KR20150048299A - Anthranilate Synthase Mutant Having Activity of Increasing Amino Acid Content in Plant and Use Thereof - Google Patents

Abstract

The present invention relates to a manufacturing method of an anthranilate synthase mutant for increasing amino acid content in a plant, a nucleic acid molecule coding the mutant, genetically modified organisms transformed by the nucleic acid, and the genetically modified organisms. The anthranilate synthase mutant can be effectively used for increasing amino acid content in a plant, and particularly, developing genetically modified rice which produces high quality rice containing amino acid at high content.

Description

Anthranilate synthase mutants which increase amino acid content in plants and uses thereof <br> Anthranilate Synthase Mutant Having Activity Amounts of Amino Acid Content in Plant and Use Thereof

The present invention relates to anthranilate synthase mutants that increase the amino acid content in plants and uses thereof.

Anthranilate Synthase (AS, EC 4.1.3.27) is an enzyme that converts chorismate, the initial reaction of tryptophan biosynthesis from aromatic amino acid shikimate, to anthranilate Catalyzing enzyme. In higher plants, the tryptophan biosynthetic pathway is used to generate precursors necessary for the synthesis of important and diverse secondary metabolites, in addition to the primary purpose of providing amino acids for protein synthesis. Anthranilate synthase, an enzyme that catalyzes the initial reaction of tryptophan biosynthesis, undergoes feedback inhibition by tryptophan.

Essential amino acids such as lysine, methionine, and tryptophan are very important for improving the nutritional value of foods such as rice. However, since the content of these essential amino acids is limited in many crops, the use of mutants or biotechnology This is very urgent. In order to increase the competitiveness of rice, basic research for rice quality improvement is needed. In particular, analysis of feedback inhibition function of anthranilate synthase involved in tryptophan biosynthesis, which is closely related to high quality rice, Development of high-yield tryptophan-producing rice is required through mutation development. The biosynthesis of tryptophan is regulated by the α-subunit of anthranilate synthase, which is the target of feedback suppression by tryptophan, the final product of the tryptophan biosynthesis circuit. Therefore, when anthranilate synthase produces a mutant insensitive to feedback inhibition by tryptophan, a transgenic plant capable of accumulating a large amount of tryptophan, which is an essential amino acid, can be developed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 2009-0058519 US Patent Publication No. US 2003/0213010

The present inventors have made efforts to develop a modified rice which can produce rice containing a high content of free amino acid by using a molecular genetic method. As a result, anthranilate synthase mutants insensitive to the suppression of the feedback by tryptophan in the tryptophan biosynthetic pathway were induced. The mutant gene was introduced into rice to successfully produce transgenic rice, and the transgenic rice And free tryptophan such as tryptophan was contained in a high content. Thus, the present invention was completed.

It is an object of the present invention to provide anthranilate synthase mutants capable of increasing the content of amino acids in plants.

It is another object of the present invention to provide a nucleic acid molecule encoding said variant.

It is another object of the present invention to provide a recombinant vector comprising the nucleic acid molecule.

It is another object of the present invention to provide a transformed host cell or a transgenic plant comprising the nucleic acid molecule.

It is another object of the present invention to provide a method for increasing the content of amino acids in plants.

It is another object of the present invention to provide a method for producing a plant having an increased amino acid content.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an Anthranilate Synthase mutant having the amino acid sequence of SEQ ID NO: 1, which has the property of increasing the content of amino acids in plants.

Anthranilate synthase in the present invention is a key enzyme that regulates the rate of tryptophan biosynthesis in the tryptophan biosynthetic pathway of a plant. The α-subunit of this enzyme undergoes feedback suppression by tryptophan, which is the final product of the biosynthetic pathway It is known.

Anthranilate synthase mutant of the present invention includes a double mutation of S126F and L530D among the wild-type amino acid sequence (SEQ ID NO: 7), and it is presumed that the mutation of the feedback inhibitory action by tryptophan is eliminated or mitigated do.

Anthranilate synthase mutant of the present invention has the property of increasing the content of free amino acid in plants. According to a preferred embodiment of the present invention, the amino acid whose content is increased in the plant includes but is not limited to tryptophan, phenylalanine, threonine, serine, asparagine, arginine, and valine.

According to a preferred embodiment of the present invention, the plant of the present invention is a food crop such as rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats, sorghum; Vegetable crops such as Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops such as ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rape; Apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops such as ragras, red clover, orchardgrass, alpha-alpha, tall fescue and perennialla grass. The plant is more preferably rice, wheat, barley, corn, tobacco, potato, tomato, or sweet potato, most preferably rice.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding said angiotensin converting enzyme mutant.

The term nucleic acid molecule as used herein has the same meaning as "polynucleotide "," polynucleotide molecule ", or "polynucleotide sequence ". A nucleic acid molecule or a polynucleotide molecule is a polymer of a nucleotide in which a nucleotide monomer is linked by a covalent bond in a long chain, and means a DNA (deoxyribonucleic acid) or a ribonucleic acid strand of a certain length or longer.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the angiotensin converting enzyme variant has the polynucleotide sequence set forth in SEQ ID NO: 2.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding said angiotensin converting enzyme mutant.

