KR101857606B1 - Use of Mutated Anthranilate Synthase Gene Resistant to 5-Methyltryptophan for Selection Marker of Plant Transformation - Google Patents

Abstract

The present invention relates to the use of anthranilate synthase mutant coding polynucleotides resistant to 5-methyltryptophan as markers for transgenic plant selection. The transgenic plant screening markers for anthranilate synthase mutant coding polynucleotides which are resistant to 5-methyltryptophan of the present invention can replace the antibiotic markers used in the past, and GM (Genetically modified) crops.

Description

{Use of Mutant Anthranilate Synthase Gene Resistant to 5-Methyltryptophan for Selective Marker of Plant Transformation of 5-Methyltryptophan Resistant Anthranilate Synthase Mutant Gene [

The present invention relates to the use of 5-methyltryptophan-resistant anthranilate synthase mutant genes as transgenic plant selection markers.

Approximately 50 selectable marker genes are evaluated for efficiency, biological safety and scientific application and commercialization in transgenic plant studies or crop development. Selective marker genes can be classified according to whether they are positive or negative selection and whether they are conditional or unconditional, depending on the presence of an external substrate. The positive selection marker gene is a gene that promotes the growth of the transformant, whereas the negative selection marker gene is a marker gene that induces the death of the transformant. Initially, a positive selectable marker gene was developed and used for the use of toxic agents such as antibiotics, herbicides or drugs. Recently, a positive selectable marker gene such as a non-toxic agent which can be a substrate for inducing the growth and differentiation of the transformed tissue has been developed.

Anthranilate Synthase is an enzyme that catalyzes the conversion of chorismate to anthranilate, the initial reaction of tryptophan biosynthesis from an aromatic amino acid shikimate. 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, is subjected to feedback inhibition by tryptophan. Cells that can grow in a medium containing 5-methyltryptophan (5-MT), a tryptophan analogue, are mutated to prevent feedback inhibition due to abnormalities in the intracellular tryptophan biosynthetic system .

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.

Korea Patent Publication No. 10-2009-0050189

The present inventors have sought to develop selective markers for identifying and screening for transformation in transgenic plants. As a result, a mutant of anthranilate synthase, which imparts growth properties to a medium containing 5-methyltryptophan (5-MT), which is a tryptophan analogue, was successfully isolated. The present inventors have completed the present invention by experimentally proving that a gene encoding a lyase-synthesizing enzyme can be used as a selectable marker for selecting transgenic plants.

It is therefore an object of the present invention to provide a recombinant vector for screening transgenic plant cells comprising an anthranilate synthase mutant coding polynucleotide which confers resistance to 5-methyltryptophan.

Another object of the present invention is to provide a recombinant vector comprising (a) an anthranilate synthase mutant coding polynucleotide having resistance to 5-methyltryptophan; And (b) 5-methyltryptophan. The present invention also provides a kit for screening a transformed plant cell.

It is still another object of the present invention to provide a method for screening plant cells transformed using the recombinant vector.

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 a recombinant vector for screening transformed plant cells comprising an anthranilate synthase mutant coding polynucleotide which confers resistance to 5-methyltryptophan.

As used herein, the term " anthranilate synthase " is a key enzyme that regulates the rate of tryptophan biosynthesis in the tryptophan biosynthetic pathway of a plant, and the α-subunit of this enzyme undergoes feedback suppression by tryptophan, the final product of the biosynthetic pathway It is a known enzyme.

Variants of the anthranilate synthase of the present invention include a single mutation of F124V or a double mutation of S126F and L530D in the amino acid sequence of the wild-type enzyme. Due to such mutation, the mutation of tryptophan- Is thought to be extinguished or alleviated.

The plant cell having a variant of the anthranilate synthase of the present invention has 5-methyltryptophan resistance which can grow even in the presence of a tryptophan analog, 5-methyltryptophan.

