JP2010051310A - Modification of rice anthranilate synthetase using homologous recombination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a plant body containing high-concentration tryptophan by a gene-targeting method, and the plant body and the like produced by the method. <P>SOLUTION: In designing a vector used for a gene-targeting method, a point mutation of S126F is brought to locate in the right border (RB) side. By arranging the S126F mutation in the RB side of the T-DNA, it is confirmed that a gene-targeting individual is efficiently obtained even in the case when a homologous sequence region must be short. It is found that it is effective to use 5MT in 500 μM which is higher than the concentration ever reported in selecting an individual succeeded in gene-targeting of OASA2 gene. The gene-targeting individual thus obtained accumulates about 8.62 nmol/mg isolated tryptophan in rice leaf blades. It is about 130 times the content of wild type isolated tryptophan. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a plant containing a high concentration of tryptophan, a plant produced by the method, and use thereof.

  Anthranilate synthase (AS) is known as a key enzyme for tryptophan synthesis and is known to be negatively controlled by a high concentration of tryptophan. AS is a tetramer composed of α and β subunits, of which α subunit is subject to feedback inhibition by a high concentration of tryptophan. In rice, there are two copies of the gene encoding the α subunit of AS, which are named OASA1 and OASA2 genes, respectively (Non-patent Document 1). The OASA2 gene is more widely expressed in rice tissues than the OASA1 gene, but OASA2 is more sensitive to OASA1 than OASA1 (Non-patent Document 2). In addition, from biochemical studies using OASA2 synthetic proteins, modified OASA2 (S126F / L530D) and OASA2 (Y367A / L530D) substituted with 2 amino acids are only less sensitive to high concentrations of tryptophan. It is known that the enzyme activity itself is very high (Non-patent Document 3). For example, it has been shown that in a transformed rice callus in which a large amount of mutant OASA2 gene (S126F / L530D) is expressed, tryptophan is accumulated 300 to 400 times that of non-transformants (Non-patent Document 4).

Gene targeting (gene targeting, GT) using homologous recombination is a very useful technique capable of modifying only a target gene, unlike conventional gene introduction methods. The conventional gene transfer method inserts foreign DNA at random positions in the genome, but GT can modify only the target gene as desired, so it can be said to be a “cleaner” gene modification technique. Modification of target genes using GT is widely performed in microorganisms such as yeast and mammals such as mice. On the other hand, in higher plants such as rice, the efficiency of homologous recombination is very low. The probability that a DNA sequence introduced into a plant cell undergoes homologous recombination is said to be 10 ^-3 -10 ^-5, which is the probability that the sequence will be randomly inserted into the plant genome. Therefore, modification of target genes with GT is not easy.

  Recently, it has been reported that endogenous genes have been successfully modified by GT in model higher plants such as rice and Arabidopsis thaliana. The first successful example of GT in rice is a knockout of the Waxy gene (Non-patent Document 5). This method uses a hygromycin phosphotransferase gene as a marker for selecting GT individuals. Therefore, it can be applied to other genes, and the ADH2 gene has been successfully knocked out by the same method (Non-patent Document 6). On the other hand, if the endogenous gene itself can be used as a selection marker, it is possible to select GT individuals without using antibiotics. It has been reported that the rice ALS gene becomes resistant to the herbicide Bispyripac (BS) by introducing a point mutation. Using this, only cells that have succeeded in GT have been selected to exhibit BS resistance, and the target point mutation has been successfully introduced into the ALS gene (Non-patent Document 7). In addition, individuals who have expressed a large amount of BS-resistant ALS gene by a conventional gene recombination technique are sensitive to the treatment of a high concentration of BS because the endogenous BS-sensitive ALS gene is expressed. On the other hand, it was revealed that individuals who eliminated the wild-type BS-sensitive ALS gene by GT (that is, individuals that were homozygous by GT and had a BS-resistant ALS gene) acquired complete resistance to BS. (Non-patent document 7). This was thought to be because the endogenous ALS gene could be completely eliminated by GT.

Patent Publication 2006-246744 Gene Introduction Method, Gene Targeting Method, and Transgenic Plant Production Method WO2003 / 020940 Genome modification method of higher plant Patent Publication 2004-350692 Modified variant of DNA encoding first isozyme α subunit of rice anthranilate synthase Patent Publication 2002-101776 Method for selecting transformed plants WO99 / 011800 GENE ENCODING α SUBUNIT OF RICE ANTHRANILATE SYNTHASE AND DNA RELATED TO THE GENE Patent publication 2003-265182 Plasmid containing callus-specific expression promoter and method for selecting transformed plant cell callus Patent Publication 2006-042801 Novel modified gene of rice anthranilate synthase gene OASA2 and use thereof Patent Publication 2000-342277 Promoter of α subunit gene of second isozyme of rice anthranilate synthase

