JP2011160806A - RICE BLAST-SUSCEPTIBLE GENE Pi21 AND RESISTANT GENE pi21, AND UTILIZATION THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new lesion progression control gene of a plant, and a method for modifying disease resistance of the plant utilizing the gene. <P>SOLUTION: A field-resistant gene pi21 of rice plants is successfully isolated by a chain analysis and it is found out that the rice blast field resistance of the plant could be possibly modified by transferring the gene or inhibiting the expression thereof. Thus, the field resistance can efficiently be imparted to the plant. Furthermore, it becomes possible to select a rice plant having the field resistance against rice blast in an early stage. In addition, a variety having high resistance together with characteristics highly useful in practice can be raised by altering the tissue expression specificity or expression level of the gene associated with the field resistance. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a rice blast field resistance gene pi21 and a method for modifying the blast field resistance of plants using the gene.

  The resistance of rice to blast fungus is classified into two types: true resistance and field resistance (Non-patent Document 1). The former is a resistance based on a hypersensitive reaction, and is a qualitative resistance that is highly effective and highly specific to the race. It is empirically known that varieties introduced with one resistance gene lose their effects in several years due to the appearance of bacteria having affinity for that gene. On the other hand, field resistance is defined as the difference between resistance varieties observed under conditions where true resistance does not function. Although less effective than true resistance, it has low practicality in that it can impart persistent resistance to varieties because of its low specificity to race.

  More than 30 types of genes involved in true resistance are known, of which the Pib and Pita genes have been isolated (Non-patent Document 2). These genes have been revealed to be NBS-LRR class genes having nucleotide binding sites (NBS) and leucine-rich repeats (LRRs), which are similar to the disease resistance genes of previously reported plants. Like other disease resistance genes, plant resistance gene products are thought to function as receptors that directly or indirectly recognize the products of the corresponding pathogenic non-pathogenic genes. Yes. In fact, Pita has been shown to physically bind directly to non-pathogenic gene products.

  Regarding field resistance, it is known that Japanese upland rice varieties have excellent characteristics, and the positions on the chromosome have been clarified for a plurality of loci involved (Non-patent Document 3). However, the structure and expression mechanism of the gene have not been clarified, and it is not in a situation where it can be used for breeding selection more efficiently than intrinsic resistance. Regarding incomplete resistance, a concept similar to field resistance, the involvement of multiple chromosomal regions in West African upland rice cultivar Moroberekan has been reported (Non-Patent Document 4), but the gene is specified. It has not been.

Japanese Patent Application No. 10-271807 Japanese Patent Application No.11-153146 Patent No. 3376453 (P3376453) JP2003-88379 (P2003-88379A) JP2003-199577 (P2003-199577A) JP 2003-199448 (P2003-199448A) JP2004-329215A (P2004-329215A)

Rice Blast and Resistance Breeding p175-186 Takasaka / Yamazaki 1980 Hirotomo Wang et al. Plant J 19: 55-64, 1999, Bryan et al. Plant Cell. 12: 2033-46, 2000 Fukuoka and Okuno 2001 Theor Appl Genet. 03: 185-190 Wang et al. Genet 136: 1421-1434, 1994

  The present invention has been made in view of such a situation, and the problem to be solved by the present invention is to isolate and identify a gene related to blast field resistance by map-based cloning, and to It is intended to provide a method for modifying the field resistance of rice blast disease using plants.

  The present invention relates to a gene that controls resistance to plant blast disease. The allele pi21 of the Pi21 gene is known to exist in one of the vast regions of rice chromosome 4 as a gene that imparts a rice blast disease resistance to rice (Oryza sativa L). . The present inventors have clarified the region of existence and attempted to isolate it as a single gene.

  In order to create a genetic map of the pi21 region, the present inventors first performed a detailed linkage analysis of the pi21 region with a large-scale segregation population essential for map-based cloning. For a population obtained by continuous backcrossing rice cultivars, Nihonbare or Aichi-Asahi, with a susceptibility allele Pi21 that does not suppress lesion progression, to the Japanese upland rice variety Owari Hatamochi that has the resistance allele pi21 that suppresses lesion development An attempt was made to analyze the linkage with the RFLP marker, and it was confirmed that the pi21 locus exists between the RFLP markers G271 and G317.

  Next, in order to create a genetic map of the pi21 region with higher accuracy, the present inventors selected a recombination-type individual near the pi21 locus using RFLP markers RA3591 and 13S1 present on both sides of pi21. . At the same time, a search was conducted using the F2 population obtained by crossing a line with the susceptible allele of the Indian rice cultivar Kasalath and a line with the resistance allele from Owari Hatamochi in the genetic background of the Japanese rice cultivar, and pi21 We also selected chromosome-recombinant individuals near the locus. As a result of creating a detailed linkage map using these individuals and the created DNA marker, it was found that the pi21 locus is present in an approximately 25 kb genomic region sandwiched between the SSCP marker Pa102484 and the SNP marker P702D3_ # 12. Furthermore, by determining the nucleotide sequence of PAC clone P702D03, which is thought to contain the pi21 gene, and analyzing the nucleotide sequences of the resistant cultivar Owari Hatamochi and the susceptible varieties Aichi Asahi and Kasalath in the 25 kb genome candidate region, It was revealed that the gene exists in a genomic region of about 1.8 kb sandwiched between SNP markers P702D03_ # 38 and P702D03_ # 80.

Therefore, the present inventors designed primers that can amplify the corresponding portion using the already obtained Nipponbare base sequence information, and used genomic PCR and RT-PCR products as susceptible varieties Nipponbare and AichiAsahi and resistant varieties. The base sequence was compared between Owari Hatamochi. As a result, in the exon region of the gene, it was clarified that there were two DNA mutations between the resistant variety and the susceptible variety. In varieties that are resistant to susceptible varieties, deletions of 7 and 16 amino acids have occurred, demonstrating that these mutations are associated with the development of blast disease infecting blast. That is, the present invention relates to the pi21 gene that controls resistance to blast disease in plants, and specifically provides the following inventions.
[1] DNA according to any one of (a) to (h) below;
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 22,
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1, 2, 20 or 21;
(C) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 22, DNA encoding a protein having a function equivalent to that of the protein comprising the described amino acid sequence,
(D) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 2, 20 or 21, comprising a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 or 22; DNA encoding a protein having an equivalent function,
(E) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(F) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or 5,
(G) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence set forth in SEQ ID NO: 6 DNA encoding a protein having the same function as the protein consisting of
(H) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 5, and having a function equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 6 DNA encoding
[2] DNA according to any one of (i) to (iv) below, which has a blast disease field resistance performance in a plant;
(I) DNA encoding an RNA complementary to the transcription product of the DNA according to any one of (1) to (1),
(Ii) DNA encoding an RNA having ribozyme activity that specifically cleaves the transcript of the DNA according to any one of (1) to (1),
(Iii) DNA encoding RNA that inhibits the expression of the DNA according to any one of (1) to (d) by co-suppression effect,
(Iv) DNA encoding RNA having RNAi activity that specifically cleaves the transcription product of DNA according to any one of (a) to (d) in [1].
[3] The DNA according to [2], wherein the plant is rice, wheat, barley, oat, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, or sugar cane.
[4] A vector comprising the DNA according to any one of [1] to [3].
[5] A transformed cell that retains the DNA of any one of [1] to [3] in an expressible manner.
[6] A transformed plant cell into which the DNA according to any one of (a) to (d) of [1] is introduced.
[7] A transformed plant cell into which the DNA according to [2] or [3] is introduced.
[8] The transformed plant cell according to [6] or [7], wherein the plant is rice, wheat, barley, oat, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane.
[9] A transformed plant comprising the transformed cell according to [6] to [8].
[10] A transformed plant that is a descendant or clone of the transformed plant according to [8].
[11] A propagation material for the transformed plant according to any one of [9] and [10].
[12] A method for producing a transformed plant according to any one of [9] or [10], wherein the DNA according to (1) (a) to (d), [2] or [3] A method comprising the steps of introducing a plant cell into a plant cell and regenerating the plant body from the plant cell.
[13] A method for imparting blast disease field resistance to a plant, comprising a step of expressing the DNA according to [2] or [3] in a cell of the plant body.
[14] The method according to [13], wherein the plant is rice, wheat, barley, oat, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane.
[15] A protein encoded by the DNA according to any one of (a) to (d) in [1].
[16] A step of culturing a transformed cell containing the vector containing the DNA according to any one of (a) to (d) in [1] and recovering the recombinant protein from the cell or the culture supernatant thereof. [15] The method for producing a protein according to [15].
[17] An antibody that binds to the protein of [15].
[18] A DNA comprising at least 15 consecutive bases complementary to the DNA according to [1] or a complementary sequence thereof.
[19] An agent for increasing the blast field resistance of a plant, comprising any one of the DNA according to [2] or [3] or a vector containing the DNA.
[20] A primer set for amplifying all or part of the base sequence described in SEQ ID NO: 1, 4 or 20.
[21] at least one primer set shown in any one of (a) to (c) (a) DNA consisting of the base sequence set forth in SEQ ID NO: 8 and DNA consisting of the base sequence set forth in SEQ ID NO: 9;
(B) DNA consisting of the base sequence set forth in SEQ ID NO: 16 and DNA consisting of the base sequence set forth in SEQ ID NO: 17
(C) DNA consisting of the base sequence set forth in SEQ ID NO: 26 and DNA consisting of the base sequence set forth in SEQ ID NO: 27.
[22] DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7, 10, 18, 19, 23 or 25
[23] A method comprising the following steps (a) to (c), wherein the test plant is determined to be blast field resistant when the molecular weight or the base sequence matches:
(A) a step of preparing a DNA sample from a test plant;
(B) a step of amplifying the DNA region according to [1] from the DNA sample;
(C) A step of comparing the molecular weight or base sequence of the amplified DNA fragment with that of the DNA according to either (e) or (f) of [1].
[24] A method comprising the following steps (a) to (d), wherein the test plant is determined to be blast field resistant when the mobility on the gel matches:
(A) a step of preparing a DNA sample from a test plant;
(B) a step of amplifying the DNA region according to [1] from the DNA sample;
(C) separating the amplified double-stranded DNA on a non-denaturing gel;
(D) A step of comparing the mobility of the separated double-stranded DNA on the gel with that of the DNA according to either (e) or (f) of [1].
[25] A method comprising the following steps (a) to (e), wherein when the mobility on the gel matches, the test plant is determined to be blast field resistant;
(A) a step of preparing a DNA sample from a test plant;
(B) a step of amplifying the DNA region according to [1] from the DNA sample;
(C) dissociating the amplified DNA into single-stranded DNA;
(D) separating the dissociated single-stranded DNA on a non-denaturing gel;
(E) A step of comparing the mobility of the separated single-stranded DNA on the gel with that of the DNA described in either (e) or (f) of [1].
[26] A method comprising the following steps (a) to (d), wherein when the mobility on the gel matches, the test plant is judged to be blast field resistant;
(A) a step of preparing a DNA sample from a test plant;
(B) a step of amplifying the DNA region according to [1] from the DNA sample;
(C) separating the amplified DNA on a gel in which the concentration of the DNA denaturant is gradually increased,
(D) A step of comparing the mobility of the separated DNA on the gel with that of the DNA according to either (e) or (f) of [1].
[27] A method of selecting a plant having resistance to blast disease field, comprising the following steps (a) and (b):
(A) a step of producing a variety in which a plant having a rice blast field resistance performance and a plant having an arbitrary function are crossed,
(B) The process of determining whether the plant produced at the process (a) is rice blast field resistance by the method in any one of [23]-[26].
[28] A molecular marker linked to the DNA of [1], wherein the test rice is a blast disease field when the molecular marker exhibits a genotype similar to that of rice having a trait resistant to blast disease field A method for determining resistance.
[29] The method according to [28], wherein the molecular marker is a molecular marker comprising the DNA of SEQ ID NO: 10.
[30] A method for selecting rice that is resistant to blast field, comprising the steps of (a) and (b) below:
(A) a step of producing a variety in which rice having rice blast field resistance performance and rice having an arbitrary function are crossed,
(B) A step of determining whether or not the rice produced in the step (a) is blast field resistant by the method according to any one of [28] and [29].
[31] A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (c):
(A) contacting the test compound with the transcription product of the DNA according to any one of (a) to (d) in [1],
(B) a step of detecting the binding between the transcription product of the DNA according to any one of (a) to (d) of [1] and a test compound;
(C) A step of selecting a test compound that binds to the transcription product of the DNA according to any one of (a) to (d) in [1].
[32] A method for screening a drug for preventing or ameliorating blast of plants, comprising the following steps (a) to (c):
(A) a step of bringing a test compound into contact with a cell collected from a plant;
(B) a step of measuring the expression level of the transcription product of the DNA according to any one of (a) to (d) in [1],
(C) A step of selecting a test compound in which the expression level of the transcript is reduced as compared with the case where the test compound is not contacted.
[33] A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (d):
(A) a step of providing a cell or a cell extract having a DNA to which a reporter gene is operably linked downstream of the promoter region of the DNA of any one of (a) to (d);
(B) contacting the test compound with the cells or the cell extract;
(C) measuring the expression level of the reporter gene in the cell or cell extract,
(D) A step of selecting a test compound in which the expression level of the reporter gene is reduced as compared with the case where the test compound is not contacted.
[34] A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (d):
(A) a step of regenerating a transformed plant from the transformed plant cell according to [6],
(B) contacting the transformed plant with a blast fungus and a test compound;
(C) The process of selecting the test compound which suppresses the blast disease of this transformed plant body compared with the case where the test compound is not made to contact.
[35] A kit for use in the screening method according to any one of [31] to [34].