According to one embodiment of the present invention, the anthranilate synthase mutant which imparts resistance to 5-methyltryptophan in the present invention comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

According to another embodiment of the present invention, the recombinant vector in the present invention has the structure of the vector shown in Fig.

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

According to another embodiment of the present invention, the polynucleotide encoding the anthranilate synthase mutant has the polynucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

The recombinant vector of the present invention includes a polynucleotide (nucleic acid molecule) encoding the anthranilate synthase mutant as a marker for screening transgenic plant cells or transformed plants.

The recombinant 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, But are not limited to, eukaryotic vectors such as vectors.

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.

A polynucleotide sequence encoding an anthranilate synthase variant in the recombinant vector of the present invention may be operably linked to an expression control sequence (e.g., a promoter sequence). As used herein, the term " operably linked " means that an expression control sequence is linked to regulate transcription and translation of a polynucleotide sequence encoding an anthranilate synthase variant, and the regulation of the expression control sequence In which the polynucleotide sequence is expressed so that an anthranilate synthase variant encoded by the polynucleotide sequence is generated.

Promoters that may be used in the recombinant vectors of the invention include constitutive or inducible promoters in plant cells. 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).

The recombinant vector of the present invention may typically comprise an expression cassette or transcription unit containing all the additional elements required to express the nucleic acid in a prokaryotic or eukaryotic host cell. Thus, the recombinant vector of the present invention may contain, in addition to a promoter operably linked to a polynucleotide sequence encoding an anthranilate synthase, an efficient polyadenylation sequence, transcription termination, ribosome binding site, or signal required for translation termination of the transcript 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, there is provided a recombinant vector comprising (a) a recombinant vector comprising an anthranilate synthase mutant coding polynucleotide having resistance to 5-methyltryptophan; And (b) 5-methyltryptophan.

According to an embodiment of the present invention, the anthranilate synthase mutant having resistance to 5-methyltryptophan comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The contents of the recombinant vector of the present invention are the same as those described in another recombinant vector for selecting transgenic plant cells, which is another aspect of the present invention, and thus are not duplicated in order to avoid excessive complexity of the specification.

According to another aspect of the present invention, the present invention provides a method for screening a transformed plant comprising the steps of: (a) transforming a plant cell with the recombinant vector; (b) culturing the transformed plant cells in a medium containing 5-methyltryptophan; And (c) screening the plant cells grown in the cultured medium.

According to the method of the present invention, the recombinant vector of the present invention includes an anthranilate synthase mutant coding polynucleotide selectable marker that confers resistance to 5-methyltryptophan, so that the transformed plant cell is transformed with 5-methyltryptophan The plant cell transformed with the recombinant vector can be selectively screened by selecting only the plant cell that has grown while culturing in the contained medium.

As used herein, the terms "transformation" and "introduction" are used interchangeably and include transferring 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).

The anthranilate synthase mutant coding polynucleotides introduced in the present invention can be transiently or stably introduced into the host cell, for example, can be maintained as a non-integrative like a plasmid, or integrated into the genome of the host have. 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.

The present invention relates to the use of anthranilate synthase mutant coding polynucleotides resistant to 5-methyltryptophan as markers for transgenic plant selection. The transgenic plant screening markers for anthranilate synthase mutant coding polynucleotides which are resistant to 5-methyltryptophan of the present invention can replace the antibiotic markers used in the past, and GM (Genetically modified) crops.