Tozawa Y. et al. (2001) Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1. Plant Physiol. 126: 1493-506. Kanno T. et al. (2004) In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2.Plant Mol Biol. 54: 11-22. Kanno T. et al. (2005) Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway.Plant Physiol.138: 2260-8. Control and utilization of primary and secondary metabolism in tryptophan biosynthesis system, Satoshi Wakasa, Strategic Creative Research Promotion Project Team Type Research CREST, FY 2006 Research Completion Report (2001, FY 2003 Adopted Research Project), 247-262 page Terada, R. et al. (2002) Efficient gene targeting by homologous recombination in rice.Nature Biotech, 20, 1030-1034. Terada, R. et al. (2007) Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics.Plant Physiol., 144, 846-856. Endo, M. et al. (2007) Molecular breeding of a novel herbicide-tolerant rice by gene targeting.Plant J., 52, 157-166. Yamada T. et al. (2004) Use of a feedback-insensitive at subunit of anthranilate synthase as a selectable marker for transformation of rice and potato. Mol. Breeding 14: 363-373. Saika H, Toki S (2008) Approach for metabolic engineering of amino acid production by T-DNA mediated gene targeting, FAO / IAEA International Symposium on Induced Mutations in Plants Toki S, Endo M, Saika H, Abe K, Osakabe K (2008) Site-directed mutagenesis in plants via gene targeting, FAO / IAEA International Symposium on Induced Mutations in Plants Sayaka Saga, Haruko Onodera, Seiichi Toki (2008) Modification of rice anthranilate synthase gene by gene targeting Research in Breeding 10 (Apart 2): 129 Saika H, Onodera H, Toki S (2008) Generation of a tryptophan hyper-accumulating rice by homologous recombination mediated gene targeting, The 6th 3R (Replication, Recombination, Repair) Symposium Saika H, Onodera H, Toki S (2008) Production of the tryptophan hyper-accumulating rice by T-DNA mediated gene targeting The 6th International Symposium of Rice Functional Genomics Hiroaki Saiga, Haruko Onodera, Seiichi Toki (2009) Artificial modification of rice anthranilate synthase gene by gene targeting. Abstracts of the 50th Annual Meeting of the Japanese Society of Plant Physiology Hiroaki Saiga, Haruko Onodera, Seiichi Toki, Modification of Rice Anthranilate Synthase Gene Using Gene Targeting, Agricultural Bioresource Research Institute Research Results Presentation (October 21, 2008) Towards a highly efficient gene targeting system in higher plants H. Saika and S. Toki S, Japan Agricultural Research Quarterly. 43, 81-85 (2009)

  This invention is made | formed in view of such a condition, The subject of this invention is providing the plant body etc. which are manufactured by the manufacturing method of the plant body containing the high concentration tryptophan by gene targeting method, the said method, etc. There is to do.

The inventors first designed a vector for use in gene targeting. In designing the vector, the present inventors devised the point mutation of S126F to be located on the right border side. The mutation of S126F is located at a distance of about 0.2 kb from the end of the vector. Usually, when the homologous sequence region is set short in the gene targeting method, the homologous recombination efficiency decreases. However, the present inventors have found that by placing the S126F mutation on the right border (RB) side of T-DNA, even when the homologous sequence region has to be shortened, gene targeting individuals can be obtained efficiently. I found it.
Next, the present inventors introduced the prepared vector into rice callus by the Agrobacterium method. The present inventors conducted a 5MT concentration test in order to efficiently select individuals who succeeded in gene targeting of the OASA2 gene. As a result, it was found that the use of 500 μM 5MT higher than the concentration reported so far was effective in selecting individuals who succeeded in gene targeting of the OASA2 gene.
The present inventors have found that about 8.62 nmol / mg free tryptophan is accumulated in rice leaf blades in the gene targeting individuals thus obtained. This was about 130 times the wild-type free tryptophan content.
The present invention is based on the discovery of such findings by the present inventors, and provides the following [1] to [24].
[1] A plant transformed with the DNA according to (a) or (b) below, which contains a high concentration of tryptophan;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3,
[2] The plant according to [1], which does not contain an endogenous anthranilate synthase α subunit,
[3] The plant according to [2], wherein the plant is a monocotyledonous plant,
[4] The plant body according to [3], wherein the monocotyledonous plant is a gramineous plant,
[5] The plant body according to [4], wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, sorghum, oat, and grass.
[6] A cell isolated from the plant according to any one of [1] to [5],
[7] Plant propagation material according to any one of [1] to [5],
[8] An organ isolated from the plant according to any one of [1] to [5],
[9] A method for producing a plant body containing tryptophan, comprising the following steps (a) and (b):
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence set forth in SEQ ID NO: 1 or 3,
[10] The method according to [9], wherein the plant does not contain an endogenous anthranilate synthase α subunit,
[11] The method according to [10], wherein the plant is a monocotyledonous plant,
[12] The method according to [11], wherein the monocotyledonous plant is a gramineous plant.
[13] The method according to [12], wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, sorghum, oat, and grass.
[14] The method according to [13], wherein the vector is selected from vectors comprising the nucleotide sequence according to any of SEQ ID NOs: 7 to 10,
[15] A method of adding tryptophan to a plant, comprising the following steps (a) and (b):
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence set forth in SEQ ID NO: 1 or 3,
[16] The method according to [15], wherein the plant does not contain an endogenous anthranilate synthase α subunit,
[17] The method according to [16], wherein the plant is a monocotyledonous plant,
[18] The method according to [17], wherein the monocotyledonous plant is a gramineous plant,
[19] The method according to [18], wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, sorghum, oat, and grass.
[20] The method according to [19], wherein the vector is selected from vectors comprising the nucleotide sequence according to any of SEQ ID NOs: 7 to 10,
[21] An agent for containing a plant with a high concentration of tryptophan, comprising the DNA according to (i) or (ii) below or a vector containing the DNA as an active ingredient;
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence set forth in SEQ ID NO: 1 or 3,
[22] An agent for containing a plant with a high concentration of tryptophan, comprising as an active ingredient at least one of vectors comprising the base sequence of SEQ ID NO: 7 to 10,
[23] A method for producing tryptophan, comprising the following steps (a) and (b):
(A) A step of obtaining a plant body containing tryptophan by the method according to any one of [9] to [20], and (b) a step of recovering tryptophan from the plant body obtained in step (a). ,
[24] A transformed plant obtained by the method according to any one of [9] to [14].