  The characteristics of the Pi21 gene are particularly suitable for the production of blast-resistant varieties of plants. Until now, in order to confer rice blast field resistance, a variety that originally had field resistance but had inferior characteristics was crossed with a variety that did not have this but had excellent characteristics. It was necessary to select individuals that had superior field resistance characteristics and other excellent characteristics from their offspring. However, in order to accurately evaluate the field resistance, a great deal of labor is required, and when the exact chromosomal location of the gene to which it is applied is not clear, this is efficiently and accurately selected, It was difficult to introduce into a highly practical variety. And in fact, it has never been successful.

  According to the present invention, the position on the chromosome of the gene involved in field resistance and the structure of the gene are provided. Thereby, it became possible to provide field resistance efficiently to a plant. In addition, by changing the tissue expression specificity and expression level of genes involved in field resistance, it became possible to cultivate varieties having both resistance and high practicality. Thus, the gene of the present invention is highly practical and useful for realizing highly safe agriculture. In addition, plants produced by the method of the present invention are expected not only to stably obtain high yields for useful crops, but also to add new aesthetic value to ornamental plants.

It is a photograph showing the lesions of blast disease in lines AA-pi21 (Fig. Left) and Aichi Asahi (Fig. Right) having the pi21 gene in the genetic background of Aichi Asahi. It is a figure which shows the detailed linkage map of the pi21 gene region, and the alignment map of a genomic clone. A and B show genetic maps created by segregating populations of 72 and 1014 individuals. C shows an aligned map of Nipponbare PAC clones. D shows the detailed genetic map and candidate genomic region of the pi21 gene region. It is a figure which shows the comparison of the structure of a pi21 candidate gene, and the genome base sequence of Nipponbare / AichiAsahi and Owarihamochi. It is a photograph which shows the lesion which arose on the transformant which introduce | transduced the Pi21 gene derived from Nipponbare into the resistant strain AApi21. A: Only vector is introduced. B: 1 copy of Pi21 gene introduced. C: Introduced 3 or more copies of Pi21 gene.

  The allele pi21 gene of the susceptibility gene Pi21 that does not suppress the development of blast disease in rice is a gene that confers rice with the trait of blast disease field resistance, and has been located anywhere in the vast region of rice chromosome 4 so far. Was known to exist. The present inventors narrowed down the existing region of the pi21 gene in rice chromosome 4 by using a map-based cloning technique, and finally succeeded in identifying it as a single gene. We also succeeded in isolating the pi21 allele Pi21 gene.

  In the present invention, “blast disease” refers to a disease symptom (spot) that a plant is infected with a blast fungus and that part or whole of the plant is discolored or necrotized by its action or can be recognized. Spots of rice blast occur at various parts of the plant, and are called seedling buds, leaf blasts, ear buds, rice blasts, knotomochi, leaf nodes (leaf tongue) potatoes, etc., depending on the site of occurrence. “Blast disease” in the present invention includes blast disease that occurs in all these sites. In addition, “rice blast fungus” that causes blast disease in rice is called Magnaporthe grisea or Magnaporthe oryzae, although there is no unified scientific name at present. The blast fungus has a complete generation name and an incomplete generation name. Pyricularia oryzae is given as an incomplete generation name for the complete generation name Magnaporthe oryzae. The blast fungus in the present invention includes all these blast fungi regardless of their names.

  Further, in this specification, “susceptibility to blast” means the property that a plant is infected with blast (rarely, its symptom is remarkable). Furthermore, “Blast field resistance” refers to differences in disease symptoms recognized as differences in the number and size of lesions between varieties and lines (within the same plant species) when a plant is infected with blast fungus. Or it means the property of suppressing the number and size of lesions. “Intrinsic resistance” refers to the property that a plant causes cell death due to a hypersensitive reaction in cells invaded by blast fungus and prevents infection.

  The present invention provides a blast susceptibility gene Pi21 involved in plant blast and a blast field resistance gene pi21.

More specifically, the Pi21 gene of the present invention includes:
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 22,
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1, 2, 20 or 21;
(C) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 22, DNA encoding a protein having a function equivalent to that of the protein comprising the described amino acid sequence,
(D) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 2, 20 or 21, comprising a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 or 22; DNA encoding a protein having an equivalent function,
Is included.

The pi21 gene of the present invention more specifically includes
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 4 or 5,
(C) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in SEQ ID NO: 6, and the amino acid sequence described in SEQ ID NO: 6 DNA encoding a protein having the same function as the protein consisting of
(D) a protein that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 5, and having a function equivalent to that of the protein comprising the amino acid sequence set forth in SEQ ID NO: 6. DNA encoding,
Is included.

  By using the Pi21 gene or the pi21 gene of the present invention, for example, it becomes possible to prepare a recombinant protein, or to produce a transformed plant having a modified rice blast field resistance.

  In the present invention, the plant from which the gene of the present invention is derived is not particularly limited, for example, rice, corn, wheat, barley, oat, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane, Examples thereof include monocotyledonous plants such as pearl millet and dicotyledonous plants such as rapeseed, soybean, cotton, tomato and potato. Examples of flowering plants include chrysanthemum, roses, carnations, and cyclamen, but are not limited thereto.

  The “Pi21 gene” and “pi21 gene” of the present invention are not particularly limited as long as they can encode “Pi21 protein” and “pi21 protein”, respectively. “Pi21 gene” and “pi21 gene” In addition to cDNA, each includes genomic DNA, chemically synthesized DNA, and the like. In addition, as long as the Pi21 gene encodes the Pi21 protein and the pi21 gene encodes the pi21 protein, DNA having an arbitrary base sequence based on the degeneracy of the genetic code is included.

  Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from a plant, a genomic library (a vector such as a plasmid, phage, cosmid, BAC, PAC, etc. can be used) is expanded and Pi21 gene or pi21 It can be prepared by performing colony hybridization or plaque hybridization using a probe prepared based on a gene (for example, the DNA described in any one of SEQ ID NOs: 1, 2, 4, 5, 20, or 21). Is possible. It is also possible to prepare a primer specific to the Pi21 gene or pi21 gene and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from a plant, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization in the same manner as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

  Furthermore, since the Pi21 gene or pi21 gene is considered to exist widely in the plant kingdom, the Pi21 gene or pi21 gene includes not only rice but also homologous genes existing in various plants. Here, the “homologous gene” refers to a gene encoding a protein having physiological functions similar to those of the Pi21 gene or pi21 gene product in rice (for example, rice blast susceptibility or blast field resistance) in various plants.

  Methods well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, EM, Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki). , RK, et al. Science, vol. 230, 1350-1354, 1985, Saiki, RK et al. Science, vol. 239, 487-491, 1988). That is, for those skilled in the art, the nucleotide sequence of rice Pi21 gene or pi21 gene (for example, the sequence described in any one of SEQ ID NOs: 1, 2, 4, 5, 20, or 21) or a part thereof is used as a probe, Isolation of a Pi21 gene or a homologous gene of the pi21 gene from various plants using an oligonucleotide that specifically hybridizes to the Pi21 gene or the pi21 gene as a primer is usually possible.

