion of word hits in each direction is due to the accumulation of the sequence of residues whose cumulative score is one or more negatively scored when the cumulative sequence score is reduced from the maximum achieved by the quantity X Is stopped when it reaches zero or less, or when the end of either sequence reaches the target. The BLAST algorithm parameters W, T and X determine the detection sensitivity of the sequence and the speed of the sequence. In the BLASTN program (for nucleotide sequences), word length (W) 11 is used as the default value, 10 is the expected value (E), M = 5, N = -4, and comparison of both strands. Regarding the amino acid sequence, the BLASTP program uses a word length of 3 as a default value, 10 as an expected value (E), and 50 as a BLOSUM62 score matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989). ) Arrangement (B), 10 is used as expected value (E), M = 5, N = -4, and comparison of both strands.

  The BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, eg, Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787). One similarity measure provided by the BLAST algorithm is the minimum total probability (P (N)). This minimum total probability indicates an indication of the probability that a match between two nucleotide sequences or between two amino acid sequences may occur by chance. For example, a nucleic acid is similar to a reference sequence when the minimum total probability is about 0.2 or less, more preferably about 0.01 or less, and most preferably about 0.001 or less in comparison of a test nucleic acid to a reference nucleic acid. Is considered to be.

  The term “stringent hybridization conditions” typically refers to conditions in a probe that hybridize to its target subsequence in a complex mixture of nucleic acids but not to another sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. Long sequences hybridize at particularly high temperatures. Extensive guidance for nucleic acid hybridization can be found in Tijssen (Technology in Biochemistry and Molecular Biology-Hybridization with Nucleic Acids, "Introduction to Hybridization Principles and Nucleic Acid Assays" (1993)). In general, at high stringency conditions, a particular sequence is selected to be about 5-10 ° C. below the melting point (Tm) at a defined ionic strength pH. Tm is the temperature complementary to target hybridization to a target sequence with 50% of the probes in equilibrium (when the target sequence is present excessively and 50% of the probes are in equilibrium at Tm). Stringent conditions are salt concentrations of about 1.0 M sodium ion or less, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, The condition is that the temperature is at least about 30 ° C. for short probes (eg, 10 to 50 nucleotides) and at least about 60 ° C. for long probes (eg, 50 nucleotides or more). Stringent conditions may also be achieved with the addition of destabilizing reagents (such as formamide). For selective or specific hybridization, a positive signal is at least 2-fold background, preferably 10-fold background hybridization.

  Nucleic acids that do not hybridize to each other under stringent conditions remain substantially the same if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is made by a degeneracy of the maximum amount of codons allowed by the genetic code. In such cases, the nucleic acid typically hybridizes under moderately stringent hybridization conditions.

  Genomic DNA or cDNA containing a GPT polypeptide can be identified in standard Southern blots under stringent conditions by the GPT polynucleotide sequences disclosed herein. For this purpose, the most stringent conditions for the hybridization include hybridization at 37 ° C. in a buffer of 40% formamide, 1M NaCl, 1% SDS, and at least about 50 ° C., usually about 55 ° C. And at least one wash in 0.2 X SSC for 20 minutes at a temperature of about 60 ° C.

  A further indication that two polynucleotides are substantially identical is that a cDNA or genomic library when a reference sequence (amplified by a pair of oligonucleotide primers) is used as a probe in stringent hybridization conditions. Is it possible to isolate the test sequence from or to be able to identify the test sequence, for example with a Northern or Southern blot.

(Transgenic plant)
The present invention provides novel transgenic plants with sufficiently improved growth characteristics and other agronomic properties. Fully improved agronomic properties include accelerated growth, increased live weight and total biomass of mature plants, earlier and more abundant flowering, and higher yields in fruits and seeds. It is not limited to. The transgenic plants of the present invention are generated by introducing one or more expressible genetic constructs into the plant. This genetic construct can facilitate the expression of one or more polynucleotides encoding glutamine phenylpyruvate transaminase (GPT). The present invention has been demonstrated, for example, by the generation of transgenic tobacco plants carrying and expressing heterologous Arabidopsis GPT genes (Example 2 described below). In addition, GPT homologs that function in the same metabolic pathway, ie, biosynthesis of the signal metabolite 2-hydroxy-5-oxoproline, are expected to be included in all plant species. Is done. Thus, in the practice of the present invention, any plant gene encoding a GPT homolog or functional variant thereof may be useful for the generation of the transgenic plants of the present invention.

  In the stable transformation embodiment of the present invention, one or more copies of the expressible genetic construct will be integrated into the host plant genome. This increases the GPT enzyme capacity in the plant. This serves to mediate an increase in the synthesis of 2-oxoglutaramate, in other words, signal metabolic gene expression. As a result, plant growth is increased and plant growth characteristics and other agronomic properties are improved. 2-Oxoglutaramate is a metabolite and a very powerful effector in gene expression, metabolism and plant growth (US Pat. No. 6,555,500). This metabolite can then play a pivotal role in regulating the carbon and nitrogen metabolic system (Lancien et al., 2000, Enzyme Redundancy and the Importance of 2-Oxoglutaratein Higher Plants Ammonium Assimilation, Plant Physiol. 123: 817-824). See also the schematic diagram of the 2-oxoglutarate pathway shown in FIG.

In one aspect of the invention, Applicants have identified a nucleic acid molecule encoding an Arabidopsis glutamine phenylpyruvate transaminase (GPT) enzyme (see Example 1 below). It was first shown that the expressed recombinant enzyme has activity and can promote the synthesis of the signal metabolite, 2-oxoglutaramate (Example 2 described later). In addition, Applicants have first shown that this Arabidopsis glutamine transaminase gene is overexpressed in transformed heterologous plants, resulting in improved CO ₂ fixation and increased growth characteristics ( Example 3 described later).

Also, as described herein (see Example 3 below), in a transgenic tobacco plant, overexpression of a transgene containing the full length of the GPT coding sequence of Arabidopsis accelerates CO ₂ fixation, and This results in increasing the amount of total protein, glutamine and 2-oxoglutaramate. In addition, these transgenic plants grow faster than wild-type plants (FIG. 2). Similarly, in tests on tomato plants (see Example 4 below), tomato plants transformed with the Arabidopsis GPT transgene are compared to wild type control plants for growth rate, flowering and seed yield. (Fig. 3 and Example 4 described later).

  In addition to the above-described transgenic tobacco plants, GPT-containing two types of tomato, pepper, legumes, cowpea, alfalfa, cantaloupe, pumpkin, Arabidopsis and Camilena, and exhibit improved growth characteristics A variety of transgenic plant species have been generated (US patent filed August 31, 2009, co-owned and co-pending application, the entire contents of which are incorporated herein by reference. See application specification 12 / 551,271). The transgenic plants described above were generated using various transformation methods. Various transformation methods include Agrobacterium-mediated callus, flower soaking, seed sowing, pod sowing, and direct seeding of flowers, combinations thereof, and one type using various GPT transgenes. Methods involving sexual crossbreeding in plants with multiple transgenes.

  The present invention also provides a method for producing transgenic plants with improved growth and other agronomic properties. In one embodiment, a method of generating a transgenic plant with improved growth and other agronomical properties is obtained by introducing a plant transformed with an expression cassette comprising a nucleic acid molecule encoding a GPT transgene. Producing a cell and obtaining a transgenic plant expressing the encoded GPT. The GPT transgene is under the control of a suitable promoter capable of promoting the expression of this transgene. In other embodiments, a method of generating a transgenic plant with improved growth and other agronomic properties introduces one or more nucleic acid constructs or expression cassettes comprising nucleic acid molecules encoding a GPT transgene into plant cells. Producing a transformed plant cell, and obtaining a transgenic plant that expresses a GPT transgene to produce a biologically active GPT protein. The GPT transgene is under the control of one or more suitable promoters (and optionally other control elements) that are capable of promoting the expression of the transgene.