According to the present invention, a method for producing a plant containing a high concentration of tryptophan and a plant produced by the method are provided. Since the plant body of the present invention contains tryptophan at a higher concentration than the plant body that can be obtained by a conventional gene transfer method, it is useful as a variety for feed rice or high-nutrition rice.
In the plant of the present invention, since only the target gene is modified, other regions are considered to have the same base sequence as the wild type. In contrast, conventional gene recombination techniques insert T-DNA into a random region in genomic DNA, which is likely to cause unexpected mutations. In addition, the T-DNA used in the present invention uses the rice genome sequence as it is. From the above, although genetic recombination technology is used, it is considered that individuals obtained by gene targeting are equivalent to mutant individuals. From this, it is thought that it is easy for general consumers to accept.
Conventional transgenic individuals often use genes other than rice (such as hygromycin phosphotransferase) as selection markers. When a conventional recombinant individual is crossed with a cultivated wild-type rice, a selective marker gene is introduced into the seed, so that antibiotic-resistant rice may diffuse into the environment. In contrast, in the present invention, the target gene itself was used as a selection marker. This suggests that antibiotic-resistant rice does not diffuse into the environment even when crossed with wild-type rice, and the burden on the natural environment is low.
In addition, the manufacturing method of the plant body of this invention can be widely utilized for the modification | change by gene targeting of the anthranilate synthase gene of the main grain rice, corn, and wheat.

It is a figure which shows the structure of the vector for GT. The black box indicates the coding sequence of the OASA2 gene, and the white box indicates UTR. In addition, the hatched box indicates the chloroplast transfer signal of the OASA2 gene. The GT vector used a sequence of about 7.0 kb downstream from the chloroplast transfer signal, and the positive control vector used a sequence of about 7.5 kb including 1.3 kb upstream from the translation start point. Black circles indicate the site where the point mutation was introduced. It is a figure which shows the construction of the vector used for experiment. As vectors for GT, OASA2SL-GT1 (SEQ ID NO: 7), OASA2SL-GT2 (SEQ ID NO: 8), OASA2YL-GT1 (SEQ ID NO: 9), and OASA2YL-GT2 (SEQ ID NO: 10) were used. Further, OASA2SL-PC and OASA2YL-PC were used as positive controls at the time of selection, and pPZP2028 was used as a negative control. It is a photograph which shows the growth of the rice callus which introduce | transduced positive control vector OASA2SL-PC (right) or empty vector pPZP2028 (left) on N6D culture medium containing 500 micromol 5MT. It is a photograph which shows the analysis result of the CAPS marker using the obtained 5MT resistant individual | organism | solid. (Upper) Since the HindIII site is newly added in the vicinity of S126F in individuals who have succeeded in GT and those in which the GT vector is randomly inserted, the 0.8 kb PCR product containing S126F is cut into 0.2 kb and 0.6 kb by HindIII. The (Bottom) In individuals who have succeeded in GT or individuals in which a GT vector has been randomly inserted, the EcoRV site disappears in the vicinity of L530D, so that the 0.5 kb PCR product containing L530D is not cleaved by EcoRV. It is the photograph of the plant body of the obtained GT individual. (Left) Whole plant body. (Right) Ho. It is the figure and photograph which show the result of having cut | disconnected DNA extracted from the obtained plant body with HindIII, and having performed the Southern analysis using the probe. Since the HindIII site is added as a CAPS marker to the GT vector OASA2SL-GT1, a band is confirmed to be 6.0 kb for the wild-type OASA2 gene and 1.5 kb for the OASA2 gene modified by GT. WT: non-transformed individual, M: λ HindIII marker.

The present invention is a plant transformed with a DNA encoding a variant of anthranilate synthase α subunit (also referred to herein as “OASA2”), which contains a high concentration of tryptophan To provide a plant body. More specifically, the present invention provides a plant transformed with the DNA described in (a) or (b) below, which contains a high concentration of tryptophan.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3

As a modified form of OASA2 of the present invention, in wild type OASA2 (protein containing the amino acid sequence described in SEQ ID NO: 6), an amino acid in which serine at position 126 is substituted with phenylalanine and leucine at position 530 is replaced with aspartic acid A protein containing the sequence (the amino acid sequence described in SEQ ID NO: 2) is exemplified, but not limited thereto. In addition, as a modified form of OASA2 of the present invention, in wild-type OASA2 (protein containing the amino acid sequence described in SEQ ID NO: 6), an amino acid in which tyrosine at position 367 is replaced with alanine and leucine at position 530 is replaced with aspartic acid A protein containing the sequence (the amino acid sequence described in SEQ ID NO: 4) is exemplified, but not limited thereto.
Further, in the DNA encoding wild-type OASA2 (DNA containing the nucleotide sequence described in SEQ ID NO: 5) as the DNA encoding the modified OASA2 of the present invention, cytosine at position 377 is thymine, and cytosine at position 1588 is The guanine includes, but is not limited to, a DNA containing a base sequence (base sequence described in SEQ ID NO: 1) in which thymine at position 1589 is replaced with adenine and thymine at position 1590 is replaced with cytosine. In addition, as a DNA encoding a modified form of OASA2, in the DNA encoding wild-type OASA2 (DNA containing the base sequence described in SEQ ID NO: 5), thymine at position 1099 is guanine, adenine at position 1100 is cytosine, Examples include DNA containing a base sequence (base sequence described in SEQ ID NO: 3) in which cytosine at position 1588 is replaced by guanine, thymine at position 1589 is replaced by adenine, and thymine at position 1590 is replaced by cytosine. Not.

  The “DNA encoding a modified OASA2” of the present invention is not particularly limited as long as it can encode a modified OASA2 in rice or other plants. In addition, DNA encoding an OASA2 variant includes DNA having an arbitrary base sequence based on the degeneracy of the genetic code.

  Tryptophan content can be measured, for example, by a high-speed amino acid analyzer (Wakasa, K. et al. (2006) High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile. J. Exp. Bot, 57. 3069-3078), but is not limited to this method.