  In order to isolate DNA encoding such a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5 × SSC, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, and 0.1 × SSC. The sequence of the isolated DNA can be determined by a known method. The homology of the isolated DNA is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) in the entire amino acid sequence. And the like). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. Further, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

Further, the present invention is a DNA used to suppress the expression of endogenous Pi21 gene in plants,
(A) DNA encoding RNA complementary to the transcription product of Pi21 gene,
(B) DNA encoding RNA having ribozyme activity that specifically cleaves the transcript of Pi21 gene,
(C) DNA encoding RNA that inhibits the expression of Pi21 gene by co-suppression effect,
(D) DNA encoding RNA having RNAi activity that specifically cleaves the transcription product of Pi21 gene,
I will provide a.
These DNAs can suppress the development of lesions caused by plant blast.

  In the present invention, the plant that suppresses the expression of the Pi21 gene is not particularly limited, and a desired plant that is desired to impart resistance to rice blast field can be used. Is preferred. There are no particular restrictions on useful crops, but examples include monocotyledonous plants such as rice, corn, wheat, barley, oat, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugarcane, pearl millet, and rapeseed. And dicotyledonous plants such as soybean, cotton, tomato and potato. Examples of ornamental plants include, but are not limited to, flowering plants such as chrysanthemum, roses, carnations, and cyclamen. As plants sensitive to rice blast fungus, barley and Italian rye. There are grass and corn such as ras, meadow fescue, etc., other than that, rice plants such as Ezonosyanukagusa, Makomo, etc., oats, barley, oats, etc. Many plants have been reported, such as bearded beetles such as Dalles Megaya, millet such as foxtail millet and mosquito, and these plants are also included in plants that impart blast field resistance.

  In the present specification, “suppression of Pi21 gene expression” includes suppression of gene transcription and suppression of protein translation. Also included is a decrease in expression as well as complete cessation of DNA expression.

  One embodiment of “DNA used for suppressing the expression of the Pi21 gene” is DNA encoding an antisense RNA complementary to the Pi21 gene. The antisense effect in plant cells was demonstrated for the first time by the antisense RNA introduced by electroporation using a transient gene expression method to exert an antisense effect in plants (Ecker and Davis, Proc. Natl Acad. USA, 83: 5372, 1986). Subsequently, tobacco and petunia have been reported to decrease the expression of target genes by expressing antisense RNA (Krol et. Al., Nature 333: 866, 1988), and currently suppress gene expression in plants. Established as a means to

  There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993).

  The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene is designed, it will be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.

  Antisense DNA is obtained, for example, by the phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221, 1988) based on the sequence information of DNA described in SEQ ID NO: 1, 2, 20, or 21. It is possible to prepare. The prepared DNA can be transformed into a desired plant by a known method. The sequence of the antisense DNA is preferably a sequence complementary to the transcription product of the endogenous gene of the plant to be transformed, but may not be completely complementary as long as the gene expression can be effectively inhibited. . The transcribed RNA preferably has a complementarity of 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. In order to effectively inhibit the expression of the target gene using the antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

  Suppression of endogenous Pi21 gene expression can also be carried out using a ribozyme-encoding DNA. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type or a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 35: 2191, 1990).

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (Koizumi et. Al., FEBS Lett. 228: 225, 1988). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (Koizumi et. Al., FEBS Lett. 239: 285, 1988, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 35: 2191, 1990, Koizumi et. Al., Nucleic. Acids. Res. 17: 7059, 1989).

  Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992, and Hiroshi Kikuchi, Chemistry and Biology 30: 112, 1992).

  A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells. However, at that time, if an extra sequence is added to the 5 ′ end or 3 ′ end of the transcribed RNA, the ribozyme activity may be lost. In such a case, in order to accurately cut out only the ribozyme part from the RNA containing the transcribed ribozyme, another trimming ribozyme that acts in cis for trimming is placed on the 5 'side or 3' side of the ribozyme part. (Taira et.al., Protein Eng. 3: 733, 1990, Dzianott and Bujarski, Proc. Natl. Acad. Sci. USA, 86: 4823, 1989, Grosshans and Cech, Nucleic Acids Res. 19 : 3875, 1991, Taira et. Al., Nucleic Acids Res. 19: 5125, 1991).

  In addition, such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved, thereby further enhancing the effect (Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271 , 1992). Such a ribozyme can be used to specifically cleave the transcription product of the target gene in the present invention, thereby suppressing the expression of the gene.

  Suppression of endogenous gene expression can also be achieved by “co-suppression” resulting from transformation of DNA having a sequence identical or similar to the target gene sequence. “Co-suppression” refers to a phenomenon in which the expression of both a foreign gene and a target endogenous gene to be introduced is suppressed when a gene having the same or similar sequence as that of the target endogenous gene is introduced into a plant by transformation. Say. Details of the mechanism of co-suppression are not clear, but are often observed in plants (Curr. Biol. 7: R793, 1997, Curr. Biol. 6: 810, 1996).

  For example, in order to obtain a plant body in which the Pi21 gene is co-suppressed, a plant DNA obtained by transforming a vector DNA prepared so that the Pi21 gene or a DNA having a similar sequence can be expressed into a target plant. A plant having a trait of the Pi21 mutant from the body, that is, a plant resistant to blast field can be selected. The gene used for co-suppression need not be completely identical to the target gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (eg, 95%, 96%, 97%, 98 %, 99% or more).

  Furthermore, suppression of the expression of the endogenous gene in the present invention can also be achieved by transforming a gene having a dominant negative trait of the target gene into a plant. A gene having a dominant negative trait refers to a gene having a function of eliminating or reducing the activity of an endogenous wild-type gene inherent in a plant by expressing the gene.

  Another embodiment of the “DNA used for suppressing the expression of the Pi21 gene” is a DNA encoding a dsRNA (double-stranded RNA) complementary to the transcription product of the endogenous Pi21 gene. A phenomenon called RNAi (RNA interference), in which the expression of the introduced foreign gene and target endogenous gene is both suppressed by introducing dsRNA having the same or similar sequence to the target gene into the cell. Can cause. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, a RNaseIII-like nuclease called Dicer with a helicase domain is present in the presence of ATP, and dsRNA is about 21 to 23 base pairs from the 3 ′ end. Cut out and produce siRNA (short interference RNA). A protein specific to this siRNA binds to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the transcript (mRNA) of the target gene at the center of the siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  RNAi was first discovered in nematodes (Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, 1998), but now only nematodes It has been observed in various organisms such as plants, linear animals, Drosophila and protozoa (Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-363 (1999), Sharp, PA RNA interference 2001 Genes Dev. 15, 485-490 (2001), Hammond, SM, Caudy, AA & Hannon, GJ Post-transcriptional gene silencing by double-stranded RNA.Nature Rev. Genet. 2, 110-119 (2001), Zamore , PD RNA interference: listening to the sound of silence. Nat Struct Biol. 8, 746-750 (2001)). In these organisms, it has been confirmed that the expression of the target gene is suppressed by actually introducing dsRNA from a foreign body, and is also being used as a method for creating knockout individuals.

  At the beginning of RNAi, dsRNA was thought to be ineffective unless it was longer than a certain length (40 bases), but US Rockefeller University Tuschl et al. Used a single-stranded dsRNA (siRNA) of around 21 base pairs. When introduced into cells, it has been reported that the antiviral response by PKR does not occur in mammalian cells and RNAi is effective (Tuschl, Nature, 411, 494-498 (2001)). As a technology that can be applied to mammalian cells, it has attracted a great deal of attention.

  The DNA of the present invention comprises an antisense code DNA encoding an antisense RNA for any region of a target gene transcription product (mRNA) and a sense code DNA encoding a sense RNA of any region of the mRNA. The antisense RNA and the sense RNA can be expressed from the antisense code DNA and the sense code DNA. Moreover, dsRNA can also be produced from these antisense RNA and sense RNA. The target sequence in the present invention is not particularly limited as long as the expression of the Pi21 gene is suppressed as a result of introducing dsRNA having the same or similar sequence as the target sequence into the cell. An example of the target sequence is the 3 ′ untranslated region sequence of the Pi21 gene. The 3 ′ untranslated region sequence of the Pi21 gene is shown in SEQ ID NOs: 11 and 24.

  In the case where the dsRNA expression system of the present invention is retained in a vector or the like, there are a case where antisense RNA and sense RNA are expressed from the same vector, and a case where antisense RNA and sense RNA are expressed from different vectors, respectively. is there. For example, antisense RNA and sense RNA can be expressed from the same vector as antisense RNA in which an antisense coding DNA and a promoter capable of expressing a short RNA such as polIII are linked upstream of the sense coding DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction.

  It is also possible to construct an expression system in which antisense code DNA and sense code DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. In this palindromic style expression system, the two promoter types are preferably different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the sense coding DNA. An RNA expression cassette and a sense RNA expression cassette can be constructed, and these cassettes can be held in different vectors.

  In RNAi, siRNA may be used as dsRNA. “SiRNA” means a double-stranded RNA consisting of short strands in a range that does not show toxicity in cells, and is not limited to the total length of 21 to 23 base pairs reported by Tuschl et al. The length is not particularly limited as long as the length is not shown. For example, the length can be 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. Alternatively, the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. be able to.

  As the DNA of the present invention, an appropriate sequence (preferably an intron sequence) is inserted between inverted repeats of a target sequence to produce a double-stranded RNA having a hairpin structure (self-complementary 'hairpin' RNA (hpRNA)). Such constructs (Smith, NA, et al. Nature, 407: 319, 2000, Wesley, SV et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29: E55, 2001 ) Can also be used.

  The DNA used for RNAi need not be completely identical to the target gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98% , 99% or more). The sequence identity can be determined by the method described above.