  A number of GPT polynucleotides can be used to generate the transgenic plants according to the present invention. The experimental data described herein indicate that GPT proteins are highly conserved among various plant species, and closely related non-plant GPTs (eg, zebrafish GPT) can also be used. It is clear from With respect to GPT, numerous GPT polynucleotides from different species have been shown to be active and useful as GPT transgenes.

  In certain embodiments, the GPT transgene is a GPT polynucleotide that encodes a GPT from Arabidopsis thaliana (eg, the GPT shown in SEQ ID NO: 2, SEQ ID NO: 16, and SEQ ID NO: 25). This GPT transgene is a base sequence shown in SEQ ID NO: 1; a base sequence having at least 75% identity and more preferably at least 80% identity to SEQ ID NO: 1, and a base sequence encoding a polypeptide having GPT activity A base sequence encoding the polypeptide set forth in SEQ ID NO: 2, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and GPT activity; and amino terminal 30-56 amino acids Encoded by a base sequence that encodes the polypeptide shown in SEQ ID NO: 2, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and having GPT activity, wherein the residues are truncated. obtain.

  In another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a grape-derived GPT (eg, grape GPTs shown in SEQ ID NO: 4 and SEQ ID NO: 26). This GPT transgene is a base sequence shown in SEQ ID NO: 3; a base sequence having at least 75% and more preferably at least 80% identity with SEQ ID NO: 3, and a base sequence encoding a polypeptide having GPT activity It may be encoded by a base sequence encoding the polypeptide set forth in SEQ ID NO: 4 or SEQ ID NO: 26, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and GPT activity therewith.

  In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a rice-derived GPT (eg, rice GPTs shown in SEQ ID NO: 6 and SEQ ID NO: 27). This GPT transgene is a base sequence shown in SEQ ID NO: 5; a base sequence having at least 75% and more preferably at least 80% identity with SEQ ID NO: 5, and a base sequence encoding a polypeptide having GPT activity It may be encoded by a base sequence encoding a polypeptide set forth in SEQ ID NO: 6 or SEQ ID NO: 27, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and GPT activity therewith.

  In yet another specific embodiment, the GPT transgene encodes soybean-derived GPT (eg, soybean GPTs shown in SEQ ID NO: 8, SEQ ID NO: 28, or a sequence in which isoleucine is further added to the N-terminus of SEQ ID NO: 28). GPT polynucleotide. This GPT transgene is a base sequence shown in SEQ ID NO: 7; a base sequence having at least 75% and more preferably at least 80% identity with SEQ ID NO: 7, and a base sequence encoding a polypeptide having GPT activity A polypeptide shown in the sequence of SEQ ID NO: 8 or SEQ ID NO: 28 or SEQ ID NO: 28 with further addition of isoleucine at the N-terminus, or at least 75% and more preferably at least 80% sequence identity and GPT activity It can be encoded by a base sequence encoding a polypeptide having

  In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a barley-derived GPT (eg, barley GPTs shown in SEQ ID NO: 10 and SEQ ID NO: 29). This GPT transgene is a base sequence shown in SEQ ID NO: 9; a base sequence having at least 75% and more preferably at least 80% identity with SEQ ID NO: 9, and a base sequence encoding a polypeptide having GPT activity It may be encoded by a base sequence encoding a polypeptide set forth in SEQ ID NO: 10 or SEQ ID NO: 29, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and GPT activity with them.

  In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a zebrafish-derived GPT (eg, zebrafish GPTs shown in SEQ ID NO: 12 and SEQ ID NO: 30). This GPT transgene is a base sequence shown in SEQ ID NO: 11; a base sequence having at least 75% and more preferably at least 80% identity with SEQ ID NO: 11, and a base sequence encoding a polypeptide having GPT activity It may be encoded by a base sequence encoding a polypeptide set forth in SEQ ID NO: 12 or SEQ ID NO: 30, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and GPT activity therewith.

  In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a GPT derived from bamboo (eg, the bamboo GPT shown in SEQ ID NO: 31). This GPT transgene may be encoded by a base sequence encoding the polypeptide shown in SEQ ID NO: 31, or a polypeptide having at least 75% and more preferably at least 80% sequence identity and having GPT activity. .

  As can be appreciated by those skilled in the art, other GPT polynucleotides suitable for use as GPT transgenes in the practice of the present invention can be obtained by a variety of methods and include recombinant expression systems (ie, E. coli). ) (See Examples 20-23), transient expression systems in plants (see Example 19), or in transgenic plants (see Examples 1-18)) leading to GPT expression A method of testing for ability using GPT activity.

(Transgene construct / expression vector)
In order to generate a transgenic plant according to the present invention, the gene coding sequence for the desired transgene is a nucleic acid construct (herein referred to herein) that can direct the expression of the transgene sequence in transformed plant cells. (Transgene) must be incorporated into an expression vector / expression vector, expression cassette, expression construct or genetic construct, interchangeably cited). Nucleic acid constructs carrying a given transgene can be introduced into plant cells or various cells using a number of known methods. Known methods include, but are not limited to, electroporation, DNA bombardment or gene gun, microinjection, and various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes ) Vector)). Once introduced into the transformed plant cell, the nucleic acid construct will lead to the expression of the integrated transgene (ie, GPT) either transiently or stably. Stable expression is preferred, and stable expression is achieved by utilizing a plant transformation vector that allows the nucleic acid construct to be incorporated into the chromosome. Once the transformation into plant cells is successful, transgenic plants can be regenerated by cultivating them.

  Numerous expression vectors are known that are suitable for promoting the intrinsic expression or induction of inserted genes in transformed plants. In addition, various transient expression vectors and systems are known. In general, an appropriate expression vector is selected for use in a particular method in gene transformation (see below). In general, a typical plant expression vector for generating a transgenic plant can include a given transgene under expression control control of a promoter, a selectable marker to assist in the selection of transformants, and a transcription termination sequence. .

  More specifically, the basic elements of a nucleic acid construct for use in generating a transgenic plant according to the present invention are as follows: Functional expression of the transgene (s) in transformed plant cells A suitable promoter (s) operably linked to the promoter (or GPT coding sequence), preferably a suitable promoter operably linked to the transgene A transcription termination sequence (ie, a terminator of the nopaline synthase gene), and sometimes other elements useful for controlling the expression of this transgene, as well as one or more selections suitable for selecting the desired transgenic product Marker gene (ie antibiotic resistance gene).

Since Agrobacterium tumefaciens is an early transformation system used to generate transgenic plants, there are numerous vectors designed for Agrobacterium transformation. For stable transformation, Agrobacterium systems utilize “binary” vectors, which are plasmids that can be manipulated in both E. coli and Agrobacterium. This vector typically contains 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 used in Agrobacterium transformation systems typically include T-DNA border regions, multiple cloning sites, replication functions for E. coli and Agrobacterium tumefaciens, and selectable markers and reporter genes.

  So-called “super-binary” vectors provide high transformation efficiency and generally contain another virulence gene from Ti (Komari et al., 2006, Methods Mol. Biol. 343: 15-41). Super binary vectors are generally used for plants that exhibit low transformation efficiency (eg, cereals). Other virulence genes include, but are not limited to 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 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 genesimprove Agrobacterium-mediated transformation. Theor. Appl. Genet. 101, 1015-1020; Jin et al., 1987, Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. 169: 4417-4425).