The vector of the present invention may be a polynucleotide (nucleic acid molecule) encoding the antranaate synthase mutant, which may be cloned into a vector for transformation into prokaryotic or eukaryotic cells for its replication or expression. The vector of the present invention may be, for example, a plasmid or a shuttle vector, a vector for prokaryotic cells for bacterial expression or cloning, and may be a vector for yeast cells, a vector for insect cells, a vector for plant cells, But are not limited to, eukaryotic vectors such as &lt; RTI ID = 0.0 &gt; The vector of the present invention can be easily produced by a person skilled in the art according to a known method using DNA recombinant technology, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989 ; 3rd ed., 2001); "Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990)"; And Bacillus sp., And Salmonella (Palva et al., Gene 22: 229-235), for example, in Bacillus spp., And in Current Protocols in Molecular Biology (Ausubel et al., Supra. (1983)), which are incorporated herein by reference.

The polynucleotide sequence encoding anthranilate synthase variant in the vector of the present invention is operably linked to an expression control sequence (e.g., a promoter sequence). As used herein, the term "operably linked" means that the expression control sequence is linked to regulate transcription and translation of polynucleotide sequences encoding anthranilate synthase variants, Includes the precise maintenance of the translation frame so that the polynucleotide sequence is expressed under control to produce anthranilate synthase variants that are encoded by the polynucleotide sequence.

According to a preferred embodiment of the present invention, the vector of the present invention can be used for the regulation of plant genes, in which case constitutive or inducible promoters in plant cells are used. Promoters that can be used in plant cells include, for example, the ubiquitin-3 promoter sequences of A. thaliana (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493) , The promoter sequence derived from the A. tumefaciens mannopin synthase-derived promoter sequence (US Patent No. 6,730,824), and / or the CsVMV (Cassava vein mosaic virus) -derived promoter sequence (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129-1139).

Expression vectors of the invention typically include expression cassettes or transcription units containing all the additional elements required to express the nucleic acid in prokaryotic or eukaryotic host cells. Therefore, the vector of the present invention may contain, in addition to the promoter operably linked to the polynucleotide sequence encoding anthranilate synthase, an efficient polyadenylation sequence of the transcript, a transcription termination, a ribosome binding site, or a signal required for translation termination Sequence, and may include, as an additional element, an enhancer element, a heterologous splicing signal or a nuclear localization signal (NLS).

According to another aspect of the present invention, the present invention provides a transformed host cell comprising a nucleic acid molecule encoding said angiotensin converting enzyme mutant.

According to another aspect of the present invention, the present invention provides a transgenic plant comprising a nucleic acid molecule encoding said anganatyl synthase mutant.

The transformed host cell is preferably a bacterial cell or a plant cell, and the bacterial cell is preferably an Agrobacterium cell.

As used herein, the terms "transformation" and "introduction" are used interchangeably and include transfer of an exogenous polynucleotide sequence to a host cell, regardless of the method used.

The plant tissue that can be used for transformation using the recombinant vector of the present invention is a tissue capable of cloning propagation by organogenesis or embryogenesis and allowing the entire plant to be regenerated from the tissue. The particular tissue used for the transformation will be available in a particular species to be transformed and will be selected according to the most suitable clonal propagation system. For example, typical transgenic target tissues include, but are not limited to, leaf discs, seeds, pollen, stomach, cotyledon, hypocotyl, large gammgut, callus tissue, fissured tissue (e.g., apical mitotic tissue, (E.g., cotyledonary tissue and hypocotyledonous tissue).

Anthranilate synthase mutant coding polynucleotides introduced in the present invention can be transiently or stably introduced into a host cell and can be maintained non-integrally, for example, as a plasmid, or integrated into a host's genome . The transgenic plant cells produced are used to regenerate transgenic plants in a manner known to those skilled in the art.

Transformation methods that can be used in the present invention include, but are not limited to, liposomes, electroporation, chemicals that increase absorption of free DNA, direct injection of DNA into plants, particle gun stunting, transformation or microprojection using viruses or pollen, (Krenset et al, 1982; Negrutiu et al, 1987), electroporation of protoplasm (Shillito et al, 1985), microinjection (Crossway et al, 1986), DNA Or RNA coated particle impact method (Klein et al, 1987), methods by virus infection, and Agrobacterium tumefaciens-mediated transformation methods.

In addition to transgenic somatic cells that are reproduced as whole plants, it is possible to transform cells that develop into the splitting tissue of plants, especially seeds. In this case, the transformed seed is transformed through natural plant development. For example, seeds of the plant to be transformed are treated with Agrobacterium tumefaciens having a recombinant expression vector to obtain transformed seeds, and the transformed seeds are grown into transgenic plants.

The transgenic plants of the present invention are preferably food crops such as rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats, sorghum; Vegetable crops such as Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops such as ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rape; Apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops such as ragras, red clover, orchardgrass, alpha-alpha, tall fescue and perennialla grass. The transgenic plants are preferably rice, wheat, barley, corn, tobacco, potatoes, tomatoes, or sweet potatoes, most preferably rice.

According to another aspect of the present invention, there is provided a method for increasing the content of amino acids in a plant, comprising introducing a nucleic acid molecule encoding the anthranilate synthase mutant into a plant.

According to another aspect of the present invention, the present invention provides a method of producing a plant having an increased amino acid content comprising the steps of: (a) preparing the above recombinant vector; (b) introducing the recombinant vector prepared in step (a) into a plant cell to produce a transgenic plant; And (c) selecting transgenic plants into which the anthranilate synthase mutant gene has been introduced, among the transgenic plants prepared in the step (b).

The method for producing the recombinant vector of the present invention in the method of the present invention and the method for producing a transgenic plant by introducing the recombinant vector into a plant cell are the same as the recombinant vector of the present invention and the recombinant vector Cells and transgenic plants are identical to those described in the previous section, and thus are not duplicated.