  The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

In addition, the present invention provides a vector and a transformed cell containing any one of Pi21 gene, pi21 gene, or DNA that suppresses expression of Pi21 gene.
As the vector, for example, when Escherichia coli is used as a host, the vector is amplified in Escherichia coli in order to amplify it in large quantities in Escherichia coli (for example, JM109, DH5α, HB101, XL1Blue) and the like. There is no particular limitation as long as it has an ori ”and a transformed gene of E. coli transformed (for example, any drug (drug resistance gene that can be distinguished by ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. For the purpose of cDNA subcloning and excision, in addition to the above vectors, for example, pGEM-T, pDIRECT PT7, etc. A vector is used for the purpose of producing DNA that suppresses the expression of Pi21 gene, pi21 gene, or Pi21 gene. In particular, an expression vector is useful for the purpose of expression, for example, for the purpose of expression in E. coli, in addition to the above characteristics that the vector is amplified in E. coli. In the case of E. coli such as JM109, DH5α, HB101, XL1-Blue, etc., a promoter that can be expressed efficiently in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. ( 1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter, etc. Other examples include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET.

  The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for polypeptide secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the polypeptide is produced in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli, examples of vectors for producing DNA that suppresses expression of Pi21 gene, pi21 gene, or Pi21 gene include mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF, etc. -BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), insect cell-derived expression vectors (for example, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8) Plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “ Pichia Expression Kit "(manufactured by Invitrogen), pNV11, SP-Q01), Bacillus subtilis-derived expression vectors (for example, pPL608, pKTH50) and the like.

  When intended for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

  Those skilled in the art can introduce the DNA of the present invention into cells by a known method such as electroporation (electroporation).

  Furthermore, the present invention relates to transformed plant cells into which DNA encoding Pi21 protein or DNA that suppresses expression of Pi21 gene is introduced, transformed plants thereof, transformed plants that are descendants or clones of the transformed plants And a propagation material for the transformed plant body. Moreover, the manufacturing method of the said transformant and propagation material is provided.

  Introduction of DNA encoding Pi21 protein or DNA that suppresses expression of Pi21 gene into plant cells can be performed by the above-described method.

  The regeneration of the plant body can be performed by a method known to those skilled in the art depending on the type of plant cell (Toki et. Al., Plant Physiol. 100: 1503-1507, 1995). For example, in rice, a method for producing a transformed plant is to introduce a gene into protoplasts using polyethylene glycol and regenerate the plant (indian rice varieties are suitable) (Datta et. Al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) Pp66-74, 1995). Gene transfer to protoplasts by electric pulses to regenerate plants (Japanese rice varieties are suitable) (Toki et. Al. , Plant Physiol. 100: 1503-1507, 1992), a method of regenerating plants by directly introducing genes into cells by the particle gun method (Christou et. Al., Bio / technology, 9: 957-962, 1991) And several techniques have already been established, such as a method for introducing a gene via Agrobacterium and regenerating a plant (Hiei et. Al., Plant J. 6: 271-282, 1994). Widely used in the technical fieldIn 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 includes an expression promoter so that, for example, after introduction into a plant body, the DNA encoding the Pi21 protein of the present invention or the DNA that suppresses the expression of the Pi21 gene is expressed in the plant body. In general, a DNA encoding the Pi21 protein of the present invention or a DNA that suppresses the expression of the Pi21 gene 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 DNA encoding the Pi21 protein of the present invention or DNA that suppresses the expression of the Pi21 gene may be explants, and cultured cells obtained by preparing cultured cells from these plants are obtained. May be introduced. Examples of the “plant cell” of the present invention include plant cells such as scutellum in leaves, roots, stems, flowers and seeds, callus, suspension culture cells and the like.

  In addition, in order to efficiently select cells transformed by introduction of DNA encoding the Pi21 protein of the present invention or DNA that suppresses the expression of the Pi21 gene, the above recombinant vector contains an appropriate selection marker gene or a selection marker. It is preferably introduced into a plant cell together with a plasmid vector containing the gene. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene that is resistant to the antibiotic hygromycin, the neomycin phosphotransferase that is resistant to kanamycin or gentamicin, and the acetyltransferase that is resistant to the herbicide phosphinothricin. Genes and the like.

  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 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 plant body resistant to blast field can be obtained, and matured and fruited to obtain seeds.

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 the foreign gene comprising the DNA of the present invention present in the regenerated plant body is analyzed using the PCR method, the amplification reaction is performed using the DNA extracted from the regenerated plant body as a template as described above. In addition, an amplification reaction is carried out in a reaction mixture in which the DNA of the present invention or a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA modified according to the present invention is used as a primer. You can also In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the DNA sequence of the present invention. 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 Pi21 protein of the present invention or a DNA that suppresses the expression of the Pi21 gene is introduced into the chromosome, progeny can be generated from the plant by sexual reproduction or asexual reproduction. It is possible to obtain. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant and its descendants or clones, and mass-produce the plant based on them. In the present invention, a plant cell into which DNA encoding Pi21 protein or DNA that suppresses expression of the Pi21 gene is introduced, a plant body containing the cell, progeny and clones of the plant body, and the plant body, These plant cells, plant bodies containing the cells, the progeny and clones of the plant, and the propagation material of the plants, the progeny and the clones are used for plant blast disease fields. It can be used in a method for imparting resistance.

  The present invention further provides a method for imparting blast disease field resistance to a plant, comprising the step of expressing a DNA that suppresses the expression of the Pi21 gene in cells of the plant body. It is possible to impart blast disease field resistance to a plant by introducing into the plant cell a vector containing a DNA capable of expressing the Pi21 gene that can suppress the expression of the Pi21 gene and regenerating it.

In this specification, “providing” blast field resistance means not only giving a blast field resistance performance to a plant that does not have blast field resistance performance at all, but also providing blast field resistance performance already. It also means that the blast resistance performance of the plant they have is further increased.
In the present invention, the plant that suppresses the expression of the Pi21 gene and imparts disease field resistance is not particularly limited, and examples thereof include the above-mentioned plants.

The present invention also provides a protein encoded by the Pi21 gene of the present invention, a method for producing the protein, and an antibody that binds to the protein.
When preparing a recombinant protein, usually, a DNA encoding the protein of the present invention is inserted into an appropriate expression vector, the vector is introduced into an appropriate cell, and the transformed cell is cultured and expressed. Purify. The recombinant protein can be expressed as a fusion protein with another protein for the purpose of facilitating purification or the like. For example, a method for preparing a fusion protein with maltose binding protein using E. coli as a host (vector pMAL series released by New England BioLabs, USA), a method for preparing a fusion protein with glutathione-S-transferase (GST) (Amersham Pharmacia Biotech Vector pGEX series released by the company), a method of preparing by adding a histidine tag (pET series from Novagen), etc. can be used. The host cell is not particularly limited as long as it is a cell suitable for the expression of a recombinant protein. For example, yeast, various animal and plant cells, insect cells and the like can be used in addition to the above-mentioned E. coli. Various methods known to those skilled in the art can be used to introduce a vector into a host cell. For example, for introduction into E. coli, introduction methods using calcium ions (Mandel, M. & Higa, A. Journal of Molecular Biology, Vol. 53, 158-162, (1970), Hanahan, D. Journal of Molecular Biology, Vol. 166, 557-580, (1983)) can be used. The recombinant protein expressed in the host cell can be purified and recovered from the host cell or its culture supernatant by methods known to those skilled in the art. When the recombinant protein is expressed as a fusion protein with the above-described maltose binding protein, affinity purification can be easily performed.

  If the obtained recombinant protein is used, an antibody that binds to the protein can be prepared. For example, a polyclonal antibody can be obtained as follows. Serum is obtained by immunizing a small animal such as a rabbit with a recombinant protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a fusion protein with Pi21 protein or GST. This is prepared by, for example, purification by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with Pi21 protein or synthetic peptide, or the like. In the case of a monoclonal antibody, for example, a Pi21 protein or a partial peptide thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate the cells. Are fused using a reagent such as polyethylene glycol, and a clone producing an antibody that binds to the Pi21 protein is selected from the resulting fused cells (hybridoma). Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, Pi21 protein, It can be prepared by purification with an affinity column coupled with a synthetic peptide. The antibody thus obtained can be used for purification and detection of the protein of the present invention. The present invention includes antibodies that bind to the proteins of the present invention.

The present invention also provides an oligonucleotide having a chain length of at least 15 nucleotides complementary to DNA consisting of Pi21 gene or pi21 gene or a complementary strand thereof.
Here, “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs. Further, the term “complementary” is not limited to a case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. An algorithm for determining homology may be one known to those skilled in the art.

  The oligonucleotide of the present invention can be used as a probe or primer used for detection or amplification of DNA consisting of the base sequence described in SEQ ID NO: 1, 2, 4, 5, 20 or 21. Moreover, the oligonucleotide of the present invention can be used in the form of a substrate of a DNA array.

  When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least part of the DNA of the present invention or its complementary strand. When used as a primer, the 3 ′ region can be complementary, and a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ side.

  When the above oligonucleotide is used as a probe, the probe is specific to at least a part of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 5, 20, or 21 or a complementary strand thereof. There is no particular limitation as long as it hybridizes. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp.

When the oligonucleotide of the present invention is used as a probe, it is preferably used after appropriately labeling. As a labeling method, a method of labeling by phosphorylating the 5 ′ end of the oligonucleotide with ³² P using T4 polynucleotide kinase, and a random hexamer oligonucleotide or the like using a DNA polymerase such as Klenow enzyme are used. Examples of the primer include a method of incorporating a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin (random prime method or the like).

  The oligonucleotide of the present invention can be prepared, for example, by a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.

  Furthermore, the present invention provides use of DNA that suppresses the expression of the Pi21 gene and a vector containing the DNA. That is, the present invention relates to a drug for increasing resistance to blast field in plants, which contains any one of DNA that suppresses the expression of the Pi21 gene and a vector containing the DNA as an active ingredient. The present invention also relates to the use of a DNA that suppresses the expression of the Pi21 gene and a vector containing the DNA in the manufacture of a medicament for increasing the field resistance of blast disease in plants.

  In the pharmaceutical agent for increasing blast field resistance in plants of the present invention, in addition to the active ingredient oligonucleotide, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer Agents, preservatives and the like may be mixed as necessary.

The present invention also provides molecular markers linked to the susceptibility gene Pi21 or the resistance gene pi21.
The “molecular marker” in the present invention refers to a DNA region that is genetically linked to the Pi21 gene or the pi21 gene and is distinguishable from other DNA regions.