The transformed plant of the present invention contains a high concentration of free tryptophan compared to the wild type. The transformed plant of the present invention is not particularly limited as long as the free tryptophan content is increased compared to the wild type, but for example, at least 100 times, 110 times, 120 times, 130x, 140x, 150x, 160x, 170x, 180x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x 300 times, 310 times, 320 times, 330 times, 340 times, 350 times, 360 times, 370 times, 380 times, 390 times, 400 times, 410 times, 420 times, 430 times, 440 times, 450 times, 460 times Examples include transformed plants having double or more free tryptophan content.
In the present invention, the site where tryptophan content increases includes, but is not particularly limited to, leaves and seeds. It is known that tryptophan in plants is translocated to a new tissue, and it is considered that tryptophan content is higher in new leaves than in old leaves. Tryptophan will eventually accumulate in seeds at high concentrations.

  The transformed plant of the present invention does not have an endogenous anthranilate synthase. In the present invention, the endogenous anthranilate synthase means a wild-type anthranilate synthase (for example, a protein containing the amino acid sequence shown in SEQ ID NO: 6) inherent in a wild-type plant. Whether or not the transformed plant body has endogenous anthranilate synthase can be confirmed, for example, by a known PCR method or Southern hybridization method, or by analyzing the base sequence of DNA in the plant body. Yes, but not limited to these methods.

  The transformant of the present invention is characterized by being produced by a gene targeting (GT) method using homologous recombination. By using the gene targeting (GT) method, it is possible to modify only the target gene. Moreover, in the transformed plant produced by the gene targeting (GT) method, the expression level of the recombinant gene is stable because it is affected by the endogenous promoter. Furthermore, transformants generated by using the gene targeting (GT) method do not have an endogenous gene. Therefore, it contains a higher concentration of tryptophan compared to a transformed plant produced by the conventional gene transfer method.

  As long as the transformed plant of the present invention contains a high concentration of tryptophan, other plant traits may be modified. Examples of other trait modifications include, but are not limited to, an increase in auxin content, a change in the content and composition of alkaloid compounds, a change in the content and composition of amino acids other than tryptophan, and the like.

Preparation of DNA encoding OASA2 can be performed by those skilled in the art using conventional means. For example, genomic DNA is extracted from a plant, a genomic library (including but not limited to plasmids, phages, cosmids, BACs, PACs, etc.) is created and expanded to encode OASA2. DNA encoding OASA2 can be obtained by performing colony hybridization or plaque hybridization using a probe prepared based on the DNA to be prepared (for example, the DNA comprising the base sequence described in SEQ ID NO: 5) . Moreover, it is possible to obtain DNA encoding OASA2 by preparing a primer specific to DNA encoding OASA2 and performing PCR using this primer.
In addition, when preparing cDNA encoding OASA2, for example, cDNA is synthesized based on mRNA extracted from rice or plants other than rice and inserted into a vector such as λZAP to create a cDNA library. Can be prepared and colony hybridization or plaque hybridization can be performed in the same manner as described above, or by performing PCR.

  The DNA encoding the modified OASA2 of the present invention can be obtained by introducing a mutation into the DNA encoding OASA2 obtained by the method described above. Mutation can be introduced into DNA encoding OASA2 by a known method such as kunkel method or PCR-based method. It is also possible to introduce mutations to DNA encoding OASA2 using commercially available kits (including but not limited to Mutan-K, Mutan-Super Express Km, LA-PCR in vitro mutanegenesis Kit, etc.). is there.

To create a transformed plant using DNA encoding a modified OASA2, introduce the vector into which the DNA has been inserted, and regenerate the plant from the resulting transformed plant cell. You can do it.
The vector in the present invention is preferably a vector capable of expressing an inserted gene in plant cells, and examples thereof include vectors such as pMH1 and pMH2, but are not particularly limited. The vector of the present invention may have, for example, a promoter for constitutive gene expression in plant cells (for example, potato chitinase gene SK2 promoter, cauliflower mosaic virus 35S promoter, etc.). When such a promoter is used, a DNA is designed in which a DNA encoding a modified OASA2 of the present invention is functionally linked downstream of the promoter. Then, a vector containing the designed DNA is introduced into plant cells. By regenerating the transformed plant cell thus obtained, a transformed plant in which the modified OASA2 of the present invention is expressed can be obtained. Thus, the present invention also provides a DNA in which a DNA encoding a modified OASA2 is functionally linked downstream of a promoter. Here, “functionally bound” means that the promoter sequence and the DNA are bound so that expression of DNA encoding the OASA2 variant of the present invention is induced by binding of a transcription factor to the promoter sequence. Say that you are.
In addition to these, it is also possible to use a vector having a promoter that is inducibly activated by an external stimulus.

  For those skilled in the art, the DNA of the present invention is introduced into cells by a known method such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, particle gun method and the like. Can do.

  Furthermore, regeneration of plant bodies can be performed by methods known to those skilled in the art depending on the type of plant cells (Toki et. Al., Plant Physiol. 100: 1503-1507, 1992). For example, as a method for producing a transformed plant, a method of introducing a gene into a protoplast with polyethylene glycol and regenerating the plant (Datta et. Al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) Pp66- 74, 1995), a method of introducing a gene into a protoplast by an electric pulse to regenerate the plant body (Toki et. Al., Plant Physiol. 100: 1503-1507, 1992), a gene directly introduced into a cell by the particle gun method. A method of regenerating a plant (Christou et. Al., Bio / technology, 9: 957-962, 1991) and a method of regenerating a plant by introducing a gene through Agrobacterium (Hiei et. Al. , Plant J. 6: 271-282, 1994) have already been established and widely used in the technical field of the present invention. In the present invention, these methods can be suitably used.