Among the transgenic plants produced by the present invention, selection of transgenic plants containing nucleic acid molecules coding for anthranilate synthase mutants can be carried out by selecting transgenic plants which are known to those skilled in the art For example, by using a genomic gene extracted from transformant cells using a polymerase chain reaction method, a base sequence analysis method, a restriction enzyme treatment method, or a combination of these methods .

The present invention relates to a mutant of anthranilate synthase capable of increasing the content of amino acids in plants, a nucleic acid molecule encoding the mutant, a transgenic plant transformed with the nucleic acid molecule, and a method for producing the transgenic plant . Anthranilate synthase mutant of the present invention can be usefully used for increasing the content of amino acids in plants, and particularly useful for the development of transgenic rice producing high quality rice containing high amino acid content.

Fig. 1 schematically shows the pathway of tryptophan biosynthesis in plants. Anthranilate synthase is a key enzyme in tryptophan biosynthesis.
Figure 2 shows more specifically the pathway of tryptophan biosynthesis in plants. The relevant path referred to in the present invention is shown. The curve from tryptophan to anthranilate synthase indicates inhibitory feedback regulation.
Figure 3 shows the phenotype of wild type and mutant rice. The upper photograph shows the photograph of rice at 14 days after 5-methyltryptophan treatment, and the lower photograph shows the relative weight, stem and root length of each of rice varieties # 2 and # 4 against 5-methyltryptophan As shown in FIG. Mean measurements and standard horses were shown for five plants from representative experiments.
FIG. 4 is a diagram showing gene structure and transcript analysis of anthranilate synthase (OsASA1) gene (top) and anthranilate synthase (OsASA2) gene (bottom). The red arrows indicate the location of the gene on the chromosome in rice.
FIG. 5 shows the results of PCR amplification of a site containing double mutation positions of anthranilate synthase (AS). The size of the amplified product was 1.2 Kb. Lane M; DNA ladder, Lane 1 and 2: PCR amplification product of double mutation site by primers 1 and 2.
FIG. 6 shows the nucleotide sequence and amino acid sequence of the OsASA2 gene of anthranilate synthase α-2 subunit having the S126F / L530D double mutation of the present invention. The sequence shown in red indicates the position of each mutation. The underlined sequence represents the primer sequence for cloning and the restriction enzyme site is shown in bold.
Figure 7 shows the structure of a Ti-plasmid vector for overexpression of the OsASA2 gene coding for the S126F / L530D double mutation in rice. The PGD1 promoter and CaMV 35s promoter gene, 3'PINII: protease inhibitor II terminator gene, phosphinotricine acetyltransferase gene and 3 'nos (nopaline synthase terminator) gene are shown. (A) The OsASA2 gene encoding the S126F / L530D double mutation was cloned into a pGEM T-easy vector. (B) The structure of the pPZP-Bar binary vector containing the OsASA2 gene encoding the S126F / L530D double mutation in a form capable of expression.
FIG. 8 shows the process of introducing the OsASA2 gene coding for the S126F / L530D double mutation through Agrobacterium-mediated transformation. Seeds were seeded in N6D medium, inoculated, co-cultured in 2N6-AS medium, callus growth in 2N6-C medium, shoot formation and growth, roots formation and growth, and purification procedures were performed according to the protocol.
Fig. 9 shows the results of PCR amplification of the gene (Bar, OsASA2) introduced in the transgenic rice line. The amplified product was isolated on a 1.5% agarose gel. Lane M; DNA ladder, Lane P; PCR products generated from plasmid pPZP-Bar containing OsASA2 as a DNA template, Lane W; Wild type plants, Lane 1-20; Individual transgenic rice lines.
Figure 10 shows the results of TaqMan PCR analysis using a TaqMan probe for single copy selection in transgenic rice plants.
FIG. 11 shows the results of reverse transcription PCR analysis of the expression of the OsASA2 gene introduced in the transformant strain. Total RNA was isolated from each plant and 0.5 μg of RNA was amplified using gene specific primers. As a control, samples were amplified using specific primers for rice actin gene. Lane W; PCR products from wild-type cDNA templates, Lane 1-3, 5-7, 9-14, 17-20; Each transformant line selected to have a single copy introduced.
Figure 12 shows the results of quantitative RT-PCR analysis of RNA extracted from the transformant strain and the wild-type control. CT values were calculated using actin expression as a control. The error bars show the standard deviation of the mean for three repeated measurements.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Cultivation of 5-methyltryptophan resistant rice line

Rice mutants were induced by EMS treatment. Rice mutants were cultured on MS medium containing 25 ppm of 5-methylpryptopane to select 5-methyltryptophan-resistant rice strain. FIG. 3 shows photographs of rice cultivated in MS medium containing 50 ppm of 5-methyltryptophan for 14 days after confirming the 5-methyltryptophan resistance-resistant strain # 2 and # 4. As shown in FIG. 3, the growth of roots and stems of the 5-methyltryptophan resistant strains # 2 and # 4 lines was normal, and the growth was not observed in the control. The above results are due to the fact that the rice that can be grown in the medium containing 5-methyltryptophan has an abnormality in the intracellular tryptophan biosynthetic system and the mutation is induced so as not to inhibit the feedback, thereby increasing the content of tryptophan in the cell. The content of tryptophan in the initial 5-methyltryptophan-resistant mutant was 8 times higher than that of the control.

Example 2: Isolation and characterization of OsASA1 and OsASA2 genes

The genes encoding anthranilate synthase, a key enzyme in the tryptophan biosynthetic pathway, are known as OsASA1 and OsASA2 (NCBI database). These genes are located on chromosome 3, OsASA1 is in short arm and consists of 11 exons, and consists of a total of 1831 bp ORF. The OsASA2 gene is located on the long arm of chromosome 3, and consists of 10 exons and 9 introns with an 1821 bp ORF (see FIG. 4).