  In general, a molecular marker is more useful because the shorter the map distance expressed in units cM, the closer to the gene and the inheritance of the gene is. It was shown that pi21 exists between the marker “Pa102484” and the marker “P702D3_ # 12” (FIG. 2c). Therefore, in the method of the present invention, among the molecular markers shown in FIG. 2c, markers existing between the two markers and between the two markers ("P702D03_ # 38", "P702D03_ # 79", "P702D03_ #" 80 ") is a preferred marker. Among them, “P702D03_ # 79” is a particularly preferable marker, and can be exemplified as DNA described in SEQ ID NO: 7 or 23 (linked to the susceptibility gene Pi21) or SEQ ID NO: 10 (linked to the resistance gene pi21).

  Examples of the molecular marker of the present invention include an STS (Sequence Tagged Site) marker. An STS marker refers to a DNA region that can be used to determine the presence or absence of a polymorphism in a sequence tag site (STS) on DNA, and STS refers to a sequence site specific to a specific position on DNA. A polymorphism of STS is obtained by amplifying a DNA region containing the specific sequence site by a nucleic acid amplification method such as PCR method, and subjecting the amplified product to agarose or polyacrylamide gel electrophoresis. Can be detected.

  When an STS marker is used as the molecular marker of the present invention, the identification method of the present invention can be performed, for example, as follows. First, a DNA sample is prepared by a well-known method from a test rice and a rice having a blast disease resistance trait. Next, a nucleic acid amplification reaction (for example, PCR method) is performed using the prepared DNA as a template and a primer DNA. The size of the amplified DNA fragment is compared between the test rice and a marker linked to the rice blast field resistance gene (for example, the marker described in SEQ ID NO: 10) by electrophoresis or the like, and the same genotype is determined. In the case of showing, it is determined that the test plant has a rice blast field resistant trait.

  As a primer DNA used in the identification method of the present invention, those skilled in the art can appropriately design an optimal primer in consideration of sequence information on various molecular markers. Usually, the above-mentioned primers are a pair of primer sets for amplifying the base sequences that are specifically present in rice and designed to sandwich a base sequence linked to the Pi21 gene or the pi21 gene.

Specifically, as a primer set of the STS marker,
(A) Primer set consisting of primers 5'-AGA AGG TGG AGT ACG ACG TGA AGA-3 '(SEQ ID NO: 8) and 5'-AGT TTA GTG AGC CTC TCC ACG ATT A-3' (SEQ ID NO: 9) ,
(B) a primer set consisting of primers 5′-GTA CGA CGT GAA GAA CAA CAG G-3 ′ (SEQ ID NO: 16) and 5′-GCT TGG GCT TGC AGT CC-3 ′ (SEQ ID NO: 17);
(C) Example of primer set consisting of primers 5'-GAT CCT CAT CGT CGA CGT CTG GC-3 '(SEQ ID NO: 26) and 5'-AGG GTA CGG CAC CAG CTT G-3' (SEQ ID NO: 27) I can do it. By comparing the information characterized by the DNA sequence amplified by these primer sets with the molecular marker of the present invention, it is possible to determine whether or not the test rice is field resistant to blast.

  In addition to the primers described above, those skilled in the art can create a primer set having the same function using the nucleotide sequence set forth in SEQ ID NO: 1, 4 or 20. Such primers are also included in the primer of the present invention.

  The PCR primer of the present invention can be prepared by those skilled in the art using, for example, an automatic oligonucleotide synthesizer. Those skilled in the art can also carry out the method of the present invention by a well-known polymorphism detection method, for example, the PCR-SSCP method described later using the above PCR primers.

  When the molecular marker of the present invention is present in an exon of genomic DNA, RT-PCR using mRNA as a template can be used. Further, if a Taqman (quantitative PCR detection) system (Roche) is used, the presence or absence of an amplification product can be detected by fluorescence. According to this system, it is possible to perform the identification method of the present invention in a short time since the labor of electrophoresis can be saved.

Furthermore, the present invention provides a method comprising the following steps (a) to (c), wherein when a molecular weight or a base sequence matches, a test plant is determined to be blast field resistant: To do.
(A) a step of preparing a DNA sample from a test plant (b) a step of amplifying the Pi21 gene or pi21 gene region from the DNA sample (c) comparing the molecular weight or base sequence of the amplified DNA fragment with that of the pi21 gene Process

A person skilled in the art can prepare (extract) the above-described DNA sample of the present invention by a known method. As a preferable preparation method, for example, a method of extracting DNA using the CTAB method can be mentioned.
The DNA sample used in the identification method of the present invention is not particularly limited, but usually genomic DNA extracted from rice as a test plant is used. Further, the collection source of genomic DNA is not particularly limited, and it can be extracted from any tissue of rice. For example, it can be extracted from ear, leaf, root, stem, seed, endosperm part, bran, embryo and the like.

  In the method for identifying the blast field resistance of a plant of the present invention, next, a nucleic acid amplification reaction (for example, PCR method) is performed using the prepared DNA as a template and a primer DNA. The amplified DNA fragment is cleaved with a restriction enzyme, and the size of the cleaved DNA fragment is compared between the test plant and the blast field resistant plant by electrophoresis, etc., and the molecular weight or base sequence matches. In this case, it is determined that the test plant has a blast field resistant trait. Examples of the “rice-resistant field-resistant plant” include, but are not limited to, Owari Hatamochi described in Examples.

In the method of determining that the test plant is blast field resistant in the present invention, “matching” means that the molecular weight or base sequence of both genes of the allele matches that of the blast field resistant plant. Sometimes or in amino acid sequence. Therefore, if the molecular weight, base sequence or amino acid sequence of one of the alleles is different from that of the blast field resistant plant, but the other is the same as that of the blast field resistant plant, it “matches” Not included.
The electrophoretic analysis may be performed according to a conventional method. For example, electrophoresis is performed by applying voltage in an agarose or polyacrylamide gel, and the separated DNA pattern is analyzed.

In addition, the present invention is a method including the following steps (a) to (d), wherein when the mobility on the gel matches, the test plant is determined to be blast field resistant: I will provide a.
(A) a step of preparing a DNA sample from a test plant (b) a step of amplifying a Pi21 gene or pi21 gene region from the DNA sample (c) a step of separating amplified double-stranded DNA on a non-denaturing gel (D) A step of comparing the mobility of the separated double-stranded DNA on the gel with that of the pi21 gene

Furthermore, the present invention is a method comprising the following steps (a) to (e), wherein the test plant is determined to be blast field resistant when the mobility on the gel matches. I will provide a.
(A) a step of preparing a DNA sample from the test plant (b) a step of amplifying the Pi21 gene or pi21 gene region from the DNA sample (c) a step of dissociating the amplified DNA into single-stranded DNA (d) dissociation (E) a step of comparing the mobility of the separated single-stranded DNA on the gel with that of the pi21 gene

  One such method is the PCR-SSCP (single-strand conformation polymorphism) method (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11. Genomics. 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products.Oncogene. 1991 Aug 1; 6 (8): 1313-1318. , Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). This method is particularly suitable for screening a large number of DNA samples because it is relatively simple to operate and has advantages such as a small amount of sample. The principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When the dissociated DNA strand is electrophoresed in a polyacrylamide gel containing no denaturing agent, single-stranded DNA having the same complementary strand length moves to a different position according to the difference in each higher-order structure. The higher-order structure of this single-stranded DNA also changes by substitution of a single base, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of mutations due to point mutations, deletions or insertions in DNA fragments.

Specifically, first, a region containing the target site of Pi21 gene or pi21 gene is amplified by PCR or the like. The amplification range is preferably about 100 to 600 bp. During PCR by gene fragment amplification, isotope such as ³² P or fluorescent dyes and isotopes such as depending using labeled primers, or a PCR reaction solution including ³² P biotin or substrate bases labeled with a fluorescent dye or biotin,, Is added to label the DNA fragment synthesized. Alternatively, labeling can also be carried out by adding an isotope such as ³² P using a Klenow enzyme or a substrate base labeled with a fluorescent dye or biotin after the PCR reaction to the synthesized DNA fragment. The DNA fragment obtained in this manner is subjected to electrophoresis with a polyacrylamide gel containing no denaturing agent such as urea, while remaining double-stranded. Alternatively, such a DNA fragment may be denatured by applying heat or the like, and then subjected to electrophoresis using a polyacrylamide gel that does not contain a denaturing agent such as urea. At this time, conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. Electrophoretic conditions vary depending on the nature of each DNA fragment, but are usually performed at room temperature (20 to 25 ° C). If favorable separation cannot be obtained, the temperature at which the optimal mobility is obtained at temperatures from 4 to 30 ° C. Review. After electrophoresis, the mobility of the DNA fragment is detected and analyzed by autoradiography using an X-ray film or a scanner that detects fluorescence. When a band with a difference in mobility is detected, this band can be directly excised from the gel, amplified again by PCR, and directly sequenced to confirm the presence of the mutation. Even when the labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide or silver staining.

Furthermore, the present invention is a method including the following steps (a) to (e), and when the detected DNA fragments have the same size, it is determined that the test plant is blast field resistant: Provide a way to do it.
(A) a step of preparing a DNA sample from a test plant (b) a step of amplifying a Pi21 gene or pi21 gene region from the DNA sample (c) a step of cleaving the prepared DNA sample with a restriction enzyme (d) a DNA fragment (E) a step of comparing the size of the detected DNA fragment with that of the pi21 gene

  Examples of such a method include RFLP method and PCR-RFLP method using restriction fragment length polymorphism / RFLP. A restriction enzyme is usually used as an enzyme that cleaves DNA. Specifically, if there are base additions or deletions in the restriction enzyme recognition site, or if there are base insertions or deletions in the DNA fragment produced by the restriction enzyme treatment, the size of the fragment produced after the restriction enzyme treatment However, it varies between blast-affected plants and blast-field-resistant plants. These polymorphic sites can be detected as a difference in mobility of bands after electrophoresis by amplifying the portion containing the mutation site by PCR and treating with each restriction enzyme. Alternatively, the polymorphic site can be detected by treating chromosomal DNA with these restriction enzymes, performing electrophoresis, and performing Southern blotting using the probe DNA. The restriction enzyme used can be appropriately selected according to each mutation site. In this method, in addition to genomic DNA, RNA prepared from a subject can be converted to cDNA using reverse transcriptase, and this can be cleaved as it is with restriction enzyme and then subjected to Southern blotting. In addition, a part or all of the Pi21 gene or pi21 gene can be amplified by PCR using this cDNA as a template, cleaved with a restriction enzyme, and the difference in mobility can be examined.