Fig. 1 schematically shows the pathway of tryptophan biosynthesis in plants. Anthranilate synthase is a key enzyme in tryptophan biosynthesis.
Figure 2 shows the phenotype of wild type and mutant rice. 14 days after treatment with 5-methyltryptophan.
FIG. 3 shows a Ti-plasmid vector structure for overexpression of a phosphinotricine acetyltransferase gene in rice. The PGD1 promoter and the CaMV 35s promoter gene, the OsASA2 gene used as a selection marker, the 3'PINII: protease inhibitor II terminator gene, and the 3'nos (nopaline synthase terminator) gene.
Fig. 4 shows the process of introducing the mutant OsASA2 gene 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.
Panel A of Figure 5 shows the PCR amplification results of the transformed gene (Bar, PGD1 :: OsASA2) introduced in rice lines. The amplified product was isolated on a 1.5% agarose gel. Lane M; DNA ladder, Lane WT; Wild type plant, Lane MC; Mock control, Lane 1-2; Individual transgenic rice lines (S-TG; single point mutant, D-TG; double point mutant). Panel B shows the results of analysis of OsASA2 expression in the transformant strain obtained by the reverse transcription PCR method. Total RNA was isolated from each plant and 0.5 μg of RNA was amplified with gene-specific primers. As a loading control, samples amplified with primers specific for the actin gene were also loaded.
Figure 6 shows the results of a TaqMan PCR analysis performed using a TaqMan probe for single copy selection in transgenic rice plants.
Figure 7 shows the phenotype of wild type, mock control and OsASA2 transformants in rice. (S-TG; single point mutant, D-TG; double point mutant). Panel A is T1 generation, Panel B is T2 generation.
Figure 8 shows representative chromatograms in wild-type (WT) and OsASA2 transformants (S-TG and D-TG) rice seed lines. Metabolic profiling by HPLC combined with PDA detection was performed on the extract. Panel A is tryptophan and IAN (Indole-3-acetonitrile) and Panel B is IAA (Indole-3-acetic acid).
FIG. 9 shows the results of measurement of Trp, IAN and IAA contents of the seeds derived from the transformants grown in rice field. The amounts of free Trp, IAN, and IAA were quantified in the same seeds of S-TG, D-TG or wild-type (WT) plants. Data are expressed as nanomoles of the analyte per gram of dry seed weight and expressed as the mean ± SD measured from three groups of six seeds.
Panel A of Figure 10 shows the biosynthetic pathway of tryptophan in plants. The relevant path referred to in the present invention is shown. The curve from tryptophan to anthranilate synthase indicates inhibitory feedback regulation. Panel B shows the expression levels of several genes associated with tryptophan metabolism.
FIG. 11 shows the results of screening various strains of seeds by growing seeds in Murashige and Skoog medium (1 / 2MS) containing 25 mg / L 5-methyltryptophan. The mutant strains 5MT-4 and 5MT-5 were well grown in stem and root (panels A and B). Wild type was Dongjin rice.
Fig. 12 shows the results of culturing wild type Dongjin rice calli and the transformed Dongjin beetle callus transformed with the pSP1-5MT vector prepared in Example 3 in a medium containing 25 ppm of 5MT.

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: Screening of 5-methyltryptophan resistant rice mutants

Rice mutants were induced by EMS treatment. Rice mutants were cultured in MS medium containing 25 ppm of 5-methyltryptophan to select 5-methyltryptophan-resistant rice strain. Mice were observed for 14 days after culturing in # 2 strain (Mutant line 2) and # 4 strain (Mutant line 4) identified as 5-methyltryptophan resistance strain in MS medium containing 50 ppm of 5-methyltryptophan Respectively. As shown in Fig. 2, 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: Structural analysis of 5-methyltryptophan-resistant mutant ASA2 gene

Genes coding for 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. The 5-methyltryptophan-resistant mutant was presumed to have a low feedback inhibition by the final product, tryptophan, 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 anthranilate synthase gene of the resistant mutant, a primer set for cloning of the enzyme was prepared. The prepared primers are as follows: Primer: [forward direction: 5'-ATGGAGTCCATCGCCGCCGCCA-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 leaf was collected from the plant and finely pulverized using liquid nitrogen and pulverized into a 1.5 ml centrifuge tube containing 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. Then, 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 PCI (Phenol / Chloroform / Isoamylalchol (25: 24: 1)) to the mixture, the mixture was stirred at room temperature for 30 times. Proteins were separated by centrifugation at 12,000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 ml tube and equilibrated with isoprophanol, stored in a -20 ° C freezer for 20 minutes, and 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 TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) 50 to which RNase (1 mg / Lt; 0 > C for 1 hour.