The 5-methyltryptophan-resistant mutant was presumed to have a low level of feedback inhibition by tryptophan, the final product, due to mutation of the anthranilate synthase α-subunit gene, which is a key enzyme in the synthesis regulation of tryptophan biosynthesis pathway. Therefore, in order to confirm the mutation of the angiathanilate synthase gene of the resistant mutant, a primer set for cloning of the enzyme was prepared. The prepared primers were as follows: Primer 1: [forward direction: 5'-ATGGAGTCCATCGCCGCCGCCA-3 '(SEQ ID NO: 3), reverse direction: 5'-AGAGGTTTGAGAGGCGAAC-3' (SEQ ID NO: 4)]; Primer 2: [forward direction: 5'-GATGATGTTCTCGTCTTCGA-3 '(SEQ ID NO: 5), reverse direction: 5'-AGCTTTCGTAGACAAGGAATAG-3' (SEQ ID NO: 6)].

Separation of genomic DNA was performed by CTAB (Cetyltrimethyl ammonium bromide) method. 0.5 g of the leaves were collected from the plants, finely pulverized using liquid nitrogen, and pulverized in a 1.5 ml centrifuge tube containing 400 μl of DNA extraction buffer [100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl] Tissue was added and shaken 20 times up and down. Thereafter, 200 μl of 2X CTAB buffer [2% (w / v) CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 1% pvp-40 (polyvinylpyrrolidine) 10% SDS was added, and the mixture was shaken up and down about 10 times, and then left in a constant temperature water bath at 65 ° C for 20 minutes. After adding 200 μl of 5M potassium acetate (pH 7.5) and shaking it up and down 50 times, 700 μl of PCI [Phenol / Chloroform / Isoamylalchol (25: 24: 1)] was added, Proteins were separated by centrifugation at 12,000 rpm for 10 minutes. About 400 μl of the upper layer was transferred to a new 1.5 ml tube, and an equal amount of isoprophanol was added. The mixture was stored in a -20 ° C. freezer for 20 minutes and then centrifuged at 12,000 rpm for 4 minutes at 4 ° C. for 15 minutes to precipitate the DNA. The precipitated DNA was washed with 1 ml of 70% ethanol, completely dried at room temperature, sufficiently dissolved in 50 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing 2 μl of RNase (1 mg / ml) Lt; 0 &gt; C for 1 hour.

PCR was carried out at 95 ° C for 5 minutes, followed by denaturation at 94 ° C for 1 minute, annealing at 58 ° C for 1 minute and extension at 72 ° C for 2 minutes. Extension was performed for 5 minutes. The PCR product was electrophoresed on 0.8% agarose gel and the expected 1.2 kb band was detected (see FIG. 5). These bands were recovered from the gel, inserted into pGEM T-easy vector, and analyzed for nucleotide sequences (see FIG. 6). The total length of the gene was 2248 bp, consisting of 1824 bp ORF, 35 bp 5UTR, and 349 bp 3UTR. In the 5-methyltryptophan-resistant mutant, two point mutations appeared as TCC (Serine) -> TTC (Phenylalanine) and CTT (Leucine) -> GAC (Aspartic acid) at amino acid positions 126 and 530 respectively, and double mutant S126F / L530D has occurred.

Example 3: Construction of Ti-plasmid vector for overexpressing OsASA2 gene including S126F / L530D double mutation

The pPDG1 vector (MYONGJI UNIVERSITY) was used as a parent vector for transfection of plants, and the OsASA2 gene cloned by the present inventors was connected to the CaMV 35S and PDG1 promoters in order to test the expression of the target gene. For the selection of transformed calli and plants, the Bar gene regulated by the CaMV 35S promoter was used. A schematic diagram of the carrier used in the experiment is shown in Fig. After constructing a vector for expression of the OsASA2 gene coding for the S126F / L530D double mutation, this plasmid vector was transformed into Agrobacterium tumefaciens LBA4404. Agrobacterium tumefaciens competent cell LBA4404 prepared by the same method at 28 &lt; 0 &gt; C was dissolved in ice, mixed with 1 mu L of plasmid DNA, and injected into an electric shock cuvette. Then, the cells were transformed by electric shock at 1,440 V, mixed with 1 mL of SOC medium, and placed in a sterilized test tube and cultured at 28 ° C for 1 hour at 200 rpm. The culture broth was dropped on an AB agar medium containing 50 mg / L of kanamycin by pipette, and colonies were observed on a Petri dish for 2 to 3 days at 28 ° C in a thermostat. Colony PCR method was used to confirm the transformation. Cultures of the identified strains were inoculated into AB liquid medium supplemented with kanamycin 50 mg / L and cultured at 200 rpm in a 28 ° C incubator for 2-3 days. The culture was added to the same amount of 50% glycerol and stored in a cryogenic freezer (-80 ° C).