  In the embodiments demonstrated herein (see the examples below), an expression vector is used in which the inserted transgene is placed under the control of the CaMV 35S promoter. A number of expression vectors utilizing the CaMV 35S promoter are known and / or commercially available. However, many promoters suitable for directing the expression of the transgene are known, and such promoters may be used in the practice of the invention as further described below.

(Plant promoter)
A number of promoters that function in plants are known. When constructing a GPT transgene construct, the promoter selected may be a constitutive non-specific promoter, such as the cauliflower mosaic virus 35S ribosomal promoter (CaMV 35S promoter). This promoter is widely used for transgene expression in plants. Examples of other strong constitutive promoters include, but are not limited to, rice actin 1 promoter, CaMV 19S promoter, Ti plasmid nopaline synthase promoter, alcohol dehydrogenase promoter and sucrose synthase promoter.

  Alternatively, in certain embodiments, a promoter is selected based on the plant cell that is to be transformed by the transgene construct, the desired expression level of the transgene, the tissue or intracellular compartment in which the transgene is to be expressed, the target developmental stage, etc. It is desirable to do.

  For example, when it is desired to express in photosynthetic tissues and compartments, the promoter of ribulose bisphosphate carboxylase (RuBisCo) gene can be used. When it is desired to express in seed, promoters of various storage protein genes can be used. For expression in fruits, a fruit-specific promoter (eg tomato 2A11) can be used. Specific examples of other tissue-specific promoters include lectin-encoding promoters (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), Light-harvesting complex (Simpson, 1986, Science, 233: 34-38; Bansal et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 3654-3658), maize heat shock protein (Odell et al. 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 Phosphorus 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 cells (Conkling et al., 1990, Plant Physiol. 93: 1203-1211), corn 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 synthase promoter (US 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).

  In addition to the constitutive promoter, various inducible promoter sequences can be used if it is desired to control the expression of the transgene as the transgenic plant regenerates, matures, blooms, etc. Examples of such inducible promoters are heat shock genes, defense response genes (ie phenylalanine ammonia lyase; see eg Bevan et al., 1989, EMBO J. 8 (7): 899-906), wound response Genes (ie, cell wall protein genes), chemically inducible genes (ie, nitrate reductase, chitinase) and dark inducible genes (ie, asparagine synthase; see, eg, US Pat. No. 5,256,558) Including promoters. Also, in the nucleus of many plants, including the gene family that encodes the major chlorophyll a / b binding protein (cab) and the gene family that encodes the small subunit of ribulose-1,5-2 phosphate carboxylase (rbcS) Genes are activated by light (eg Tobin and Silverthorne, 1985, Annu. Rev. Plant Physiol. 36: 569-593; Dean et al., 1989, Annu. Rev. Plant Physiol. 40: 415-439 See.)

  Other inducible promoters include ABA inducible promoter and turgor inducible promoter, auxin binding protein gene promoter (Schwob et al., 1993, Plant J. 4 (3): 423-432), UDP glucose flavonoid glycosyltransferase gene Promoter (Ralston et al., 1988, Genetics 119 (1): 185-197); MPI proteinase inhibitor promoter (Cordero et al., 1994, Plant J. 6 (2): 141-150), 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 the light-induced plastid glutamine synthase gene from peas (US Pat. No. 5,391,725; Edwards et al. ., 1990, supra).

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

(Glutamine phenylpyruvate transaminase (GPT) transgene)
The present invention first discloses a plant comprising a glutamine phenylpyruvate transaminase (GPT) enzyme that functions directly in the synthesis of the signal metabolite 2-hydroxy-5-oxoproline. So far, no transaminases of plants whose functions have been clarified have been described. Applicants have identified and tested GPT polynucleotide coding sequences from several plant and animal species. Then, this gene is incorporated into the host plant to produce a heterogeneous transgenic plant with faster growth, improved leaf protein content, faster CO ₂ fixation rate, and significantly improved growth characteristics. succeeded in.

GPTs that function in similar metabolic pathways including the biosynthesis of the signal metabolite 2-hydroxy-5-oxoproline are expected to be included in all plant species. Thus, in the practice of the present invention, any plant gene encoding a GPT homolog or a functional variant thereof may be useful for generating a transgenic plant according to the present invention. Furthermore, considering the structural identity between various plant GPT protein structures and putative (and biologically active) GPT homologues from zebrafish (Danio rerio (Zebrafish)) ( See Example 22), other non-plant GPT homologues can be used for the preparation of GPT transgenes for use in the production of transgenic plants according to the invention. When compared individually (by BLAST alignment) to the Arabidopsis mature protein sequence shown in SEQ ID NO: 25, the following sequence identity and homology (including BLAST “positive”, similar amino acids) for the following mature GPT protein sequences: was gotten.

  As emphasized that the GPT protein structure preserves features across many plant species, this conservation is not only within the plant species mentioned above, but also to putative non-human GPTs from zebrafish and Chlamydomonas. Can be seen. In the case of zebrafish, the range of identity is very high (83% identity in amino acid sequence with mature Arabidopsis GPT shown in SEQ ID NO: 25, and 92% homology if similar amino acid residues are added to the calculation). Zebrafish mature GPT was confirmed by expressing it in E. coli and demonstrating biological activity (2-oxoglutaramate synthesis).

  In order to determine whether a putative GPT homolog is suitable for generating transgenic plants with improved growth in the present invention, its coding sequence is expressed in E. coli or other suitable host, and It may be examined whether the amount of 2-oxoglutaramate signal metabolite synthesized increases (see Examples 19 to 23). If such an increase is shown, then the growth characteristics may be assessed after the coding sequence has been introduced into both homologous and heterologous plant hosts. For this purpose, any assay that can specifically detect 2-oxoglutaramate can be used. All assays include, but are not limited to, the NMR and HPLC assays described in Example 2 below. An assay for directly measuring mature GPT activity may also be used.

  Any plant GPT having 2-oxoglutaramate synthesis activity can be used to transform plant cells to produce transgenic plants according to the present invention. The level of structural homology between plant species is thought to be high, as evidenced by the close homology between the various plant GPT proteins and the zebrafish putative GPT homolog. Is considered to extend beyond plants. Accordingly, various plant GPT genes can be used to generate transgenic plants with enhanced growth in various heterologous plant species. Furthermore, if a GPT transgene is expressed in a homologous plant, control within the homologous cell may reduce the expression of the transgene in some way that would not occur in a heterologous cell. However, most would be expected to result in the desired improvement in growth characteristics (ie, over-expressing rice glutamine transaminase in transgenic rice plants).

(End of transcription)
In a preferred embodiment, a 3 ′ transcription termination sequence is incorporated downstream of the transgene to direct transcription termination and normalize polyadenylation of the mRNA transcript. Suitable transcription terminators are those known to function in plants, including but not limited to Agrobacterium tumefaciens nopaline synthase (NOS) and octopine synthase: OCS) gene, T7 transcript from octopine synthase gene, 3 ′ end of protease inhibitor I or II gene from potato or tomato, CaMV 35S terminator, tml terminator and pea rbcS E9 terminator. In addition, transcription termination factors in natural genes can be used. In one embodiment, a transcription terminator of nopaline synthase is used, which is described below as an example.