PCR was carried out by pre-denaturation at 95 ° C for 5 minutes, followed by denaturation at 94 ° C for 1 minute, annealing at 58 ° C for 1 minute, extension at 72 ° C for 2 minutes extension) was performed for 35 cycles, followed by extension at 72 ° C for 5 minutes. The PCR product was electrophoresed on 0.8% agarose gel and the expected 1.2 kb band was detected. These bands were recovered from the gel and inserted into the pGEM T-easy vector, and the nucleotide sequences were analyzed. As a result, the total length of the gene was 2248 bp, consisting of 1824 bp ORF, 35 bp 5UTR and 349 bp 3UTR. At the 124th amino acid position, the amino acid F (phenylalanie) was changed to V (Valine) in the 5-methyltryptophan-resistant 7 strains, and the 2 point mutations in the other strains were 126 (TAC) -> TTC (phenylalanine) at amino acid position and CTT (Leucine) -> GAC (aspartic acid) at amino acid position 530 (Table 1).

Example  3: OsASA2 Mutant  Gene As a selection marker  Included Ti - Construction of plasmid vectors

The pPDG1 vector (MYONGJI UNIVERSITY) was used as the parent vector for transfection of the plant, and Bar gene was constructed by connecting to the CaMV 35S promoter in order to test the expression of the target gene. For selection of transformed calli and plants, the mutant OsASA2 (anthranilate synthase) gene, which is controlled by the PDG1 promoter, was used. A schematic diagram of the carrier used in the experiment is shown in Fig. Plasmid confirmed for vector construction for plant expression was transformed into Agrobacterium tumefaciens LBA4404. Agrobacterium tumefaciens competent cell LBA4404 prepared by the same method at 28 < 0 > 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: Production of 5-methyltryptophan-resistant OsASA2 mutant transgenic rice

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 placed 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 increase the influence of the medium component, the filter paper was dentated to the medium in which the filter paper was removed, and co-cultured at 25 ° C for 4 days. 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 the hormones used may enable efficient callus formation and introduction of target genes by Agrobacterium in co-culture Respectively. 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 > C for 1 week to proliferate callus. After 1 week of callus proliferation, the cells were transferred to N6D medium containing 25 mg / L 5-methyltryptophan (5-MT) and 400 mg / L carbenicillin for callus insertion with the calli using a healthy callus, And cultured for 2 weeks with continuous light.

After cultivation, the calli propagating proliferately from the blastocyst were transferred to SF (shoot formation) medium to induce plant regeneration. Green spots began to form on the callus after two weeks in the medium containing antibiotics (see FIG. 4), and shoot formation was initiated in the callus after approximately 3 weeks of culture 4). After transferring from antibiotic medium to SF medium, the efficiency of green spot formation and regeneration was different depending on the gene inoculated. The ground regeneration plant was transferred to a root formation medium (FIG. 4) to induce root development, and the purified rice transgenic plants were transplanted from the greenhouse into the pot (FIG. 4). A total of 35 transgenic plants were obtained from 115 transgenic calli.

Example 5: Identification of transgenic OsASA2 gene and gene expression analysis

The transgenic plants obtained in the transgenic experiments were examined for the introduction of the OsASA2 gene and the gene expression patterns in the rice genome, and the results are shown in FIG. In order to confirm the gene transfer, seeds were obtained from T0 plants and seeds were seeded at a rate of 20 per plant in rice seedlings box. After germination, seeds with resistance of 4ppm bastar treatment were used at 14 days and transgenic Bar and OsASA2 PCR analysis was performed using a primer set for gene-specific amplification. Bar and OsASA2 genes were detected in the transgenic lines transfected with the ASA2 gene causing the single mutation and the ASA2 gene causing the double mutation (panel A in FIG. 5). Also, transgenes were more expressed in these plants than in control and Mock (panel B of FIG. 4). This suggests that the OsASA2 gene was introduced into the rice genome well and transferred to the later stage, and the gene was expressed well.