  When the Agrobacterium method is used, for example, the method of Nagel et al. (Microbiol. Lett. 67: 325, 1990) is used. According to this method, the recombinant vector is transformed into Agrobacterium, and then the transformed Agrobacterium is introduced into the cell by a known method such as a leaf disk method. The vector contains an expression promoter so that, for example, after introduction into a plant body, the DNA encoding the modified OASA2 of the present invention is expressed in the plant body. In general, a DNA encoding a modified OASA2 of the present invention is located downstream of the promoter, and a terminator is located downstream of the DNA. The recombinant vector used for this purpose is appropriately selected by those skilled in the art depending on the method of introduction into the plant or the type of plant. Examples of the promoter include CaMV35S derived from cauliflower mosaic virus, corn ubiquitin promoter (Japanese Patent Laid-Open No. 2-79983), and the like.

  Examples of the terminator include a terminator derived from cauliflower mosaic virus or a terminator derived from a nopaline synthase gene. However, the terminator is not limited to these as long as it is a promoter or terminator that functions in a plant body.

  Further, the plant into which the DNA encoding the modified OASA2 of the present invention is introduced may be an explant, and cultured cells may be prepared from these plants and introduced into the obtained cultured cells. The plant of the present invention includes plant cells such as scutellum in leaves, roots, stems, flowers and seeds, callus, suspension culture cells and the like in addition to individual plants.

  In addition, in order to efficiently select cells transformed by introduction of DNA encoding the modified OASA2 of the present invention, the recombinant vector is a plant cell together with an appropriate selection marker gene or a plasmid vector containing the selection marker gene. May be introduced. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene resistant to the antibiotic hygromycin, the neomycin phosphotransferase gene resistant to kanamycin or gentamicin, and the acetyl resistant to the herbicide phosphinothricin. Examples include transferase genes.

  Cells into which the recombinant vector has been introduced are placed on a known selection medium containing an appropriate selection agent according to the type of the introduced selection marker gene and cultured. As a result, transformed plant cultured cells can be obtained.

The T-DNA encoding the modified OASA2 of the present invention can be used as a marker gene for GT. Since the cells into which the T-DNA encoding the OASA2 variant of the present invention has been introduced into the endogenous OASA2 gene are resistant to tryptophan, the OASA2 variant of the present invention can be obtained by adding a tryptophan analog to the medium. It is possible to select only cells in which a DNA encoding is introduced and GT is produced.
Moreover, since the T-DNA of the present invention does not contain a chloroplast translocation signal, the modified OASA2 does not function when the T-DNA is randomly inserted into the genome. That is, the T-DNA of the present invention acts as a negative marker in GT experiments using 5MT selection, and transformed cells into which T-DNA is randomly inserted on the genome are killed. As a result, it is possible to obtain only cells that have succeeded in GT.

The plant body regenerated from the transformed cells is then cultured in an acclimation medium. Thereafter, when the acclimatized regenerated plant body is cultivated under normal cultivation conditions, a transformed plant body containing a high concentration of tryptophan is obtained, and seeds can be obtained by maturing and fruiting. That is, this invention provides the manufacturing method of the transformed plant body containing a tryptophan including the process of following (a) and (b), and the transformed plant body obtained by the said method.
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A step of regenerating a plant from cells (i) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence described in SEQ ID NO: 1 or 3.
Moreover, this invention provides the manufacturing method of the transformed plant body containing a tryptophan including the process of following (a) and (b), and the transformed plant body obtained by the said method.
(A) a step of introducing a vector comprising the nucleotide sequence of any one of SEQ ID NOs: 7 to 10 into a plant cell by gene targeting method; and (b) a plant from the plant cell into which the vector was introduced in step (a). Step of Regenerating Body A transformed plant containing a high concentration of tryptophan can be obtained by these methods.

  In addition, the presence of introduced foreign DNA in a transformed plant that has been regenerated and cultivated in this way can be analyzed by a known PCR method or Southern hybridization method, or by analyzing the base sequence of the DNA in the plant body. Can be confirmed. In this case, extraction of DNA from the transformed plant body can be performed according to a known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, 1989). When a foreign gene comprising DNA encoding a modified OASA2 present in a regenerated plant body is analyzed using the PCR method, an amplification reaction is performed using the DNA extracted from the regenerated plant body as a template. Alternatively, a synthetic oligonucleotide having a base sequence appropriately selected according to the base sequence of DNA encoding a modified OASA2 can be used as a primer, and an amplification reaction can be carried out in a reaction mixture in which these are mixed. In the amplification reaction, when DNA denaturation, annealing, and extension reaction are repeated several tens of times, an amplification product of a DNA fragment containing a DNA sequence encoding a modified OASA2 can be obtained. When the reaction solution containing the amplification product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and the DNA fragments correspond to the DNA of the present invention.

  Once a transformed plant is obtained in which a DNA encoding the OASA2 variant of the present invention is introduced into the chromosome, progeny can be obtained from the plant by sexual reproduction or asexual reproduction. In addition, cells, organs, and propagation materials (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) are isolated from the plant body, its progeny or clones, and the plant body is based on them. Mass production is also possible. The present invention includes a plant cell into which a DNA encoding a modified OASA2 is introduced, a plant body containing the cell, an organ of the plant body (for example, a flower, a leaf, a root, a stem, etc.), a progeny of the plant body, and The clone and the propagation material of the plant, its progeny and clones are included. These plant cells, plants containing the cells, organs of the plant (eg, flowers, leaves, roots, stems, etc.), progeny and clones of the plant, and propagation materials of the plants, progeny and clones thereof It can be used to produce transformed plants containing high concentrations of tryptophan.

  The plant of the present invention includes monocotyledonous plants. Such plants include gramineous plants. Examples of grasses include, but are not limited to, rice, corn, wheat, barley, sorghum, oats, and grass.

The present invention further provides a method for causing a plant to contain tryptophan, which comprises the following steps (a) and (b).
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence described in SEQ ID NO: 1 or 3.
By this method, a transformed plant containing a high concentration of tryptophan can be obtained.