Example 4: Construction of OsASA2 gene transgenic rice with S126F / L530D double mutation

A common method commonly used to transform rice plants is to inoculate the callus in rice seeds and then inoculate the callus with Agrobacterium spp. It usually takes about 4 months to obtain the transformant . In this experiment, agar bacterium was inoculated with seeds that had been swollen at the blastocyst stage after washing rice seed matured seeds to shorten the time consumed for transplanting the rice, and soaking them in N6 liquid medium for 24 hours. The surface-sterilized seeds were cultured in N6 liquid medium containing 2 mg / L of 2,4-D (Chu CC, Wang CS, Sun CC, Hsu C, Yin KC, and Chu CY (1975) rice through comparative experiments on thenitrogen sources. Sci Sinica 18: 659-668] and germinated for 24 hours at 30 ° C in a dark state. The seeds in which the abdomen swelled and the plant began to differentiate were immersed in a tube containing 15 mL of Agrobacterium suspension, The seeds were placed on sterilized filter paper after inoculation for a minute to remove excess Agrobacterium. The seeds inoculated with Agrobacterium were seeded on filter paper on 2N6-AS medium containing 1 mM DTT (dithiothreitol), 3 mg / L AgNO ₃ (silver nitrate) and 0.5% gelrite, For 3 days. Then, in order to enhance the influence of the medium components, the cells were cocultured for 4 days under the dark condition at 25 ° C in the medium on which the filter paper was removed. 1 mM DTT (dithiothreitol) and 3 mg / L AgNO3 (silver nitrate) were added to prevent the excessive growth of Agrobacterium during co-cultivation and to reduce the substances that could be produced by the defense mechanism of plant cells due to the introduction of Agrobacterium. Were included in the medium. After 7 days of coculture, the callus formation rate after hormone treatment was less effective than the callus formation and growth after infection with bacterium. In addition to the addition of antioxidants that can inhibit excessive growth of Agrobacterium during co-culture, proper regulation of hormones used may enable efficient callus formation and introduction of target genes by Agrobacterium in co-culture . Since the seeds used in this experiment are not yet sufficient for callus proliferation, they are transferred to N6D medium containing only 400 mg / L of carbenicillin for propagation of healthy calli and inhibiting Agrobacterium growth. Lt; 0 &gt; C for 1 week (Fig. 8). One week after the callus proliferation, the callus was transferred to a N6D medium containing 50 mg / L hygromycin and 400 mg / L carbenicillin for a callus inserted with a healthy callus. And cultured for 2 weeks.

After cultivation, the calli propagating proliferately from the blastocyst were transferred to SF (shoot formation) medium to induce plant regeneration. After 2 weeks in the medium containing antibiotics, a green spot in the callus began to form (see FIG. 8), and shoot formation began to take place in the callus after about 3 weeks of culture 8). After transferring from antibiotic medium to SF medium, the efficiency of green spot formation and regeneration was different depending on the gene inoculated. The regenerated plant was transferred to a root formation medium (FIG. 8) to induce root development, and the purified rice transgenic plants were transplanted from the greenhouse into a pot (FIG. 8). A total of 35 transgenic plants were obtained from 115 transgenic calli.

Example 5: Identification of OsASA2 gene introduced by PCR

In order to investigate the copy number of OsASA2 gene in the rice genome of the regenerated plant obtained from the transfection experiment, genomic DNA was isolated from the regenerated leaves, and a primer set for the transgene Bar and OsASA2 gene specific amplification PCR analysis was performed. Bar and OsASA2 genes were detected in the 20 regenerated plants examined (see FIG. 9). This means that the OsASA2 gene is well introduced into the rice genome, and these transformants are numbered from the T0 generation.

Example 6: Selection of a single copy introduced transformant

Selection of transgenic rice with single copy was carried out by TaqMan PCR method. In order to select a single copy introduced transfectant by TaqMan PCR method, a labeled probe primer that binds to a specific sequence of the nos terminator region at the terminal of OsASA2 gene was used according to the protocol provided by Takara Co. . Transformants were able to calculate the absolute standard concentration for the quantification of the OsASA2 gene in the genome in 20 plant genomes identified by PCR analysis with bar and OsASA2 genes. Of the total 20 transgenic T0 individuals, 16 plants were inserted with a single copy of the transgene (see FIG. 10).

Example 7: Analysis of expression amount of OsSA2 gene transfected with S126F / L530D double mutant

The results of performing RT-PCR and qRT-PCR for the expression amount of the OsSA2 gene-transfected transformant encoding the S126F / L530D double mutation are shown in FIGS. 11 and 12. The RT-PCR analysis revealed that the PCR product appeared as a band, but the difference in the expression level was not clearly known. However, OsASA2 gene, which is the transgene in most of the transformants, was well expressed in the cells. When the results of quantification of OsASA2 gene by real time-PCR method were compared with those of the control, the expression level was found to be high in most transformants. Relatively high expression levels were obtained in # 5, # 9, # 11, # 12, # 13, # 19, and # 20 of the transformant strain.

Example 8 Analysis of Amino Acids of Transgenic Transgenic Rice Overexpressing OsASA2 Gene Containing S126F / L530D Double Mutant

Transformants # 5 and # 20, which were transfected with a single copy of the transgenic lines and high in OsASA2 gene expression, were analyzed for free amino acids. As a result, the total amount of free amino acids of Transformants # 5 and # And 1.8 times higher than the control. In particular, tryptophan, phenylalanine, threonine, serine, and valine contents were 6.7, 4.6, 3.6, 2.8, and 2.0 times higher than the control, respectively (Table 1). Thus, overexpression of the OsASA2 mutant gene containing the S126F / L530D double mutation resulted in insensitivity to the feedback inhibition mechanism on the tryptophan biosynthesis circuit, resulting in an overall increase in the amino acid content, and in particular, an increase in the tryptophan amino acid content. Table 1 shows the content of free amino acids in transgenic rice and wild type rice. In Table 1, the unit of numerical value is mole / g fresh weight.