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 (Fig. 6).

Example 7: Amino acid analysis of transformants

The total amount of free amino acids in ASA2 gene which caused single mutation and ASA2 gene which caused double mutation was about 10-30 times higher than that of the control. The single mutation gene was higher than the double mutation. The contents of tryptophan, phenylalanine, threonine, serine and valine were 6.7, 4.6, 3.6, 2.8 and 2.0 times higher than the control, respectively (Table 2). Overexpression of the OsASA2 gene resulted in insensitivity to feedback inhibition on the tryptophan biosynthesis circuit, resulting in an overall increase in amino acid content and a high specific amino acid yield. In Table 2 below, # data are expressed as amino acid nano-mol / g dry seed weight and are based on the wild type (Dongjinbara), Mock control (Ti-plasmid vector) or 5-MT resistant transgenic plants (single point mutants and double point mutants) Mean ± SD of 6 seeds. Mean values indicated by the same letter were not significantly different at the 5% level (Student's t test).

Example 8: Analysis of agricultural traits of transformants

Homozygous and intergenic transgenic plants were harvested by sowing on a GMO packaging basis at the Biotech Institute of Biotechnology with a single copy selected (Fig. 7). The agronomic traits of the transgenic crops were harvested on October 2, 2013, including the number of days of heading, number of shoots, number of shoots, number of tillers, lambing rate, densities / The results are shown in Table 3. The number of days of heading was about 100 days, the soybean was 78-81 cm, and the head was 18-20 cm. This means that the transgenic plants show similar traits to the control in T2 generation and are considered to have little effect on gene transfer.

a: Spikelet fertility is recorded for all spikes of 10 plants in 2012 and 3 spikes in 60 stems of 2013 plants.

b: From the average dry weight of air-dried harvested seeds, seeds are estimated to contain approximately 10-15% moisture content.

c: Pollen fertility was recorded for three small heads of each of three randomly selected plants.

d: 1000 seed weights were measured from 20 or 100 grains.

Example 9: Control analysis of tryptophan biosynthesis system using ASA transgenic transformant

(T3 generation) in which the free Trp content was increased by the introduction of the ASA2 mutation gene. The free Trp content of the seeds was increased by 15-33 times compared with non-recombinant, and the difference in the seeds of the plants that controlled the expression by the rice seed protein promoter reached about 0.4%. High Trp content seeds germinated and grown normally and had finesse. Using the system in which the amino acid content was most changed among the plants, the extract was extracted from 80% acetone for each portion shown in FIG. 7, and the extract was purified by using a reverse-phase solid cartridge, followed by LC / MS / MS analysis ).

Trp, IAN and IAA amino acid contents of single mutant transgenic transformants (S-TG) and double mutant transgenic transformants (D-TG) were measured by root, stem, first leaf, second leaf and third leaf As a result, Trp was found to be high in all the transformants, especially in leaf 3. IAN and IAA contents showed similar patterns. From these results, the IAA content of OASA2 transgenic rice plants was significantly increased by plant growth compared to the control. Interestingly, the distribution of biosynthesis in the plant body of IAA showed the same tendency as Trp accumulation. In addition, IAN is also increasing with the same trend as these two components, and accumulating IAA is biosynthesized by IAN through Trp (FIG. 9).