(Selection marker)
A selectable marker is usually included in the transgene expression vector to allow a means of selecting transformants. Various types of markers can be used, but various negative selection markers are usually utilized. Negative selection markers include markers that confer resistance to selective agents that inhibit or kill untransformed cells, such as antibiotics (eg, kanamycin, gentamicin, anamycin, hygromycin and hygromycin B). ), Or genes that confer resistance to herbicides (eg, sulfonylureas, gulfosinate, phosphinotricin and glyphosate). Screenable markers include, for example, a gene encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405), a gene 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, eg, US Pat. No. 6,573,432). The E. coli glucuronidase gene (gus, gusA or uidA) is a selectable marker that has been widely used in plant gene transfer. The main reason for this is the stability, high sensitivity and ease of detection of the glucuronidase enzyme (eg, fluorometric methods, spectrophotometric methods, various histochemical methods). Furthermore, there is essentially no detectable glucuronidase in the highest plant species.

(Transformation methods and systems)
Various methods for introducing the transgene expression vector construct of the present invention into plants or plant cells are well known to those skilled in the art, and any transformable plant or plant cell can be used as a target.

  Agrobacterium-mediated transformation is perhaps the most common method utilized for plant gene transfer. And Agrobacterium-mediated transformation protocols in many plants have been extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2nd edition, 2006). Agrobacterium tumefaciens is found in a large number of dicotyledonous species through the insertion of a small fragment of DNA that induces the mass (“T-DNA”, “transfer DNA”) into plant cells. Gram-negative soil bacteria that cause disease). T-DNA is integrated at semi-random locations in the plant genome and can eventually be stably integrated there. Directly repetitive DNA sequences called T-DNA borders define the left and right ends of T-DNA. T-DNA can be physically separated from the rest of the Ti plasmid to create a “binary vector” system.

  Agrobacterium transformation can be used to stably transform dicotyledonous, monocotyledonous plants, and their cells (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 of introducing DNA into Agrobacterium are known, including electroporation, freeze-thaw methods, and triparental mating. The most efficient way to place external DNA in 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, NJ, pp. 43-53). Furthermore, given that most of the T-DNA is not integrated, the use of Agrobacterium-mediated transformation allows the transgene to be mediated through the transcriptional ability of the non-integrated transgene construct molecule. Transient expression can be obtained (Helens et al., 2005, Plant Methods 1:13).

  A number of Agrobacterium transformation vectors and methods have been described (Karimi et al., 2002, Trends Plant Sci. 7 (5): 193-5). Many of these vectors are then commercially available (eg, Invitrogen, Carlsbad, CA). In addition, more “open source” Agrobacterium transformation vectors are available (eg, pCambia vectors; Cambia, Canberra, Australia). See also the section “Transgene Constructs” described hereinabove. In particular embodiments described in the Examples, the Horsch et al. Leaf disc transformation system (Horsch et al., 1995, Science 227: 1229-) is used to produce transgenic tobacco and tomato plants with improved growth. 1231), a vector derived from pMON316 was used.

  Other commonly used transformation methods that can be used to generate transgenic plants according to the present invention include, but are not limited to, microprojectile bombardment or biolistic transformation methods, protoplast transformation of naked DNA with 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).

  Microparticle gun transformation involves injecting millions of metal particles coated with DNA into target cells or tissues using a microparticle gun apparatus (or “gene gun”). Various types of particle guns are commercially available. Once in the cell, the DNA is eluted from the particle and a portion can be stably incorporated into one or more chromosomes of the cell (for review, 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, NJ).

  Electroporation is a technique that reversibly permeates the lipid bilayer of cell membranes using a short, high-intensity electric field (eg, 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, NJ, 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 ass ays, in Cell Biology, Vol. 4, ed., Celis, Academic Press, London, UK, pp. 92-99). This technique is performed by creating water-soluble pores in the cell membrane. The pores are large enough to allow DNA molecules (and other macromolecules) to enter the cell. A transgene expression construct (such as T-DNA) can then be stably integrated into plant genomic DNA, leading to the generation of transformed cells, which can then be regenerated into transgenic plants. .

Newer transformation methods include the so-called “floral dip” method. The floral dip method, like all other commonly used transformation methods, does not require plant tissue culture and is expected to be simple (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, NJ, 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, except for Arabidopsis, these methods are not widely used across a wide variety of plant species. Briefly, transformation by floral dip is accomplished by dipping a flowering plant or spraying the flowering plant with a suitable strain of Agrobacterium tumefaciens. Thereafter, seeds collected from the three T ₀ plants were germinated, and transgenic T ₁ individuals were identified and selected. Example 16 shows that Arabidopsis plants were inoculated by dipping flowers to produce transgenic Arabidopsis plants.

  Other transformation methods include a method of transforming seeds or seedlings of a growing plant using an Agrobacterium vector or the like. For example, such vectors can be used to transform growing seeds by directly injecting a suspension or mixture of the vector (ie, Agrobacterium) into a cavity in the growing seed pod. Can be used (Wang and Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). Seedlings can be transformed as described in Yasseem, 2009, Plant Mol. Biol. Reporter 27: 20-28. Germinated seed may be transformed as described in Chee et al., 1989, Plant Pysiol. 91: 1212-1218. A fruit internal method in which the vector is injected into the fruit or growing fruit may also be used. Other transformation methods also include methods that target floral organs for vector inoculation, such as flower inoculation methods.

  The plant transformation methods described above can be used to introduce transgenes into many types of plant cells and tissues. Plant cells and tissues include, but are not limited to, whole plants, transplants of tissues and organs including chloroplasts, transplants of tissues and organs including chloroplasts, flowering tissues and cells, protoplasts, meristem cells, callus, This includes immature embryos and gametes, such as microspores, pollen, sperm and egg cells, cultured cells of the tissues described above, and any other cells from which regenerated, reproductive transgenic plants can be generated. Callus arises from tissue sources. Tissue sources include, but are not limited to, immature embryos, seedling apical meristems, microspores, and the like. Cells that can grow as callus are also cells that undergo genetic transformation.

  Methods for regenerating individual plants from transformed plant cells, tissues and organs are known and described for many plant species.

  By way of example, transformed plantlets (derived from transformed cells or tissues) are cultured in an implantable growth medium supplemented with a selective agent (ie, an antibiotic such as kanamycin) used in the transformation method. This transformed plantlet is transferred to soil after rooting and allowed to grow fully. After flowering, mature plants are preferably self-pollinated. The resulting seed is then collected and used to grow the next generation. Examples 3-6 describe the regeneration of transgenic tobacco plants and transgenic tomato plants.

The T ₀ transgenic plant can be produced by self-pollination of the first or second transformant or of the first or second transformant of another plant (transformed or non-transformed plant). It can be used to generate the next generation (eg, T ₁ , T _2, etc.) by sexing.

(Selection of transgenic plants with improved growth)
Transgenic plants can be selected, screened and identified using standard methods. Preferred transgenic plants of the present invention may exhibit one or more phenotypic characteristics that exhibit improved growth and / or other desired agronomic properties. Transgenic plants are usually regenerated under selective pressure to select for transformants, followed by transgenic plant production. Further, the selection pressure used in order to confirm the presence of desired transgene expression construct or cassette may be used in excess of T ₀ generation.

A T ₀ transformed plant cell, callus, tissue or plant selects for the genetic composition of the marker gene contained in the transgene expression construct used for transformation and / or the phenotypic characteristics encoded by the marker gene Or can be identified and isolated by screening. For example, by growing a potentially transformed plant, tissue or cell in a growth medium containing a growth-suppressing amount of an antibiotic or herbicide that can be tolerated by a genetic construct Selection can be made. In addition, transformed plant cells, tissues and plants can also be identified by screening for the activity of a marker gene (ie, β-glucuronidase) that may be present in the transgene expression construct.