summary

Specific mutants of the anthranilate synthase (AS) gene, which regulates tryptophan biosynthesis, were cultivated and the mutants grown on MS medium containing 50 ppm 5-methylpryptopane, a tryptophan analogue, were analyzed. As a result, the 5-methyltryptophan resistance system , Two mutations of TCC (Serine) -> TTC (Phenylalanine) and CTT (Leucine) -> GAC (Aspartic acid) appeared at amino acid positions 126 and 530 in the amino acid sequence, and the mutation S126F / L530D occurred . The mutant OsA2 gene, which has a double mutation (S126F / L530D), is located on the long arm of chromosome 3, and consists of 18 exon regions and 9 intron regions with an 1821 bp ORF. The OsASA2 gene with double mutation (S126F / L530D) was transformed by constructing a plant-overexpressing Ti-plasmid vector controlled by the PDG1 promoter for introduction into the rice genome. The transgenic plants were constructed by preparing specific primers of Bar selection gene and promoter region and OsASA2 gene region, and OsASA2 gene was detected by PCR. The transgenic plants transfected with a single copy by TaqMan PCR were selected and their expression levels were analyzed. As a result of RT-PCR and qRT-PCR, high expression levels of the genes introduced in most transformants were confirmed. The free amino acids were analyzed by free amino acid analysis of # 5 and # 20, which were introduced as a single copy of the transformant strains. The free amino acids were 1.8 times higher than the control, and the tryptophan content 6.7 times higher. Thus, overexpression of the OsASA2 gene with the double mutation (S126F / L530D) resulted in insensitivity to feedback suppression on the tryptophan biosynthetic pathway, resulting in an overall increase in amino acid content and a high specific amino acid, particularly tryptophan. Using the mutant OsASA2 gene of the present invention, it is possible to develop rice with a high content of tryptophan.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Anthranilate Synthase Mutant Having Activity of Increasing Amino          Acid Content in Plant and Use <130> PN130580 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 606 <212> PRT <213> Oryza sativa <400> 1 Met Glu Ser Ile Ala Ala Thr Phe Thr Pro Ser Arg Leu Ala Ala   1 5 10 15 Arg Pro Ala Thr Pro Ala Ala Ala Ala Ala Pro Val Arg Ala Arg Ala              20 25 30 Ala Val Ala Ala Gly Arg Arg Arg Thr Ser Arg Arg Gly Gly Val          35 40 45 Arg Cys Ser Ala Gly Lys Pro Glu Ala Ser Ala Val Ile Asn Gly Ser      50 55 60 Ala Ala Ala Arg Ala Ala Glu Glu Asp Arg Arg Phe Phe Glu Ala  65 70 75 80 Ala Glu Arg Gly Ser Gly Lys Gly Asn Leu Val Pro Met Trp Glu Cys                  85 90 95 Ile Val Ser Asp His Leu Thr Pro Val Leu Ala Tyr Arg Cys Leu Val             100 105 110 Pro Glu Asp Asn Met Glu Thr Pro Ser Phe Leu Phe Glu Phe Val Glu         115 120 125 Gln Gly Pro Glu Gly Thr Thr Asn Val Gly Arg Tyr Ser Met Val Gly     130 135 140 Ala His Pro Val Met Glu Val Val Ala Lys Glu His Lys Val Thr Ile 145 150 155 160 Met Asp His Glu Lys Gly Lys Val Thr Glu Gln Val Val Asp Asp Pro                 165 170 175 Met Gln Ile Pro Arg Ser Met Met Glu Gly Trp His Pro Gln Gln Ile             180 185 190 Asp Gln Leu Pro Asp Ser Phe Thr Gly Gly Trp Val Gly Phe Phe Ser         195 200 205 Tyr Asp Thr Val Arg Tyr Val Glu Lys Lys Lys Leu Pro Phe Ser Gly     210 215 220 Ala Pro Gln Asp Asp Arg Asn Leu Pro Asp Val His Leu Gly Leu Tyr 225 230 235 240 Asp Asp Val Leu Val Phe Asp Asn Val Glu Lys Lys Val Tyr Val Ile                 245 250 255 His Trp Val Asn Leu Asp Arg His Ala Thr Thr Glu Asp Ala Phe Gln             260 265 270 Asp Gly Lys Ser Arg Leu Asn Leu Leu Leu Ser Lys Val His Asn Ser         275 280 285 Asn Val Pro Lys Leu Ser Pro Gly Phe Val Lys Leu His Thr Arg Gln     290 295 300 Phe Gly Thr Pro Leu Asn Lys Ser Thr Met Thr Ser Asp Glu Tyr Lys 305 310 315 320 Asn Ala Val Met Gln Ala Lys Glu His Ile Met Ala Gly Asp Ile Phe                 325 330 335 Gln Ile Val Leu Ser Gln Arg Phe Glu Arg Arg Thr Tyr Ala Asn Pro             340 345 350 Phe Glu Val Tyr Arg Ala Leu Arg Ile Val Asn Pro Ser Pro Tyr Met         355 360 365 Ala Tyr Val Gln Ala