Furthermore, the present invention is a method comprising the following steps (a) to (d), wherein the test plant is judged to be blast field resistant when the mobility on the gel matches. I will provide a.
(A) a step of preparing a DNA sample from a test plant (b) a step of amplifying the Pi21 gene or pi21 gene region from the DNA sample (c) the amplified DNA on a gel in which the concentration of the DNA denaturant is gradually increased Step of separating (d) Step of comparing the mobility of the separated DNA on the gel with that of the pi21 gene

  Examples of such a method include denaturant gradient gel electrophoresis (DGGE). A region containing the Pi21 gene or the target site of the pi21 gene is amplified by a PCR method using the primer of the present invention, etc., and this is gradually increased as the concentration of denaturing agents such as urea moves. Electrophoresis with and compare with healthy subjects. In the case of a DNA fragment with mutation, the DNA fragment becomes single-stranded at a lower denaturant concentration position and the migration speed becomes extremely slow, so the presence of polymorphism is detected by detecting this difference in mobility. can do.

  In addition to these methods, allele specific oligonucleotide (ASO) hybridization methods can be used. When an oligonucleotide containing a base sequence that is considered to have a polymorphism is prepared and hybridization is performed with sample DNA, if a polymorphic base different from the oligonucleotide is present in the sample DNA to be hybridized, the hybrid The efficiency of formation is reduced. It can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap.

  Moreover, detection by the ribonuclease A mismatch cleavage method is also possible. Specifically, the region containing the target site of the Pi21 gene or pi21 gene is amplified by PCR, etc., and this is hybridized with labeled RNA prepared from normal cDNA etc. Close. Since the hybrid has a single-stranded structure in the part where a base different from the normal type exists, this part is cleaved with ribonuclease A and the presence of the polymorphism is detected by autoradiography etc. Can do.

  In the present invention, the “test plant” is not particularly limited, and includes all plants that can be infected with the blast fungus. A preferable example is rice. It is not particularly limited as long as it is rice, and it may be Indica or Japonica, or a breed / line of Indica and Japonica, wild rice, or a cultivar and wild rice It may be a hybrid variety.

  Furthermore, the present invention relates to a method for determining rice blast field resistance, using as a marker a molecular marker linked to the pi21 gene and comprising at least a molecular marker of SEQ ID NO: 7, 10, or 23. provide. Preferred molecular markers of the present invention include “P702D03_ # 38”, “P702D03_ # 79”, and “P702D03_ # 80” as described above. Among them, “P702D03_ # 79” is a particularly preferred marker, and can be exemplified as DNA described in SEQ ID NOs: 7, 23 (linked to the susceptibility gene Pi21) or 10 (linked to the resistance gene pi21). In the identification method of the present invention, among these molecular markers, at least “P702D03_ # 79” is used as an index. Therefore, in the identification method of the present invention, “P702D03_ # 79” may be used alone, or “P702D03_ # 79” and another marker may be used in combination. Examples of combinations of “P702D03_ # 79” and other markers include “P702D03_ # 38”, “P702D03_ # 79” and “P702D03_ # 80”, and “P702D03_ # 79” and any other marker. It is done.

  In the identification method of the present invention, it is possible to specifically and efficiently determine the rice blast field resistance of a test rice by examining whether or not it has a molecular marker linked to the pi21 gene. In the determination method of the present invention, in the desired rice for which it is desired to determine the presence or absence of blast disease field resistance, when the rice has the base sequence set forth in SEQ ID NO: 10, the test rice is a rice that is resistant to blast disease field. When it is determined that there is no nucleotide sequence described in SEQ ID NO: 10 (when it has the nucleotide sequence described in SEQ ID NO: 7 or 23), the rice to be tested is a rice susceptible to rice blast Determined.

The comparison between the molecular marker in the test rice and the molecular marker of the present invention can be performed not only by comparing the DNA sequence of the molecular marker but also by comparing information characterized by the DNA sequence. Information characterized by the DNA sequence of the molecular marker includes information on the molecular weight of the molecular marker and information on the presence or absence of a mutation site or polymorphic site contained in the molecular marker. A person skilled in the art can compare the polymorphic site (deletion site and single base substitution site) by comparing the sequence number: 10 with the base sequence described in SEQ ID NO: 7 or SEQ ID NO: 23 by a known method. ) Can be identified.
The determination method of the present invention can also be performed by detecting information on the presence / absence of a mutation site or polymorphic site contained in the molecular marker.

  Detection of information on the presence or absence of mutation sites and polymorphic sites as described above can be performed by directly sequencing the sequence or by using a primer that can amplify the region containing the mutation site or polymorphic site. Alternatively, it can be detected by using a probe that can hybridize to a mutation site or polymorphism site (for example, DNA containing all or part of the base sequence described in SEQ ID NO: 18, 19 or 25). .

Moreover, if the determination method of this invention is utilized, it will become possible to select the plant (for example, rice) identified as having rice blast field resistance at an early stage.
That is, this invention provides the method of selecting the plant which is blast field resistant including the process as described in the following (a) and (b).
(A) a step of producing a variety in which a plant resistant to blast field (for example, rice) and a plant having an arbitrary function (for example, rice) are crossed (b) the test plant described herein is Step of determining whether or not the plant obtained in step (a) is blast field resistant by the method of determining whether or not it is blast field resistant In the present invention, the plant is particularly limited. Although it is not a thing, Preferably it is a rice. The specific mode of rice is as described above.

  If the selection method of this invention is utilized, it will become possible to select the plant (for example, rice) identified as rice blast field resistance at an early stage. The present invention also provides a method for early selection of plants identified as such a rice blast field resistance. Here, “early” refers to, for example, a state before the heading of rice, and preferably refers to a state immediately after germination. By using the selection method of the present invention, it is possible to cultivate varieties having a rice blast field resistant trait in a shorter period of time than before.

The present invention relates to a method for screening a drug for preventing or ameliorating plant blast.
The first aspect of the screening method of the present invention includes a method for screening a drug for preventing or ameliorating plant blast, which comprises the following steps (a) to (c).
(A) contacting the test compound with the transcription product of Pi21 gene (b) detecting the binding between the transcription product of Pi21 gene and the test compound (c) selecting a test compound that binds to the transcription product of Pi21 gene Process

  In the first embodiment, first, a test compound is brought into contact with the transcription product of the Pi21 gene. The “Pi21 gene transcription product” in the screening method of the present invention includes a translation product translated from the transcription product in addition to the Pi21 gene transcription product.

  There is no restriction | limiting in particular as "test compound" in the method of this invention, For example, a single compound, such as a natural compound, an organic compound, an inorganic compound, protein, a peptide, and an expression product of a compound library and a gene library And cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, animal cell extracts, and the like. The test compound can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label.

  In the present invention, “contact” is performed as follows. For example, if the transcription product of the Pi21 gene is in a purified state, it can be carried out by adding a test compound to the purified sample. Moreover, if it is the state expressed in the cell or the state expressed in the cell extract, it can be carried out by adding a test compound to the cell culture solution or the cell extract, respectively. Although it does not restrict | limit especially as a cell in this invention, The plant-derived cell containing rice is preferable. When the test compound is a protein, for example, a vector containing DNA encoding the protein is introduced into a cell in which the Pi21 gene is expressed, or the vector is introduced into a cell extract in which the Pi21 gene is expressed. It is also possible to carry out by adding. For example, a two-hybrid method using yeast or animal cells can be used.

  In the first embodiment, the binding between the Pi21 gene transcript and the test compound is then detected. The means for detecting or measuring the binding between proteins can be performed, for example, by using a label attached to the protein. Examples of the type of label include a fluorescent label and a radiolabel. Moreover, it can also measure by a known method such as a yeast two-hybrid method or a measurement method using BIACORE. In this method, a test compound bound to the biosynthetic enzyme is then selected. The selected test compound includes an agent for preventing or ameliorating plant blast. In addition, the selected test compound may be used as a test compound for the following screening.

In addition, as a second aspect of the screening method of the present invention, there is provided a method for screening a drug for preventing or ameliorating plant blast, which comprises the following steps (a) to (c).
(A) a step of contacting a test compound with a cell collected from a plant (b) a step of measuring the expression level of a transcription product of Pi21 gene (c) as compared with the case where the test compound is not contacted A step of selecting a test compound having a reduced expression level of the transcript

  In the second embodiment, first, a test compound is brought into contact with cells collected from a plant. Here, the “cell collected from the plant” may be any plant that clearly has the blast susceptibility gene. The description of “test compound” and “contact” is as described above.

  In the second embodiment, the expression level of “Pi21 protein” is then measured. The expression level of Pi21 protein can be measured by methods known to those skilled in the art. For example, mRNA level encoding the Pi21 protein can be extracted according to a standard method, and the transcription level of the gene can be measured by performing Northern hybridization or RT-PCR using this mRNA as a template. Furthermore, it is possible to measure the expression level of Pi21 protein using DNA array technology.

  Alternatively, the fraction containing Pi21 protein can be collected according to a standard method, and the expression level of Pi21 protein can be detected by electrophoresis such as SDS-PAGE to measure the translation level of the gene. Moreover, it is also possible to measure the translation level of a gene by performing Western blotting using an antibody against the Pi21 protein and detecting the expression of the Pi21 protein.

  The antibody used for detection of Pi21 protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used. The antibody can be prepared by methods known to those skilled in the art as described above.

  In the second embodiment, the test compound is then selected as an agent for preventing or ameliorating plant blast if the expression level of Pi21 protein is reduced compared to when the test compound is not contacted. .

The third aspect of the screening method of the present invention is a method for screening a drug for preventing or ameliorating plant blast disease, which comprises the following steps (a) to (d).
(A) a step of providing a cell or a cell extract having DNA functionally linked to a reporter gene downstream of the promoter region of the Pi21 gene (b) a step of bringing a test compound into contact with the cell or the cell extract ( c) a step of measuring the expression level of the reporter gene in the cell or the cell extract (d) a test compound in which the expression level of the reporter gene is reduced as compared with the case where the test compound is not contacted The process of selecting

In the third embodiment, first, a cell or cell extract having DNA to which a reporter gene is operably linked downstream of the Pi21 gene promoter region is provided.
In the third embodiment, “functionally linked” refers to the promoter region of the Pi21 gene and the reporter so that expression of the reporter gene is induced by binding of a transcription factor to the promoter region of the Pi21 gene. It means that the gene is linked. Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, the transcription factor binds to the promoter region of the Pi21 gene, so that the fusion protein Any expression that is induced is included in the meaning of “functionally linked”.