  As is well known, various physical and biochemical methods can be used to identify plants containing the desired transgene expression construct. Specific examples of these methods include, for example, Southern blot analysis or various nucleic acid amplification methods (so-called PCR) to identify transgenes, transgene expression constructs or elements thereof, for detecting and determining RNA transcripts. Northern blotting, S1 RNase protection, reverse transcriptase PCR (RT-PCR) amplification, protein gel electrophoresis to identify proteins encoded and expressed by transgenes, Western blotting, immunoprecipitation, and enzyme immunoassays Etc. can be used.

  In other means, the expression levels of genes, proteins and / or metabolic compounds known to be modified by transgene expression in the target plant can be used to identify transformants. In one embodiment according to the invention, an increase in the amount of signal metabolite 2-oxoglutaramate can be used to screen for desired transformants.

  Finally, the transformed plants of the present invention can be screened using improved growth and / or other desired agronomic properties. In fact, especially when identifying plants by subsequent self-pollination, cross-breeding and backcrossing, to identify transformation lines with the fastest growth rate, highest seed yield, etc. Screening is usually desirable. Various parameters can be used for this purpose. Parameters include, but are not limited to, growth rate, total body weight, dry weight, seed and fruit yield (number, weight), seed and / or seed pod size, seed pod yield (eg, number, weight), Leaf size, plant size, flowering improvement, timing of flowering, overall protein content (seed, fruit, in plant tissue), specific protein content (ie GS), nitrogen content, free amino acids, and specific metabolic compounds Amount (ie 2-oxoglutarate). In general, these phenotypic measurements are taken from the same or similar plant line as the parent, the same or similar plant as the non-transformed plant, or the same or similar plant as the wild type plant (ie, standard Compared to measurements obtained from plants or parent plants). Preferably, and at least initially, the measurement of the selected phenotypic characteristic in the target transgenic plant is performed in parallel with the measurement of the same characteristic in the standard plant or the parent plant. Typically, multiple plants are used to establish phenotypic preferences and / or dominance in transgenic plants for specific phenotypic characteristics.

Preferably, the first transformant will autologize T ₁ and subsequent generations after selection until the transgene genotype maintains the ancestral trait (ie, the plant is homozygous for the transgene). Used to produce by pollination. In practice, this is accomplished by self-pollination for each generation, screening for the desired traits in each generation, and self-pollination of each of them (usually repeated (ie, 3 or 4 generations)).

  Stable transgenic lines may be crossed and backcrossed to produce variants with any number of desired traits. Variants include variants with stacked transgenes, variants with multiple copies of the transgene, and the like. Various general breeding methods are known to those skilled in the art (see, for example, Breeding Method for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)). In addition, stable transgenic plants may be further genetically modified by further transforming these plants with additional copies of the transgene or parental transgene. It is also contemplated to create a transgenic plant by performing a single transformation that introduces a given transgene or multiple copies of a plurality of transgenes.

〔Example〕
Various aspects of the invention are further described and illustrated by means of several examples, which are not intended to limit the scope of the invention.

<Example 1: Isolation of Arabidopsis glutamine phenylpyruvate transaminase (GPT) gene>
In an attempt to find a plant enzyme that is directly involved in the synthesis of the signal metabolite 2-oxoglutaramate, Applicants have found that the putative plant enzyme is involved in the synthesis of 2-oxoglutaramate. It was hypothesized that it might have some degree of structural association with the human protein that is characterized as Glutamine transaminase K (EC 2.6.1.64) which is a human protein (also referred to in the literature as cysteine-conjugated β-lyase, kynurenine aminotransferase, glutamine phenylpyruvate transaminase, etc.) It has been shown to be involved in the processing of cysteine conjugation of halogenated xenobiotics (Perry et al., 1995, FEBS Letters 360: 277-280). Human cysteine-conjugated β-lyase has detoxifying activity in humans and animals rather than having an activity involved in nitrogen assimilation (Cooper and Meister, 1977, supra). Nevertheless, the potential involvement of this protein in the synthesis of 2-oxoglutaramate was important.

  Using the protein sequence of human cysteine-coupled β-lyase, a database of protein sequences of TIGR Arabidopsis plants is searched to find one possible related sequence, a partial located at At1q77670 of the Arabidopsis gene. The polypeptide encoded by the sequence was identified. This polypeptide shares approximately 36% sequence homology in the aligned region.

  The full-length gene coding region was amplified from an Arabidopsis thaliana cDNA library using the following primers.

5'- CCCATCGATGTACC TGGACATAAATGGTGTGATG-3 '[SEQ ID NO: 32]
5'-GATGGTACCTCAGACTTTTCTCTTAAGCTTCTGCTTC-3 '[SEQ ID NO: 33]
These primers were designed to include ClaI (ATCGAT) and KpnI (GGTACC) restriction enzyme sites in order to facilitate subcloning with expression vectors to create the transgenic plants described below. High performance PCR was performed using TaKaRa ExTaq DNA polymerase enzyme under the following conditions: first denatured at 94 ° C. for 4 minutes, denatured at 94 ° C. for 30 seconds, annealed at 55 ° C. for 30 seconds, and at 72 ° C. A 90 second extension reaction is performed for 30 cycles, and finally an extension reaction is performed at 72 ° C. for 7 minutes. The amplified product was cleaved with ClaI and KpnI restriction enzymes, isolated by agarose gel electrophoresis, and then cauliflower mosaic virus (CaMV) 35S constitutive promoter and nopaline synthase (NOS) 3 ′ terminator. Was ligated to the vector pMon316 (Rogers, et. Al. 1987 Methods in Enzymology 153: 253-277) containing The ligation product was transformed into DH5α cells and the transformant sequence was determined to confirm insertion.

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

Example 2: Production of biologically active Arabidopsis glutamine phenylpyruvate transaminase
As described in Example 1 above, to determine whether the protein encoded by the isolated cDNA can catalyze the synthesis of 2-oxoglutaramate, the cDNA is expressed in E. coli. Thus, the ability to synthesize 2-oxoglutaramate was assayed by standard methods.

(NMR assay of 2-oxoglutaramate)
Briefly, the purified protein product was added to a reaction containing 150 mM Tris-HCl (pH 8.5), 1 mM β-mercaptoethanol, 200 mM glutamine, 100 mM glyoxylic acid and 200 μM pyridoxal 5′-phosphate. A reaction solution to which the protein to be tested was not added was used as a control. The test reaction solution and the control reaction solution were incubated at 37 ° C. for 20 hours and purified by centrifugation to remove precipitates. The supernatant was tested to analyze the presence and amount of 2-oxoglutaramate using ¹³ C NMR, with reference to chemically synthesized standard 2-oxoglutaramate. The reaction products were 2-oxoglutaramate and glycine, while the substrate (glutamine and glyoxylic acid) decreased in abundance. Cyclic 2-oxoglutaramate produced an entirely different signal that was easily distinguishable from the open chain glutamine precursor.

(HPLC assay of 2-oxoglutaramate)
An alternative assay for GPT activity measured 2-oxoglutaramate product using HPLC following denaturation of Calderon et al., 1985, J Bacteriol 161 (2): 807-809. Briefly, a denaturing extraction buffer containing 25 mM Tris-HCl (pH 8.5), 1 mM EDTA, 20 μM FAD, 10 mM cysteine and ˜1.5% (v / v) mercaptoethanol was used. Tissue samples from the test substances (ie plant tissues) were added to the extraction buffer at a ratio of about 1/3 (w / v), incubated for 30 minutes at 37 ° C. and stopped with 200 μM 20% TCA. After about 5 minutes, the assay solution is centrifuged and the supernatant is used to HPLC (ION-300 at a mobile phase of 0.01 NH ₂ SO ₄ , flow rate of about 0.2 ml / min, 40 ° C.) 2-oxoglutaramate was quantified by using 8 mm ID X 30 cm L column). The injection volume is about 20 μl and the retention time is approximately 38 to 39 minutes. Detection is performed with 210 nm UV light.