The method of adding tryptophan to the plant of the present invention includes not only a method of adding tryptophan to a wild type plant but also a method of adding tryptophan to a plant in which some mutation has already been introduced. Here, the kind and degree of the mutation are not limited with respect to the plant in which some mutation has already been introduced.
In the present invention, the plant to be tryptophan-containing is not particularly limited, and examples thereof include the above-mentioned plants.

  In the above method, examples of the vector containing a DNA encoding a modified OASA2 include, but are not limited to, a vector containing the base sequence described in any of SEQ ID NOs: 7 to 10. In addition, introduction of a vector containing DNA encoding a modified OASA2 into a plant cell and regeneration from the plant cell into the plant can be performed by the above-described methods.

The present invention also provides an agent for containing a high concentration of tryptophan in a plant, containing DNA encoding a modified OASA2 or a vector containing the DNA as an active ingredient. As DNA encoding the modified OASA2,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence set forth in SEQ ID NO: 1 or 3,
However, it is not limited to these. Examples of the vector containing DNA encoding a modified OASA2 include, but are not limited to, a vector containing the base sequence described in any of SEQ ID NOs: 7 to 10.
The agent of the present invention can be used, for example, when a plant containing a high concentration of tryptophan is produced or when a plant contains a high concentration of tryptophan. Plants include, but are not limited to, those mentioned above.

The present invention also provides a method for producing tryptophan comprising the following steps (a) to (c).
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A step of regenerating the plant from the cells, and (c) a step of recovering tryptophan from the plant produced in step (b).
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence described in SEQ ID NO: 1 or 3.
In the method for producing tryptophan of the present invention, first, a transformed plant containing tryptophan is obtained by a gene targeting method. A transformed plant containing tryptophan can be obtained by the method described in this specification (for example, the method described in Examples).
Next, in the method for producing tryptophan of the present invention, tryptophan is recovered from a transformed plant containing tryptophan. The recovery of tryptophan is described, for example, in Wakasa K and Widholm J. (A 5-methyltryptophan resistant rice mutant, MTR1, selected in tissue culture. Theor. Appl. Genet. 74: 49-54. (1987)). Furthermore, it is possible to carry out by centrifuging and then recovering the soluble fraction after disrupting the tissue, but it is not limited thereto.
The plant of the present invention is a plant having an anthranilate synthase α subunit having reduced sensitivity to tryptophan and not having an endogenous anthranilate synthase α subunit exhibiting sensitivity to tryptophan. It can be expressed as “body”. In addition, “an endogenous anthranilate synthase α-substance that is resistant to tryptophan feedback inhibition in the tryptophan biosynthetic pathway and that is subject to feedback inhibition by tryptophan (not resistant to tryptophan feedback inhibition). It can be expressed as “a plant without a unit”.

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples.
1. Materials and methods
1.1 Production of GT vector
The OASA2SL (ΔCP) or OASA2YL (ΔCP) region shown in FIG. 1 was used as the GT vector, and the Pro :: OASA2SL or Pro :: OASA2YL region shown in FIG. 1 was used as the positive control.
Using the genomic DNA of rice cultivar Nipponrye (Oryza sativa L. cv Nipponbare) as a template, an approximately 4.4 kb fragment of the OASA2 gene was amplified by PCR. PCR primer
5′-CCCATCGAGGAAGAGTAAACCCAAAAA-3 ′ (SEQ ID NO: 11)
5'-TAAACATGCAGCGAGAAGAACCATGAA-3 '(SEQ ID NO: 12)
And the enzyme KOD-plus- (Toyobo), following the instructions of the kit. The obtained PCR fragment was cleaved with EcoRI and XhoI and then cloned into a pBS vector. Mutations such as S126F / L530D and Y367A / L530D were introduced into the obtained clones using PCR. PCR includes primers
S126F;
5'-GGAGACGCC [aAGCTT] CTCTTCGAG TtC GTCGAGC-3 '(SEQ ID NO: 13)
5′-GCTCGAC GaA CTCGAAGAGG [AAGCTt] GGCGTCTCC-3 ′ (SEQ ID NO: 14)
Y367A;
5′-TTGTGAACCCAAGTCCA g [cC ATGG] CATATGTACAGGTAT-3 ′ (SEQ ID NO: 15)
5′-ATACCTGTACATATG [CCAT Gg] c TGGACTTGGGTTCACAA-3 ′ (SEQ ID NO: 16)
L530D;
5'-GCCTTGGAGG [GATtTC] ATTTGACGGAGACATG gac ATCGC-3 '(SEQ ID NO: 17)
5'-GCGAT gtc CATGTCTCCGTCAAAT [GAaATC] CCTCCAAGGC-3 '( SEQ ID NO: 18)
(Lower case letters indicate bases into which mutations have been introduced. The underlined portion is an amino acid substitution, and the portion between [] is a non-synonymous substitution and is a sequence converted to a CAPS marker for efficient detection of GT individuals.) QuickChange II XL Site-directed Mutagenesis kit (STRATAGENE) was used. The obtained clones were designated as SL-1 or YL-1. In addition, an approximately 9.7 kb fragment containing the OASA2 gene was amplified by PCR using genomic DNA of rice varieties Nipponbare as a template. PCR primer
5'-ATGGACTTGTTTACTCGGGAGTGGTTG-3 '(SEQ ID NO: 19)
5'-TCCCAACCTCTCATCTCTCATTTTTCG-3 '(SEQ ID NO: 20)
And the enzyme KOD-plus- were performed according to the kit instructions. The obtained PCR fragment was cleaved with KpnI and PacI and then cloned into the pPZP2028 vector (modified vector in which the AscI / PacI site was added to the MCS of pPZP202). This was digested with SacII / XhoI, and an approximately 3.8 kb fragment excised from SL-1 or YL-1 with SacII / XhoI was introduced. The resulting vector was named SL-2 or YL-2.