Arg Gly Cys Val Leu Val Ala Ser Ser Pro Glu     370 375 380 Ile Leu Thr Arg Val Arg Lys Gly Lys Ile Ile Asn Arg Pro Leu Ala 385 390 395 400 Gly Thr Val Arg Arg Gly Lys Thr Glu Lys Glu Asp Glu Met Gln Glu                 405 410 415 Gln Gln Leu Leu Ser Asp Glu Lys Gln Cys Ala Glu His Ile Met Leu             420 425 430 Val Asp Leu Gly Arg Asn Asp Val Gly Lys Val Ser Lys Pro Gly Ser         435 440 445 Val Lys Val Glu Lys Leu Met Asn Ile Glu Arg Tyr Ser His Val Met     450 455 460 His Ile Ser Ser Thr Val Ser Gly Glu Leu Asp Asp His Leu Gln Ser 465 470 475 480 Trp Asp Ala Leu Arg Ala Leu Pro Val Gly Thr Val Ser Gly Ala                 485 490 495 Pro Lys Val Lys Ala Met Glu Leu Ile Asp Glu Leu Glu Val Thr Arg             500 505 510 Arg Gly Pro Tyr Ser Gly Gly Leu Gly Gly Ile Ser Phe Asp Gly Asp         515 520 525 Met Asp Ile Ala Leu Ala Leu Arg Thr Ile Val Phe Ser Thr Ala Pro     530 535 540 Ser His Asn Thr Met Tyr Ser Tyr Lys Asp Thr Glu Arg Arg Arg Glu 545 550 555 560 Trp Val Ala His Leu Gln Ala Gly Ala Gly Ile Val Ala Asp Ser Ser                 565 570 575 Pro Asp Asp Glu Gln Arg Glu Cys Glu Asn Lys Ala Ala Ala Leu Ala             580 585 590 Arg Ala Ile Asp Leu Ala Glu Ser Ala Phe Val Asp Lys Glu         595 600 605 <210> 2 <211> 1821 <212> DNA <213> Oryza sativa <400> 2 atggagtcca tcgccgccgc cacgttcacg ccctcgcgcc tcgccgcccg ccccgccact 60 ccggcggcgg cggcggcccc ggttagagcg agggcggcgg tagcggcagg agggaggagg 120 aggacgagta ggcgcggcgg cgtgaggtgc tccgcgggga agccagaggc aagcgcggtg 180 atcaacggga gcgcggcggc gcgggcggcg gaggaggaca ggaggcgctt cttcgaggcg 240 gcggagcgtg ggagcgggaa gggcaacctg gtgcccatgt gggagtgcat cgtctccgac 300 cacctcaccc ccgtgctcgc ctaccgctgc ctcgtccccg aggacaacat ggagacgccc 360 agcttcctct tcgagttcgt cgagcagggg cccgagggca ccaccaacgt cggtcgctat 420 agcatggtgg gagcccaccc agtgatggag gtcgtggcaa aggagcacaa ggtcacaatc 480 atggaccacg agaagggcaa ggtgacggag caggtcgtgg atgatcctat gcagatcccc 540 aggagcatga tggaaggatg gcacccgcag cagatcgatc agctccccga ttccttcacc 600 ggtggatggg tcgggttctt ttcctatgat acagtccgtt atgttgaaaa gaagaagctg 660 cccttctccg gtgctcccca ggacgatagg aaccttcctg atgttcacct tgggctttat 720 gatgatgttc tcgtcttcga caatgtcgag aagaaagtat atgtcatcca ttgggtaaat 780 cttgatcggc atgcaaccac cgaggatgca ttccaagatg gcaagtcccg gctgaacctg 840 ttgctatcta aagtgcacaa ttcaaatgta cccaagcttt ctccaggatt tgtaaagtta 900 cacactcggc agtttggtac acctttgaac aaatcaacca tgacaagtga tgagtacaag 960 aatgctgtta tgcaggctaa ggagcatatt atggctggtg atattttcca gattgtttta 1020 agccagaggt ttgagaggcg aacatacgcc aatccatttg aagtctatcg agctttacga 1080 attgtgaacc caagtccata catggcatat gtacaggcaa gaggctgtgt cctggtagca 1140 tctagtccag aaattcttac tcgtgtgagg aagggtaaaa ttattaaccg tccacttgct 1200 gggactgttc gaaggggcaa gacagagaag gaagatgaaa tgcaagagca acagctacta 1260 agtgatgaaa aacagtgtgc tgaacatatt atgcttgtag atttgggaag gaatgatgtt 1320 ggaaaggtct ccaaacctgg atctgtgaag gtggagaaat taatgaacat tgaacgctac 1380 tcccatgtca tgcacatcag ttccacggtg agtggagagt tggatgatca tctccaaagt 1440 tgggatgccc tgcgagccgc gttgcctgtt ggaacagtta gtggagcacc aaaggtgaaa 1500 gccatggagc tgatagacga gctagaggtc acaagacgag gaccatacag tggcggcctt 1560 ggagggatat catttgacgg agacatggac atcgctcttg cactccgcac cattgtgttc 1620 tcaacagcgc caagccacaa cacgatgtac tcatacaaag acaccgagag gcgccgggag 1680 tgggtcgctc accttcaggc tggtgctggc attgtcgctg atagcagccc agacgacgag 1740 caacgtgaat gcgagaacaa ggcagccgct ctggctcgag ccatcgatct tgctgaatca 1800 gctttcgtag acaaggaata g 1821 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 atggagtcca tcgccgccgc ca 22 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 agaggtttga gaggcgaac 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 gatgatgttc tcgtcttcga 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 agctttcgta gacaaggaat ag 22 <210> 7 <211> 606 <212> PRT <213> Oryza sativa <400> 7 Met Glu Ser Ile Ala Ala Thr Phe Thr Pro Ser Arg Leu Ala Ala   1 5 10 15 Arg Pro Ala Thr Pro Ala Ala Ala Ala Ala Pro Val Arg Ala Arg Ala              20 25 30 Ala Val Ala Ala Gly Arg Arg Arg Thr Ser Arg Arg Gly Gly Val          35 40 45 Arg Cys Ser Ala Gly Lys Pro Glu Ala Ser Ala Val Ile Asn Gly Ser      50 55 60 Ala Ala Ala Arg Ala Ala Glu Glu Asp Arg