(Result of NMR assay)
This experiment demonstrated that the test protein can catalyze the synthesis of 2-oxoglutaramate. Thus, these data indicate that the isolated cDNA encodes a glutamine phenylpyruvate transaminase that is directly involved in the synthesis of 2-oxoglutaramate in plants. Therefore, the test protein can be said to be Arabidopsis glutamine phenylpyruvate transaminase, that is, GPT.

  The nucleotide sequence of the coding sequence of Arabidopsis GPT is shown in SEQ ID NO: 1. The translated amino acid sequence of GPT protein is shown in SEQ ID NO: 2.

<Example 3: Production of transgenic tobacco plants overexpressing Arabidopsis GPT>
(Generation of plant expression vector pMON-PJU)
Briefly, a plant expression vector pMon316-PJU was prepared as follows. The isolated cDNA encoding GPT of Arabidopsis thaliana (Experimental Example 1) was inserted into the ClaI-KpnI polylinker site of the pMon316 vector and cloned. This places the GPT gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) transcription terminator. A kanamycin resistance gene was added to provide a selectable marker.

(Plant transformation via Agrobacterium)
pMON-PJU and control vector pMon316 (without inserted DNA) were transformed into pTiTT37ASE of the Agrobacterium tumefaciens strain using standard electroporation methods (McCormac et al., 1998, Molecular Biotechnology 9: 155-159). Conversion was followed by spreading on LB plates containing the antibiotics spectinomycin (100 μg / ml) and kanamycin (50 μg / ml). Antibiotic resistant colonies of Agrobacterium were tested by PCR and confirmed to contain the plasmid.

  Nicotiana tabacum cv Xanthi plants were transformed into Agrobacterium using Horsch et al., 1995, Science 227: 1229-1231 using the leaf disc transformation method of Horsch et al. Transformation was performed with PJU. Briefly, a sterile leaf disc was inoculated, cultured for 2 days, and transferred to selective MS medium containing 100 μg / ml kanamycin and 500 μg / ml clafaran. Transformants were confirmed by their ability to form roots in selective media.

(Generation of GPT transgenic tobacco plants)
Sterile leaf sections were developed into callus on Murashige & Skog (M & S) medium from which transformed plantlets emerged. Then, these plantlets were transplanted to a selective medium capable of rooting (M & S medium containing kanamycin as a selective reagent). Thereafter, normal, rooted, transformed tobacco plantlets were transplanted to soil, grown to maturity, and flowered plants were self-fertilized and the resulting seeds were harvested. During growth, the growth phenotype of the plants was examined and the CO ₂ fixation rate was measured in many young transgenic plants.

(Generation of T ₁ and T ₂ generation GPT transgenic plants)
The seeds harvested from the T ₀ generation of transgenic tobacco plants, germinated in the M & S medium containing kanamycin (100mg / L), enhanced the quality of the transgene. At least 1/4 of the seeds do not germinate in this medium (kanamycin is expected to inhibit germination of non-tolerant seeds that can result from normal gene segregation) and more than half of the remaining seeds Was removed because it was sensitive (very weak) to kanamycin.

Grown surviving plants (T ₁ generation), these plants were self fertilized to produce T ₂ generation seeds. Seeds from T ₁ generation are the kanamycin (10 mg / L) were germinated added MS medium for strains of transformants. After 14 days, the seeds were transferred to sand and given a quarter concentration of Hogland nutrient solution supplemented with 25 mM potassium nitrate. This was grown at 24 ° C. with a light period of 16 hours light period and 8 hours dark period with a light intensity of 900 μmol / m ² / sec. They were harvested 14 days after transplanting into a sand culture.

(Characterization of GPT transgenic plants)
Harvested transgenic plants (both GPT transformants and vector control transformants) were treated with root and leaf glutamine synthetase activity, total amount of undried plants, root and leaf total protein and CO ₂ fixation rate ( Knight et al., 1988, PlantPhysiol. 88: 333). Untransformed wild type A. Tumefaciens plants were also analyzed with the same parameters to provide a baseline control.

The results of growth characteristics are shown in Table 1 below. Furthermore, a photograph of a GPT transgenic plant compared to a wild type control plant is shown in FIG. 2 (shown with the GS1 transgenic tobacco plant). Over all the parameters evaluated, GPT transgenic tobacco plants show accelerated growth characteristics. In particular, GPT transgenic plants showed a 50% increase in CO ₂ fixation rate and glutamine synthetase activity in leaf tissue was doubled compared to wild type control plants. Furthermore, the GS ratio between leaves and roots increased almost 3-fold in transgenic plants of transaminase compared to wild-type controls. Raw body weight and total protein were also increased by about 50% and 80% (leaves) in transgenic plants compared to wild type controls, respectively. These data demonstrate that tobacco plants that overexpress the Arabidopsis GPT transgene acquire significantly enhanced growth and CO ₂ fixation rates.

  Data = average value of 3 individuals.

  Wild-type control plant; not regenerated or transformed.

  The PN1 line was generated by regeneration after transformation with a construct having no transgene.

  Control over regeneration and transformation processes. The PN9 line was generated by regeneration after transformation with a construct having the Arabidopsis GPT gene.

<Example 4: Production of transgenic tomato plant having Arabidopsis GPT gene>
A transgenic tomato (Lycopersicon esculentum) (microtom tomato) plant carrying the Arabidopsis GPT gene was generated using the vectors and methods described in Example 3. T ₀ transgenic tomato plants were created and grown to maturity. Data on the initial growth characteristics of the transgenic tomato plants are shown in Table 2. Transgenic plants showed significant enhancement of growth rate, flowering and seed yield compared to wild type control plants. In addition, transgenic plants developed multiple main stems, while wild type plants developed a single main stem. A photograph of a GPT transgenic tomato plant compared to a wild type plant is shown in FIG.

<Example 5: Activity of barley GPT transgene in plant>
In this example, the predicted coding sequence of barley GPT was isolated and expressed from the transgene construct by a transient expression assay in plants. Biologically active recombinant barley GPT was generated and confirmed by HPLC to catalyze increased synthesis of 2-oxoglutaramate.

  The coding sequence of barley (Hordeum vulgare) GPT was determined and synthesized. The DNA sequence of the barley GPT coding sequence used in this example is shown in SEQ ID NO: 9, and the amino acid sequence of the encoded GPT protein is shown in SEQ ID NO: 10.

  The barley GPT coding sequence was inserted into a 1305.1 Cambia vector and LBA404 of the Agrobacterium tumefaciens strain using standard electroporation methods (McCormac et al., 1998, Molecular Biotechnology 9: 155-159). And spread on LB plates containing hygromycin (50 μg / ml). Antibiotic resistant colonies of Agrobacterium were selected for analysis.

Tobacco leaf transient expression assay was performed using transformed Agrobacterium (1.5-2.0
OD ₆₅₀ ) suspension is poured into rapidly growing tobacco leaves. Intradermal injection was performed in a lattice across the leaf surface to ensure that a significant amount of the leaf surface was exposed to Agrobacterium. Plants were grown for 3-5 days and tissues were extracted as described for all other tissue extractions and GPT activity was measured.

  The GPT activity (1217 nmol / g FWt / h) of the inoculated leaf tissue is 3 times the activity measured in the leaf tissue of the control plant (407 nmol / g FWt / h), and the soybean (Hordeum) GPT construct is a transgenic plant. Have been shown to lead to the expression of biologically active GPT.