The obtained vector SL-2 or YL-2 was used as a template, and a fragment of about 7.5 kb was amplified by PCR. PCR primer
5′-GCGTGA [GGTaCc] CCGCGGGGAAG-3 ′ (SEQ ID NO: 21)
5'-GGGCTTAGGGGTCCTGTTGGAAACTAT-3 '(SEQ ID NO: 22)
(The sequence between [] indicates a newly added KpnI site) and the enzyme KOD-plus- (Toyobo) was used according to the description of the kit. The obtained PCR fragment was cleaved with KpnI and EcoRI and then cloned into the pPZP201 vector. The obtained clone was used as a vector for GT, which was OASA2SL-GT1 or OASA2YL-GT1, respectively (FIG. 2).
The PCR fragment cleaved with KpnI and EcoRI was cloned into pCAMBIA1304. The obtained clone was used as another GT vector, which was OASA2SL-GT2 or OASA2YL-GT2, respectively (FIG. 2). Further, vector SL-2 or YL-2 was cleaved with SpeI and then cloned into pCAMBIA1304 or pCAMBIA1300 vector. The obtained clones were used as positive control vectors, and OASA2SL-PC or OASA2YL-PC, respectively (FIG. 2).

  In the GT vector prepared this time, the mutation of S126F is located at a distance of about 0.2 kb from the end of the vector. Usually, if the homologous sequence region is shortened, the homologous recombination efficiency decreases, and it is considered difficult to obtain GT individuals efficiently. This time, in order to overcome it as much as possible, the mutation of S126F was placed on the right border (RB) side of T-DNA. In contrast to the left border (LB), the T-DNA is protected by the protein on the RB side, so that the DNA is rarely scraped until reaching the nucleus. As a result, it was considered that the mutation of S126F could be introduced efficiently.

The gene transfer methods Datsu頴, sterile rice seeds cultivar Nipponbare using 1.2 Agrobacterium were plated on N6D medium and cultured 3-4 weeks at 33 ° C. under light. The obtained callus was transformed by the Agrobacterium method. Agrobacterium strain EHA105 was used for transformation. Rice callus treated with Agrobacterium was placed on 2N6AS medium and co-cultured at 25-28 ° C in the dark for 3 days. Thereafter, Agrobacterium was sterilized with sterilized water and a carbenicillin solution, and placed on a selective N6D medium containing 500 μM 5-methyltryptophan (a tryptophan analog that inhibits AS activity, 5MT) and carbenicillin. Selection of 5MT resistant callus was performed for 1-2 months under 33 ° C light conditions. On the way, callus was transplanted to a new medium several times. The obtained 5MT-resistant callus was placed on a regeneration medium containing 300 μM of 5MT and cultured under light conditions at 33 ° C. for about 1 month. The obtained redifferentiated plant was transplanted to a hormone-free medium containing 300 μM of 5MT and cultured under light conditions at 33 ° C. for about 1 month.

1.3 CAPS analysis of individuals obtained by GT
DNA was extracted from the leaves of individuals obtained by GT. The obtained DNA was used to amplify a 0.5-0.8 kb fragment containing a base into which a mutation was introduced by PCR. PCR is primer S126F;
5'-GCGTGAGGTACCCCGCGGGGAAG-3 '(SEQ ID NO: 21)
5′-ACGAGAACATCATCATAAAGCCCAAGG-3 ′ (SEQ ID NO: 23)
Y367A;
5'-ACTCGGCAGTTTGGTACACCTTTGAAC-3 '(SEQ ID NO: 24)
5'-ACAGTCCCAGCAAGTGGACGGTTAATA-3 '(SEQ ID NO: 25)
L530D;
5'-GTTGGGACAGTGTAGCCTCTTGGTAG-3 '(SEQ ID NO: 26)
5'-CTGCCTTGTTCTCGCATTCACGTT-3 '(SEQ ID NO: 27)
And the enzyme KOD-dash- (Toyobo) and performed according to the description of the kit. The obtained PCR product was subjected to restriction enzyme treatment (S126F; HindIII, Y367A; NcoI, L530D; EcoRV), and it was confirmed by electrophoresis whether the mutation introduced into the GT vector as a CAPS marker was introduced. .

1.4 Determination of the base sequence of individuals obtained by GT
DNA was extracted from the leaves of individuals obtained by GT. NucleonPhytopure (GE) was used for DNA extraction. A fragment of about 10.0 kb was amplified by PCR using the obtained DNA. Sequence reaction using the obtained PCR product
5'-GCGTGAGGTACCCCGCGGGGAAG-3 '(SEQ ID NO: 21)
5'-GTTGGGACAGTGTAGCCTCTTGGTAG-3 '(SEQ ID NO: 26)
It was performed using.

Southern analysis of individuals obtained by 1.5 GT
DNA was extracted from the leaves of individuals obtained by GT. NucleonPhytopure (GE) was used for DNA extraction. For Southern analysis, 1.5 μg of DNA subjected to restriction enzyme treatment was used. PCR DIG probe synthesis kit (Roche) was used for probe preparation. Primers to make DIG labeled probes
5'-GATAGGTGGATGGGTCGGGTTCTTTTC-3 '(SEQ ID NO: 28)
5′-ACAGGATTGAAGGAATGGGCACAGGTT-3 ′ (SEQ ID NO: 29)
Was used. Southern analysis was performed according to a conventional method.

1.6 Measurement of tryptophan amount To quantify free amino acids containing tryptophan, rice leaf blades were sampled and frozen in liquid nitrogen. The frozen sample was pulverized using a multi-bead shocker (Yasui Kikai). After adding 200 μL of 2% sulfosalicylic acid to about 2 mg of the ground sample, the sample was ground and sonicated twice for 5 minutes. 25 μL of the extract filtered through a 0.22 μm filter was used for analysis. The quantification of various amino acids was performed using a biological fluid analysis / ninhydrin coloring and using an amino acid analyzer (Hitachi, L-8800A).