  The reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase gene (GUS) and a GFP that are commonly used by those skilled in the art. A gene etc. can be mentioned.

  In the third embodiment, the test compound is then brought into contact with the cells or the cell extract. Next, the expression level of the reporter gene in the cell or the cell extract is measured. The description of “test compound” and “contact” is as described above.

  The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a β-glucuronidase gene (GUS), the luminescence of Glucuron (ICN) or 5-bromo-4-chloro-3-indolyl- By detecting the color development of β-glucuronide (X-Gluc), and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.

  In the third aspect, the test compound is then selected as an agent for preventing or ameliorating plant blast when the expression level of the reporter gene is reduced compared to when the test compound is not contacted. To do.

The fourth aspect of the screening method of the present invention is a method for screening a drug for preventing or ameliorating plant blast disease, which comprises the following steps (a) to (c).
(A) a step of regenerating a transformed plant from the transformed plant cell into which the Pi21 gene has been introduced (b) a step of bringing the transformed plant into contact with a blast fungus and a test compound (c) contacting the test compound A step of selecting a test compound that suppresses blast of the transformed plant compared to a case where the transformed plant is not used

In the fourth embodiment, first, a transformed plant body is regenerated from a transformed plant cell containing the Pi21 gene. The regeneration of the transformed plant can be performed by methods known to those skilled in the art as described above.
Next, in the fourth embodiment, the blast fungus and the test compound are contacted with the transformed plant body regenerated in step (a). “Blast fungus” and “test compound” are as described above. An example of “contact” includes a method of spraying a test compound directly onto a plant using a spray. However, the “contact” in the fourth aspect is not limited to this, and includes any method as long as the plant and the test compound can be physically touched. In the contact according to the present invention, a test compound may be contacted with a transformed plant body infected with a blast fungus, or a transformed plant body contacted with a test compound may be infected with a blast fungus.

  In the fourth embodiment, the test compound that suppresses the blast disease of the transformed plant is then selected as compared with the case where the test compound is not contacted. Whether or not rice blast is suppressed can be determined using the phenotype of the transformed plant body as an indicator. Although there is no restriction | limiting in particular as a phenotype of this transformed plant body, It can illustrate that the part or the whole discolors and necroses in each site | part of a plant. In addition, the suppression of blast of the transformed plant includes not only complete suppression but also partial suppression.

  The present invention also relates to a kit for use in the screening method described above. Such a kit may include those used in the detection step and the measurement step of the screening method described above. For example, a probe, a primer, an antibody, a staining solution, etc. required for measuring the expression level of the Pi21 gene can be mentioned. In addition, distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Creation of genetic map Detailed linkage analysis of the pi21 region was performed using a large-scale segregation population indispensable for map-based cloning. In the linkage analysis group, the Japanese upland rice cultivar Owari Hatamochi that has the resistant allele pi21 that suppresses lesion progression, followed by the rice cultivar Nipponbare or Aichi Asahi (Fig. 1) that has the susceptibility allele Pi21 that does not suppress lesion progression. 72 BC1F2 populations obtained by backcrossing were used. As a result of linkage analysis using the RFLP marker, it was found that the pi21 locus exists between the RFLP markers G271 and G317 (FIG. 2A).

  In order to create a genetic map of the pi21 region with high accuracy, the above-mentioned hybrid combination 229 individuals and progeny BC1F4 population 643 individuals were added, using a total of 1014 individuals, using RFLP markers RA3591 and 13S1 present on both sides of pi21 Thus, 27 chromosome-recombinant individuals near the pi21 locus were selected (FIG. 2B). In addition, a search was conducted using 2703 F2 populations obtained by crossing a line with a susceptible allele of the Indian rice cultivar Kasalath and a line with a resistance allele from Owari Hatamochi in the genetic background of Japanese rice cultivars. It was. Using the PCR markers 14T1 and 4S1 present on both sides of the pi21 locus, 24 chromosome recombination-type individuals near the pi21 locus could be selected. Furthermore, using these individuals, detailed linkage maps were created using the DNA markers created in the following efforts.

[Example 2] Alignment of pi21 gene region with P1-derived artificial chromosome (PAC) clone Using the alignment map of Nipponbare PAC clone created in rice genome analysis study, DNA marker RA3591 located near pi21 locus and A PAC clone having a C975 base sequence was identified (FIG. 2C). Further, the terminal fragments of the identified PAC clones P479G02, P415D09, P473G08, P703E11, P434F09, P702D03, P419B08, P472G09 and P502G01 were isolated by the cassette method, and the identified PAC clones were aligned. As a result, PAC clones P032D02, P678A02, P405D12, P689F04, P479G02, P415D09, P473G08, P703E11, P434F09, and P702D03 were found to contain the pi21 gene region (FIG. 2C).

[Example 3] Refinement of the pi21 gene region
Cloning the terminal fragments of the PAC clones aligned in the pi21 region and using them as a new RFLP marker or CAPS marker, and creating a detailed genetic map, the pi21 locus is the SSCP marker Pa102484 and the SNP marker P702D3_ # 12. It was clarified that it exists in the genomic region sandwiched. As a result, it was revealed that the pi21 locus is present in an approximately 25 kb genomic region sandwiched between two markers (FIG. 2D).

[Example 4] Identification of candidate gene region by nucleotide sequence analysis
The nucleotide sequence of the PAC clone P702D03, which is thought to contain the pi21 gene, was determined, and the nucleotide sequences of the resistant cultivar Owari Hatamochi and the susceptible varieties Aichi Asahi and Kasalath in the 25 kb genome candidate region were analyzed. The nucleotide sequence was analyzed by the dye-terminater method using DNA fragments amplified from the above three varieties using primers designed using the sequence of the candidate region of Nipponbare. Using the nucleotide polymorphism information in the candidate gene region identified by linkage analysis, the candidate regions were further narrowed down. As a result, the pi21 gene was identified as STS marker P702D03_ # 79 (primer 5'-AGA AGG TGG AGT ACG ACG TGA AGA-3 '(SEQ ID NO: 8) and AGT TTA GTG AGC CTC TCC ACG ATT A-3' (SEQ ID NO: 9)) and SNP marker P702D03_ # 38 (primer TTT TCC TGA GAA ATT TGT AAA GA-3 '(SEQ ID NO: 12) and CGT CGA CGA TGA GGA TCT-3' (SEQ ID NO: 13)) , P702D03_ # 80 (primers 5'-CTC CCA ATG TGT TTA GCA TC-3 '(SEQ ID NO: 14) and 5'-CAA CCA TAT GTC CCT AAG GAT-3' (SEQ ID NO: 15)) One recombinant individual was detected for each. From the above results, it was revealed that the pi21 gene is present in an approximately 1.8 kb genomic region sandwiched between SNP markers P702D03_ # 38 and P702D03_ # 80 (FIG. 2D).

  The base sequence of the Pi21 gene derived from the isolated rice (Oryza sativa L, Aichi Asahi and Nipponbare) is shown in SEQ ID NO: 1, the base sequence of the cDNA in SEQ ID NO: 2, and the protein encoded by the cDNA (“Pi21 The amino acid sequence of “protein”) is shown in SEQ ID NO: 3. In addition, the base sequence of the Pi21 gene derived from Kasalath corresponding to the Pi21 gene of Aichi Asahi and Nipponbare is SEQ ID NO: 20, the base sequence of cDNA is SEQ ID NO: 21, and the protein encoded by the cDNA ("Pi21 The amino acid sequence of “protein”) is shown in SEQ ID NO: 22.

[Example 5] Base sequence analysis of pi21 candidate gene
When the gene prediction and similarity search were performed for the sequence of the varietal Nipponbare in the 1.8 kb candidate genomic region, full-length cDNA clones (AK106153, AK070581, AK072320) of Nipponbare were found, but no similar genes were found in Arabidopsis etc. The function of the gene could not be predicted from the homology. However, this gene has a metal-binding site that is about 10 amino acids from the predicted translation start point, and has a function of a chaperone that transports metal in the ethylene signaling system reported in Arabidopsis (Hirayama et al 1999 Cell) In fact, the near-isogenic line AA-pi21 may have a similar function because its sensitivity to ethylene has changed compared to Aichi-Asahi. Primers that can amplify the relevant part were designed from the base sequence information of Nipponbare already obtained, and genomic PCR and RT-PCR products were compared between the susceptible strains Nipponbare and AichiAsahi and the resistant variety Owarihatamochi. However, there were two DNA mutations in the exon region of the gene between resistant and susceptible varieties. In varieties that are resistant to susceptible varieties, deletions of 7 and 16 amino acids have occurred, and these mutations are thought to be related to the development of the lesions of blast disease (Fig. 3). .

  The isolated nucleotide sequence of the gene for the pi21 gene is shown in SEQ ID NO: 4, the nucleotide sequence of the cDNA is shown in SEQ ID NO: 5, and the amino acid sequence of the protein encoded by the cDNA (“pi21 protein”) is shown in SEQ ID NO: 6. .

[Example 6] Identification of function of candidate gene by transformation (1) Introduction of susceptibility gene into AA-pi21
A genomic region XbaI 4.7 kb fragment of the susceptible cultivar Nipponbare containing the 5 ′ upstream predicted promoter region identified as a candidate for the pi21 gene was incorporated into a vector pPZP2H-lac that can be transformed via Agrobacterium. Using the vector into which this fragment was introduced and only the vector, transformation was performed by Toki's method (literature Plant Mol. Biol. Rep. 15: 16-21, 1997). The pi21 near-isogenic line AA-pi21 was used as the line to be transformed. From the vector into which the XbaI 4.7 kb fragment was introduced, 36 individuals and 12 individuals from the vector alone were obtained. Primers specific to the candidate gene (sense strand 5'-GTA CGA CGT GAA GAA CAA CAG G-3 '(SEQ ID NO: 16)) and (antisense strand 5'- GCT TGG GCT TGC AGT CC 3' (SEQ ID NO: :))) was used to investigate whether or not the introduced region was incorporated by PCR. As a result, it was found that candidate genes were incorporated in all transformants. These individuals were cultivated in an isolated greenhouse and inoculated with blast fungus (race 007) in a self-breeding progeny line. As a result, similar to the near-isogenic line AA-pi21, the progression of lesions was higher in the individuals with the vector alone and in the T1 individuals in which the transgene was not transferred due to the isolation than in the susceptible cultivar Aichi Asahi. While the progression of lesions was suppressed, the lesions progressed more in T1 individuals into which the candidate gene was introduced (Fig. 4). In particular, the degree of susceptibility increased in lines with a large number of copies of the transgene.