<Example 6: Isolation and expression of recombinant rice GPT gene coding sequence and analysis of biological activity>
In this example, a putative coding sequence for rice GPT was isolated and expressed in E. coli. Biologically active recombinant rice GPT was generated and confirmed by HPLC to catalyze increased synthesis of 2-oxoglutaramate.

[Materials and methods]
(Rice GPT coding sequence and expression in E. coli)
The rice (Oryza sativa) GPT coding sequence was determined, synthesized, introduced into the PET28 vector, and expressed in E. coli. That is, Escherichia coli cells were transformed with an expression vector, a transformant cultured overnight in an LB medium was diluted, grown to OD0.4, and induced with isopropyl-β-D-thiogalactoside (0.4 μM). And then collected by culturing for 3 hours. The biological activity of a total of 25 × 10 ⁶ cells was then analyzed by the following NMR assay. Untransformed wild type E. coli cells were analyzed as controls. For additional controls, E. coli cells transformed with an empty vector were used.

  The DNA sequence of the rice GPT coding sequence used in this example is shown in SEQ ID NO: 5, and the amino acid sequence of the encoded GPT protein is shown in SEQ ID NO: 6.

(HPLC assay of 2-oxoglutaramate)
Calderon et al., 1985, J Bacteriol 161 (2): Following denaturation of 807-809, the amount of 2-oxoglutaramate produced in E. coli cells overexpressing GPT was measured using HPLC. That is, a denatured extraction buffer containing 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 (25 × 10 ⁶ lysates of E. coli cells) were added to the extraction buffer at about 1/3 ratio (w / v), incubated at 37 ° C. for 30 minutes, and stopped with 200 μM 20% TCA. After about 5 minutes, the assay solution is centrifuged and the supernatant is used to move the mobile phase of 0.01N H ₂ SO ₄ , flow rate of about 0.2 ml / min, ION-300 7.8 mm ID X 30 cm L at 40 ° C. 2-Oxoglutaramate was quantified by HPLC using column. The injection volume is about 20 μl and the retention time is approximately 38 to 39 minutes. Detection was performed with 210 nm UV light.

  Comparison of NMR assay with standard 2-oxoglutaramate was used to demonstrate that the full-length Arabidopsis thaliana sequence expresses GPT with 2-oxoglutaramate synthesis activity. That is, to validate the above HPLC assay by confirming that the product of the assay (molecule synthesized in response to expressed GPT) and standard 2-oxoglutaramate elute with the same retention time. In addition, standard 2-oxoglutaramate (structure confirmed by NMR) was prepared by chemical synthesis. In addition, the assay product and standard compound were mixed together and eluted as a single peak. In addition, validation of the HPLC assay included monitoring the disappearance of the substrate glutamine and showing that there was a 1: 1 molar ratio between glutamine consumption and 2-oxoglutaramate production. . The analytical process always includes two controls, one with no enzyme added and one with no glutamine added. First, it shows that the product of 2-oxoglutaramate is dependent on the presence of the enzyme, and secondly, the production of 2-oxoglutaramate is dependent on the substrate glutamine.

(result)
Expression of the rice GPT coding sequence of SEQ ID NO: 5 resulted in overexpression of a recombinant GPT protein with the biological activity of a 2-oxoglutaramate synthesis catalyst. In particular, 1.72 nmol of 2-oxoglutaramate activity was found in E. coli cells overexpressing recombinant rice GPT, and only 0.02 nmol of 2-oxoglutaramate activity in control E. coli cells. Compared to the above, an 86-fold increase in activity level was observed.

<Example 7: Isolation and expression of recombinant soybean GPT gene coding sequence and analysis of biological activity>
In this example, a putative coding sequence for soybean GPT was isolated and expressed in E. coli. Biologically active recombinant soybean GPT was produced and confirmed by HPLC to catalyze increased synthesis of 2-oxoglutaramate.

(Materials and methods)
(Soybean GPT coding sequence and expression in E. coli)
The GPT coding sequence of soybean (Glycine max) was determined, synthesized, introduced into the PET28 vector, and expressed in E. coli. That is, E. coli cells were transformed with an expression vector, a transformant cultured overnight in an LB medium was diluted, cultured to OD0.4, and induced with isopropyl-β-D-thiogalacside (0.4 μM). The cells were cultured for 3 hours and collected. Biological activity was analyzed using the following HPLC assay with a total of 25 × 10 ⁶ cells. Untransformed wild type E. coli was analyzed as a control. For additional controls, E. coli cells transformed with an empty vector were used.

  The DNA sequence of the soybean GPT coding sequence used in this example is shown in SEQ ID NO: 7, and the amino acid sequence of the encoded GPT protein is shown in SEQ ID NO: 8.

(HPLC assay of 2-oxoglutaramate)
As described in Example 20 above, HPLC was used to determine the production of 2-oxoglutaramate in E. coli cells overexpressing GPT.

(result)
Expression of the soybean GPT coding sequence of SEQ ID NO: 7 resulted in overexpression of the biologically active recombinant GPT protein of the 2-oxoglutaramate synthesis catalyst. In particular, 31.9 nmol of 2-oxoglutaramate activity was found in E. coli cells overexpressing recombinant soybean GPT, and only 0.02 nmol of 2-oxoglutaramate activity in control E. coli cells. Compared to the above, an increase in activity level nearly 1600 times was observed.

<Example 8: Isolation and expression of recombinant zebrafish GPT gene coding sequence and analysis of biological activity>
In this example, the predicted coding sequence of zebrafish GPT was isolated and expressed in E. coli. Biologically active recombinant zebrafish GPT was generated and confirmed by HPLC to catalyze the increased synthesis of 2-oxoglutaramate.

(Materials and methods)
(Zebrafish GPT coding sequence and expression in E. coli)
The zebrafish (Danio rerio) GPT coding sequence was determined, synthesized, introduced into the PET28 vector and expressed in E. coli. That is, E. coli cells were transformed with an expression vector, a transformant cultured overnight in an LB medium was diluted, cultured to OD0.4, and induced with isopropyl-β-D-thiogalacside (0.4 μM). The cells were cultured for 3 hours and collected. Biological activity was analyzed using the following HPLC assay with a total of 25 × 10 ⁶ cells. Untransformed wild type E. coli was analyzed as a control. For additional controls, E. coli cells transformed with an empty vector were used.

  The DNA sequence of the coding sequence of zebrafish GPT used in this example is shown in SEQ ID NO: 11, and the amino acid sequence of the encoded GPT protein is shown in SEQ ID NO: 12.

(HPLC assay of 2-oxoglutaramate)
As described in Example 6 above, HPLC was used to determine the production of 2-oxoglutaramate in E. coli cells overexpressing GPT.

(result)
Expression of the zebrafish GPT coding sequence of SEQ ID NO: 16 resulted in overexpression of a recombinant GPT protein with the biological activity of 2-oxoglutaramate synthesis catalyst. In particular, 28.6 nmol of 2-oxoglutaramate activity was observed in E. coli cells overexpressing recombinant zebrafish GPT, and only 0.02 nmol of 2-oxoglutaramate in control E. coli cells. Compared to the activity, the activity level increased by 1400 times or more.

Example 9: Isolation and expression of truncated recombinant Arabidopsis GPT gene coding sequence and analysis of biological activity
In this example, to construct two different fragments of the predicted coding sequence of Arabidopsis GPT and to evaluate the activity of a truncated or truncated GPT protein lacking the putative chloroplast signal peptide, It was expressed in E. coli. Recombinant fragment GPT corresponding to the full-length amino acid sequence of Arabidopsis GPT of SEQ ID NO: 2, truncated to delete the first 30 amino-terminal amino acid residues or the first 45 amino-terminal amino acid residues The protein was successfully expressed and, as confirmed by HPLC, showed biological activity catalyzing increased synthesis of 2-oxoglutaramate.