2. result
2.1 Acquisition of modified OASA2 gene by GT
In order to obtain modified individuals of OASA2 gene by GT, we designed a GT experimental system. From previous reports, there was a report that 300μM 5MT was used in rice callus when selecting using modified OASA1 gene (Yamada et al, 2004). Thus, rice callus introduced with the empty vector pPZP2028 and the positive control vector OASA2SL-PC or OASA2YL-PC was placed on an N6D selection medium containing 300-600 μM of 5MT and cultured under light conditions at 33 ° C. As a result, it became clear that 5MT-resistant callus proliferates in the callus introduced with the positive control vector, while 500μM is appropriate as the 5MT concentration condition for killing the callus in the callus introduced with the empty vector (Fig. 3).

A GT experiment using a GT vector was conducted under these selection conditions. As a result of the GT experiment using OASA2SL-GT1 or OASA2YL-GT1, we succeeded in acquiring individuals that seemed to succeed in one GT each. The following explains how to acquire successful GT individuals using OASA2SL-GT1.
850 rice calluses cultured for 3 weeks were infected with Agrobacterium introduced with GT vector OASA2SL-GT1. After sterilization, selection was carried out on N6D medium containing 500 μM of 5MT. As a result, 152 5MT-resistant calli were successfully obtained. When they were placed on a regeneration medium and cultured, 25 lines of 5MT-resistant plants were successfully obtained. About them, it was confirmed whether the modified OASA2 gene was present in the redifferentiated plant using a CAPS marker. As a result, we succeeded in obtaining one strain of individuals who were expected to succeed in GT, or who were expected to have randomly inserted GT vectors.
Further, when the nucleotide sequence of S126F and L530D in the vicinity of the obtained individual was confirmed, two types of peaks, a wild type and a modified type, were confirmed for the sequence into which the mutation was introduced. The obtained individuals did not show significant morphological changes and could obtain seeds (Fig. 5).

2.2 Southern analysis of modified OASA2 gene by GT
Southern analysis was performed using T ₀ individuals obtained using the GT vector OASA2SL-GT1 and its progeny T ₁ individuals. As a result of Southern analysis, bands of 6.0 kb and 1.5 kb were confirmed in the T ₀ individuals. Therefore, OASA2 gene T ₀ individuals were shown to be heterozygous for the modified and wild-type. This was consistent with the result of the above base sequence. Further, when similar Southern analysis was performed using 7 lines of T ₁ individuals, only a 1.5 kb band was confirmed in line numbers 2 and 6. From this, it was shown that the individual obtained this time has succeeded in modifying the endogenous OASA2 gene.

2.3 Measurement of tryptophan content in gene targeting individuals obtained using OASA2SL-GT1 When free tryptophan content in the obtained GT individuals was measured, about 8.62 nmol / mg free tryptophan accumulated in rice leaf blades. It became clear that This was about 130 times the wild type free tryptophan content. This accounted for about 16% of all amino acids. From this, it was shown that the amount of tryptophan accumulated can be increased by modifying the OASA2 gene with GT.

Claims (23)

A plant transformed with the DNA according to the following (a) or (b), wherein the plant contains a high concentration of tryptophan;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3. The plant body of Claim 1 which does not contain endogenous anthranilate synthase alpha subunit. The plant body according to claim 2, wherein the plant is a monocotyledonous plant. The plant body according to claim 3, wherein the monocotyledonous plant is a gramineous plant. The plant body according to claim 4, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, sorghum, oats, and grass. A cell isolated from the plant according to any one of claims 1 to 5. The propagation material of the plant body in any one of Claim 1 to 5. An organ isolated from the plant according to any one of claims 1 to 5. A method for producing a plant containing tryptophan, comprising the following steps (a) and (b):
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence described in SEQ ID NO: 1 or 3. 10. The method of claim 9, wherein the plant does not contain an endogenous anthranilate synthase alpha subunit. The method according to claim 10, wherein the plant is a monocotyledonous plant. The method according to claim 11, wherein the monocotyledonous plant is a gramineous plant. The method according to claim 12, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, sorghum, oats, grass. The method according to claim 13, wherein the vector is selected from vectors comprising the nucleotide sequence according to any one of SEQ ID NOs: 7 to 10. A method of adding tryptophan to a plant, comprising the following steps (a) and (b):
(A) a step of introducing the DNA according to (i) or (ii) below or a vector containing the DNA into a plant cell by a gene targeting method, and (b) a plant into which the DNA or vector has been introduced in step (a) A process of regenerating plant bodies from cells,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence described in SEQ ID NO: 1 or 3. 16. The method of claim 15, wherein the plant does not contain an endogenous anthranilate synthase alpha subunit. The method according to claim 16, wherein the plant is a monocotyledonous plant. The method according to claim 17, wherein the monocotyledonous plant is a gramineous plant. The method according to claim 18, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, sorghum, oats, grass. The method according to claim 19, wherein the vector is selected from vectors comprising the nucleotide sequence according to any of SEQ ID NOs: 7 to 10. An agent for containing a plant with a high concentration of tryptophan, comprising the DNA according to the following (i) or (ii) or a vector containing the DNA as an active ingredient;
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4,
(Ii) DNA comprising the base sequence described in SEQ ID NO: 1 or 3. An agent for containing a high concentration of tryptophan in a plant, containing as an active ingredient at least one of vectors comprising the base sequence set forth in any one of SEQ ID NOs: 7 to 10. A method for producing tryptophan, comprising the following steps (a) and (b):
(A) A step of obtaining a plant body containing tryptophan by the method according to any one of claims 9 to 20, and (b) a step of recovering tryptophan from the plant body obtained in step (a).