Arg Phe Phe Glu Ala  65 70 75 80 Ala Glu Arg Gly Ser Gly Lys Gly Asn Leu Val Pro Met Trp Glu Cys                  85 90 95 Ile Val Ser Asp His Leu Thr Pro Val Leu Ala Tyr Arg Cys Leu Val             100 105 110 Pro Glu Asp Asn Met Glu Thr Pro Ser Phe Leu Phe Glu Ser Val Glu         115 120 125 Gln Gly Pro Glu Gly Thr Thr Asn Val Gly Arg Tyr Ser Met Val Gly     130 135 140 Ala His Pro Val Met Glu Val Val Ala Lys Glu His Lys Val Thr Ile 145 150 155 160 Met Asp His Glu Lys Gly Lys Val Thr Glu Gln Val Val Asp Asp Pro                 165 170 175 Met Gln Ile Pro Arg Ser Met Met Glu Gly Trp His Pro Gln Gln Ile             180 185 190 Asp Gln Leu Pro Asp Ser Phe Thr Gly Gly Trp Val Gly Phe Phe Ser         195 200 205 Tyr Asp Thr Val Arg Tyr Val Glu Lys Lys Lys Leu Pro Phe Ser Gly     210 215 220 Ala Pro Gln Asp Asp Arg Asn Leu Pro Asp Val His Leu Gly Leu Tyr 225 230 235 240 Asp Asp Val Leu Val Phe Asp Asn Val Glu Lys Lys Val Tyr Val Ile                 245 250 255 His Trp Val Asn Leu Asp Arg His Ala Thr Thr Glu Asp Ala Phe Gln             260 265 270 Asp Gly Lys Ser Arg Leu Asn Leu Leu Leu Ser Lys Val His Asn Ser         275 280 285 Asn Val Pro Lys Leu Ser Pro Gly Phe Val Lys Leu His Thr Arg Gln     290 295 300 Phe Gly Thr Pro Leu Asn Lys Ser Thr Met Thr Ser Asp Glu Tyr Lys 305 310 315 320 Asn Ala Val Met Gln Ala Lys Glu His Ile Met Ala Gly Asp Ile Phe                 325 330 335 Gln Ile Val Leu Ser Gln Arg Phe Glu Arg Arg Thr Tyr Ala Asn Pro             340 345 350 Phe Glu Val Tyr Arg Ala Leu Arg Ile Val Asn Pro Ser Pro Tyr Met         355 360 365 Ala Tyr Val Gln Ala Arg Gly Cys Val Leu Val Ala Ser Ser Pro Glu     370 375 380 Ile Leu Thr Arg Val Arg Lys Gly Lys Ile Ile Asn Arg Pro Leu Ala 385 390 395 400 Gly Thr Val Arg Arg Gly Lys Thr Glu Lys Glu Asp Glu Met Gln Glu                 405 410 415 Gln Gln Leu Leu Ser Asp Glu Lys Gln Cys Ala Glu His Ile Met Leu             420 425 430 Val Asp Leu Gly Arg Asn Asp Val Gly Lys Val Ser Lys Pro Gly Ser         435 440 445 Val Lys Val Glu Lys Leu Met Asn Ile Glu Arg Tyr Ser His Val Met     450 455 460 His Ile Ser Ser Thr Val Ser Gly Glu Leu Asp Asp His Leu Gln Ser 465 470 475 480 Trp Asp Ala Leu Arg Ala Leu Pro Val Gly Thr Val Ser Gly Ala                 485 490 495 Pro Lys Val Lys Ala Met Glu Leu Ile Asp Glu Leu Glu Val Thr Arg             500 505 510 Arg Gly Pro Tyr Ser Gly Gly Leu Gly Gly Ile Ser Phe Asp Gly Asp         515 520 525 Met Leu Ile Ala Leu Ala Leu Arg Thr Ile Val Phe Ser Thr Ala Pro     530 535 540 Ser His Asn Thr Met Tyr Ser Tyr Lys Asp Thr Glu Arg Arg Arg Glu 545 550 555 560 Trp Val Ala His Leu Gln Ala Gly Ala Gly Ile Val Ala Asp Ser Ser                 565 570 575 Pro Asp Asp Glu Gln Arg Glu Cys Glu Asn Lys Ala Ala Ala Leu Ala             580 585 590 Arg Ala Ile Asp Leu Ala Glu Ser Ala Phe Val Asp Lys Glu         595 600 605

Claims (9)

Anthranilate synthase mutant having an amino acid sequence of SEQ ID NO: 1 and having the property of increasing the content of amino acids in plants.
The mutant according to claim 1, wherein the amino acid whose content is increased in the plant is tryptophan, phenylalanine, threonine, serine, asparagine, arginine or valine.
A nucleic acid molecule encoding a variant of claim 1.
4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule has the polynucleotide sequence of SEQ ID NO: 2.
A recombinant vector comprising the nucleic acid molecule of claim 3.
A transformed host cell comprising the nucleic acid molecule of claim 3.
A transgenic plant comprising the nucleic acid molecule of claim 3.
A method for increasing the content of amino acids in a plant comprising the step of transforming a nucleic acid molecule of claim 3 into a plant.
A method for producing a plant having an increased amino acid content comprising the steps of:
(a) preparing a recombinant vector according to claim 5;
(b) introducing the recombinant vector prepared in step (a) into a plant cell to produce a transgenic plant; And
(c) selecting transgenic plants into which the anthranilate synthase mutant gene has been introduced from the transgenic plants prepared in the step (b).

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