(Materials and methods)
(Truncated Arabidopsis GPT coding sequence and expression in E. coli)
A DNA coding sequence of the Arabidopsis thaliana GPT coding sequence fragment of SEQ ID NO: 1 was constructed, synthesized, introduced into a PET28 vector and expressed in E. coli. The DNA sequence of the truncated Arabidopsis GPT coding sequence used in this example is shown in SEQ ID NO: 15 (-45AA construct), and the amino acid sequence of the corresponding truncated GPT protein is shown in SEQ ID NO: 16. That is, E. coli cells were transformed with an expression vector, a transformant cultured overnight in an LB medium was diluted, cultured to OD0.4, and induced with isopropyl-β-D-thiogalacside (0.4 μM). The cells were cultured for 3 hours and collected. A total of 25 × 10 ⁶ cells were analyzed for biological activity using an HPLC assay as described in Example 20. Untransformed wild type E. coli was analyzed as a control. For additional controls, E. coli cells transformed with an empty vector were used.

  Expression of the truncated -45 Arabidopsis GPT coding sequence of SEQ ID NO: 15 refers to overexpression of a recombinant GPT protein with biological activity (biological activity catalyzing the synthesis of 2-oxoglutaramate). The result was. In particular, the activity of 16.1 nmol of 2-oxoglutaramate was found in E. coli cells overexpressing truncated -45 GPT, and only 0.02 nmol of 2-oxoglutaramate in control E. coli cells. Compared to the activity of the mate, the activity level increased by 800 times or more. For comparison, the same E.I. In the full-length coding sequence of the Arabidopsis gene expressed in E. coli, 2.8 nmol of 2-oxoglutaramate was generated, and approximately 1/5 of the activity of the truncated recombinant GPT protein was observed.

  All publications, patents, and patent applications cited in this specification are hereby incorporated by reference so that individual publications or patent applications are clearly and individually indicated to be incorporated by reference. Incorporated into.

  The scope of the present invention is not limited by the specific examples disclosed in this specification, and the specific examples are intended to be examples of individual aspects of the invention. Within range. Various modifications of the inventive model and method, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and guidance and are intended to be within the scope of the invention as well. . Such changes or other embodiments may be practiced without departing from the proper scope and spirit of the invention.

  [Sequence Listing]

FIG. 2 shows a schematic metabolic pathway of nitrogen absorption and 2-oxoglutaramate chemical synthesis. It is a photograph which shows the comparison of the transgenic tobacco plant which overexpresses GPT with respect to a wild-type tobacco plant. It is a photograph which shows the comparison of the transgenic microtome tomato plant which overexpresses GPT with respect to a wild-type tomato plant. It is a photograph showing a comparison of leaf size between a wild-type tobacco plant (upper leaf) and a GPT transgenic tobacco plant (lower leaf).

Claims (20)

A glutamine phenylpyruvate transaminase (GPT) transgene operably linked to a plant heterologous promoter,
The GPT transgene is
Encoding a GPT polypeptide having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having GPT enzyme activity;
A transgenic plant that produces more 2-oxoglutaramate compared to a similar wild-type plant of the same species.   The transgenic plant according to claim 1, wherein the GPT transgene is incorporated in the genome of the plant.   The transgenic plant according to claim 2, which is a monocotyledonous plant.   The transgenic plant according to claim 2, which is a dicotyledonous plant. Progeny of the transgenic plant according to claim 2,
A progeny containing the GPT transgene. The seed of the transgenic plant according to claim 2,
Seeds containing the GPT transgene.   Increased growth rate, increased biomass yield, increased seed yield, increased flower or flower bud yield, increased fruit or compared to similar wild-type or non-transformed plants of the same species Spodum yield, large leaves, increased GPT activity, increased GS activity, increased 2-oxoglutaramate level, improved nitrogen utilization efficiency, and / or enhanced tolerance to salt or salinity The transgenic plant according to claim 2, wherein A method for improving the growth characteristics of a plant as compared to a similar wild type plant or non-transformed plant, comprising:
(A) having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having an amino acid sequence having glutamine phenylpyruvate transaminase (GPT) enzyme activity Transforming a plurality of plants with a GPT transgene encoding a GPT polypeptide;
(B) expressing a GPT transgene in the plurality of plants or their progeny;
(C) selecting a transgenic plant having an increased biomass yield compared to a similar wild-type plant of the same species from the plurality of plants;
And
The method wherein the transgenic plant produces more 2-oxoglutaramate compared to a similar wild type plant of the same species.   The transgenic plant has early flowering, early germination, increased plant height, increased flowering, increased germination, larger leaves, increased fruit or pod yield, and increased seed yield. 9. The method of claim 8, wherein the method exhibits at least one additional increased growth characteristic selected from the group consisting of yields. A method for improving the nitrogen utilization efficiency of a plant as compared to a similar wild type plant or non-transformed plant,
(A) having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having an amino acid sequence having glutamine phenylpyruvate transaminase (GPT) enzyme activity Transforming a plurality of plants with a GPT transgene encoding a GPT polypeptide;
(B) expressing a GPT transgene in the plurality of plants or their progeny;
(C) selecting a transgenic plant having an increased nitrogen utilization efficiency from the plurality of plants compared to a similar wild-type plant of the same species;
And
The GPT transgene is operably linked to a plant heterologous promoter;
The method wherein the transgenic plant produces more 2-oxoglutaramate compared to a similar wild type plant of the same species. A method of improving germination or growth tolerance of plant seeds in a salt or salinity environment compared to seeds produced by similar wild type plants or non-transformed plants,
(A) having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having an amino acid sequence having glutamine phenylpyruvate transaminase (GPT) enzyme activity Transforming a plurality of plants with a GPT transgene encoding a GPT polypeptide;
(B) expressing a GPT transgene in the plurality of plants or their progeny;
(C) selecting a transgenic plant having enhanced growth characteristics compared to a similar wild-type plant of the same species;
(D) harvesting seeds from the plant and selecting seeds that show improved germination in a high salinity environment;
And
The GPT transgene is operably linked to a plant heterologous promoter;
The method wherein the transgenic plant produces more 2-oxoglutaramate compared to a similar wild type plant of the same species.   The method according to claim 11, further comprising the step of propagating a plant from the selected seed and harvesting the seed from the plant. The transgenic plant according to claim 1, wherein the plant heterologous promoter is a tissue-specific promoter that is selectively expressed in photosynthetic tissue.   The transgenic plant according to claim 1, wherein the ratio of glutamine synthetase (GS) activity between leaves and roots is increased compared to a similar wild-type plant of the same species.   The GPT transgene encodes a GPT polypeptide having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having GPT enzyme activity Item 2. The transgenic plant according to Item 1. 11. The method of claim 10, wherein the plant heterologous promoter is a tissue specific promoter that is selectively expressed in photosynthetic tissue.   The GPT transgene encodes a GPT polypeptide having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having GPT enzyme activity Item 11. The method according to Item 10. 12. The method of claim 11, wherein the plant heterologous promoter is a tissue specific promoter that is selectively expressed in photosynthetic tissue.   The GPT transgene encodes a GPT polypeptide having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having GPT enzyme activity Item 12. The method according to Item 11.   The GPT transgene encodes a GPT polypeptide having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and having GPT enzyme activity Item 9. The method according to Item 8.

Images