The Trp and IAA contents of the transformants transfected with the OASA2 mutant gene were higher than those of the control. Therefore, it is clear that the accumulation amount of Trp reflects biochemical enzyme function, It has been revealed that the Trp accumulation concentration is controlled to some extent. Among the basic genes of enzymes involved in the chorismate pathway upstream of the Trp biosynthetic pathway in rice, four kinds of DAHP synthase, chorismate kinase, and EPSP synthase, which are presumed to be important for metabolic control, Function analysis. Chorismate kinase is important for the biosynthesis of aromatic amino acids and related compounds, and is also a precursor to secondary metabolites such as tannins. In the expression analysis of the enzyme of the present invention, the expression levels of S-GT, GT-ST and ATR1 genes were significantly higher in the transformants. This means that the tryptophan content in the plant is high, and the IAA content and the protein encoding the secondary metabolites S-GT, GT-ST and ATR1 gene are highly expressed (FIG. 10).

Example 10: Selection test of transformants at 5MT

Amino acid contents of 5MT-4 and 5 strains were increased by 22 and 30 fold, respectively, compared to wild-type strains. The content of tryptophan in 5MT-2 was not significantly different from that of wild-type strains. 5MT-2, 4, and 5 strains were proliferated and the resistance test was performed again on 5MT. As a result, as shown in FIG. 11, 5MT-2 showed resistance in all ages but it did not grow in the medium containing 25 mg / L 5-methyltryptophan in the same manner as the wild type.

Example 11: 5 - Methyltryptophan  Resistant mutation ASA2  Screening of transgenic plants using vectors containing genes

Fig. 12 shows the results of culturing wild type callus and callus transformed with the vector (pSP1-5MT vector) prepared in Example 3 in a medium containing 25 ppm of 5MT. Panel A of FIG. 12 shows the result of culturing the callus of wild-type Dongjin rice in a medium containing 25 ppm of 5MT. Panel B shows the results of culturing the transformed Dongjin cotton callus transfected with pSP1-5MT vector with 25 ppm of 5MT It is the result of culturing in one medium. Compared with the results of Panel A, the callus transformed with the pSP1-5MT vector of Panel B shows that the introduction of the pSP1-5MT vector containing the OsASA2 mutant gene selection marker is also possible. Thus, the pSP1-5MT vector containing the OsASA2 variant gene selection marker of the present invention was found to be very useful for screening individuals regenerated in a medium containing 5MT.

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> Use of Mutated Anthranilate Synthase Gene for Selection Marker of          Plant Transformation <130> MP14-0315 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 606 <212> PRT <213> Oryza sativa L. <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 Val 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 <210> 2 <211> 606 <212> PRT <213> Oryza sativa L. <400> 2 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> 3 <211> 1821 <212> DNA <213> Oryza sativa L. <400> 3 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 agcttcctcg tcgagtccgt 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 agacatgctt 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> 4 <211> 1821 <212> DNA <213> Oryza sativa L. <400> 4 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> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 atggagtcca tcgccgccgc ca 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 agctttcgta gacaaggaat ag 22

Claims (8)

Anthranilate Synthase mutant having the resistance to 5-methyltryptophan, which is represented by the amino acid sequence of SEQ ID NO: 1.
A recombinant vector for screening a transformed plant cell comprising a polynucleotide encoding the anthranilate synthase mutant of claim 1.
3. The recombinant vector according to claim 2, wherein the recombinant vector has the structure of the vector shown in Fig.
(a) a recombinant vector comprising a polynucleotide encoding an anthranilate synthase variant represented by the amino acid sequence of SEQ ID NO: 1 and having resistance to 5-methyltryptophan; And (b) 5-methyltryptophan.
delete A method for screening transformed plant cells comprising the steps of:
(a) transforming a plant cell with the recombinant vector according to any one of claims 2 and 3;
(b) culturing the transformed plant cells in a medium containing 5-methyltryptophan; And
(c) selecting the plant cells grown in the cultured medium.
The method according to claim 6, wherein the plant cell is a cell of rice, wheat, barley, corn, tobacco, potato, tomato, or sweet potato.
A transgenic plant comprising the anthranilate synthase mutant coding polynucleotide of claim 1.

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