(2) Introduction of Resistance Gene into Susceptible Variety On the other hand, the XbaI 4.7 kb fragment of the resistant variety Owari Hatamochi was similarly incorporated into a vector and transformed using the susceptible strain Aichi Asahi. From the vector into which the XbaI 4.7 kb fragment was introduced, 56 individuals and 24 individuals from the vector alone were obtained. As in (1), as a result of investigating whether or not the introduced region was incorporated by PCR, it was found that candidate genes were incorporated in all transformants. These individuals were cultivated in an isolated greenhouse in the same manner as in (1), and inoculated with blast fungus (race 007) in a self-bred progeny line. As a result, progress of blast disease similar to that of the susceptible cultivar Aichi Asahi was observed in all of the individuals into which only the vector was introduced, the T1 individuals into which the transgene was not transmitted, and the T1 individuals into which the candidate gene was introduced.

(3) Identification of candidate gene function From the above results, it was found that the candidate gene region derived from Nipponbare (XbaI 4.7 kb) has a function of promoting lesion formation of the near-isogenic line AA-pi21. The candidate gene was determined to be the Pi21 gene.

[Example 7] Candidate gene mutations in rice When mutations in candidate genes were searched using 79 rice varieties from around the world, insertion deletions were made in the exon region in addition to the types found in Nipponbare, AichiAsahi, and Owarihamamachi There were 10 types of mutations. These are mainly defined by the presence / absence of the insertion / deletion and the size of the two deletion sites found between Nipponbare / Aichi Asahi and Owarihamochi. This region, which is not homologous to known genes, is expected to bind to other molecules due to the similarity to the metal molecule chaperone proposed in the Arabidopsis ethylene signaling system. It is possible to subtly control the efficiency of the function, resulting in functional variation.

  From the above results, it was found that the gene pi21 that regulates the development of rice blast disease spots was selected as a candidate by the map-based cloning method. This is the first case to prove the biological function of plant quantitative resistance. When the expression of the pi21 or Pi21 gene was investigated by RT-PCR analysis, it was expected to play a fundamental role in plant growth because it was expressed constantly in all tissues above the ground. The Since the change in copy number brought about a phenotypic change, changing the expression level and expression tissue by the promoter is an important factor for functional modification. That is, it is considered possible to efficiently increase the disease resistance inherent to plants by using the isolated pi21 gene or another allele found in the species.

Claims (35)

DNA according to any one of the following (a) to (h);
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 22,
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1, 2, 20 or 21;
(C) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 22, DNA encoding a protein having a function equivalent to that of the protein comprising the described amino acid sequence,
(D) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 2, 20 or 21, comprising a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 or 22; DNA encoding a protein having an equivalent function,
(E) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(F) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or 5,
(G) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence set forth in SEQ ID NO: 6 DNA encoding a protein having the same function as the protein consisting of
(H) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 5, and having a function equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 6 DNA encoding The DNA according to any one of (i) to (iv) below, which has a blast field resistance performance in a plant;
(I) DNA encoding RNA complementary to the transcription product of DNA according to any one of claims 1 (a) to (d),
(Ii) a DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcript of the DNA according to any one of claims 1 (a) to (d),
(Iii) DNA encoding RNA that inhibits the expression of the DNA according to any one of claims 1 (a) to (d) by a co-suppression effect,
(Iv) DNA encoding RNA having RNAi activity that specifically cleaves the transcription product of DNA according to any one of claims 1 (a) to (d).   The DNA according to claim 2, wherein the plant is rice, wheat, barley, oat, corn, pearl barley, italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane.   A vector comprising the DNA according to any one of claims 1 to 3.   A transformed cell that holds the DNA according to any one of claims 1 to 3 in an expressible manner.   A transformed plant cell into which the DNA according to any one of claims 1 (a) to (d) has been introduced.   A transformed plant cell into which the DNA according to claim 2 or 3 is introduced.   The transformed plant cell according to claim 6 or 7, wherein the plant is rice, wheat, barley, oat, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, or sugar cane.   A transformed plant comprising the transformed cell according to claim 6.   A transformed plant that is a descendant or clone of the transformed plant according to claim 8.   The propagation material of the transformed plant body in any one of Claim 9 or 10.   A method for producing a transformed plant according to any one of claims 9 and 10, wherein the DNA according to claims 1 (a) to (d), claim 2 or 3 is introduced into a plant cell, A method comprising a step of regenerating a plant from plant cells.   A method for imparting blast disease field resistance to a plant, comprising the step of expressing the DNA according to claim 2 or 3 in cells of the plant body.   14. The method according to claim 13, wherein the plant is rice, wheat, barley, oat, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane.   A protein encoded by the DNA according to any one of claims 1 (a) to (d).   A step of culturing a transformed cell containing the vector containing the DNA according to any one of claims 1 (a) to (d) and recovering the recombinant protein from the cell or a culture supernatant thereof. A method for producing the protein according to 1.   An antibody that binds to the protein of claim 15.   A DNA comprising at least 15 consecutive bases complementary to the DNA according to claim 1 or a complementary sequence thereof.   An agent for increasing the blast field resistance of a plant, comprising any one of the DNA according to claim 2 or 3 or a vector containing the DNA.   A primer set for amplifying all or part of the nucleotide sequence set forth in SEQ ID NO: 1, 4 or 20. At least one primer set shown in any of (a) to (c) (a) DNA consisting of the base sequence described in SEQ ID NO: 8 and DNA consisting of the base sequence described in SEQ ID NO: 9;
(B) DNA consisting of the base sequence set forth in SEQ ID NO: 16 and DNA consisting of the base sequence set forth in SEQ ID NO: 17
(C) DNA consisting of the base sequence set forth in SEQ ID NO: 26 and DNA consisting of the base sequence set forth in SEQ ID NO: 27.   DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 7, 10, 18, 19, 23 or 25. A method comprising the following steps (a) to (c), wherein when the molecular weight or the base sequence is identical, the test plant is determined to be blast field resistant;
(A) a step of preparing a DNA sample from a test plant;
(B) amplifying the DNA region of claim 1 from the DNA sample;
(C) A step of comparing the molecular weight or base sequence of the amplified DNA fragment with that of the DNA according to any one of claims 1 (e) and (f). A method comprising the following steps (a) to (d), wherein the test plant is determined to be blast field resistant when the mobility on the gel matches:
(A) a step of preparing a DNA sample from a test plant;
(B) amplifying the DNA region of claim 1 from the DNA sample;
(C) separating the amplified double-stranded DNA on a non-denaturing gel;
(D) A step of comparing the mobility of the separated double-stranded DNA on the gel with that of the DNA according to any one of claims 1 (e) and (f). A method comprising the following steps (a) to (e), wherein the test plant is determined to be blast field resistant when the mobility on the gel matches:
(A) a step of preparing a DNA sample from a test plant;
(B) amplifying the DNA region of claim 1 from the DNA sample;
(C) dissociating the amplified DNA into single-stranded DNA;
(D) separating the dissociated single-stranded DNA on a non-denaturing gel;
(E) A step of comparing the mobility of the separated single-stranded DNA on the gel with that of the DNA according to claim 1 (e) or (f). A method comprising the following steps (a) to (d), wherein the test plant is determined to be blast field resistant when the mobility on the gel matches:
(A) a step of preparing a DNA sample from a test plant;
(B) amplifying the DNA region of claim 1 from the DNA sample;
(C) separating the amplified DNA on a gel in which the concentration of the DNA denaturant is gradually increased,
(D) A step of comparing the mobility of the separated DNA on the gel with that of the DNA according to claim 1 (e) or (f). A method for selecting a plant resistant to blast field, comprising the following steps (a) and (b):
(A) a step of producing a variety in which a plant having a rice blast field resistance performance and a plant having an arbitrary function are crossed,
(B) The process of determining whether the plant produced at the process (a) is blast field resistance by the method in any one of Claims 23-26.   A molecular marker linked to the DNA according to claim 1, wherein when the molecular marker exhibits a genotype similar to that of rice having a trait resistant to blast disease, the tested rice is resistant to blast disease field. How to determine that there is.   The method according to claim 28, wherein the molecular marker is a molecular marker comprising the DNA of SEQ ID NO: 10. A method for selecting rice that is resistant to blast disease field, comprising the following steps (a) and (b):
(A) a step of producing a variety in which rice having rice blast field resistance performance and rice having an arbitrary function are crossed,
(B) A step of determining whether or not the rice produced in the step (a) is blast field resistant by the method according to claim 28 or 29. A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (c):
(A) contacting the test compound with the transcription product of the DNA according to any one of claims 1 (a) to (d),
(B) a step of detecting the binding between the transcription product of the DNA according to any one of claims 1 (a) to (d) and a test compound;
(C) A step of selecting a test compound that binds to the transcription product of the DNA according to any one of claims 1 (a) to (d). A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (c):
(A) a step of bringing a test compound into contact with a cell collected from a plant;
(B) a step of measuring the expression level of the transcription product of the DNA according to any one of claims 1 (a) to (d),
(C) A step of selecting a test compound in which the expression level of the transcript is reduced as compared with the case where the test compound is not contacted. A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (d):
(A) providing a cell or cell extract having a DNA to which a reporter gene is operably linked downstream of the promoter region of the DNA according to any one of claims 1 (a) to (d);
(B) contacting the test compound with the cells or the cell extract;
(C) measuring the expression level of the reporter gene in the cell or cell extract,
(D) A step of selecting a test compound in which the expression level of the reporter gene is reduced as compared with the case where the test compound is not contacted. A method for screening a drug for preventing or ameliorating plant blast, comprising the following steps (a) to (d):
(A) a step of regenerating a transformed plant from the transformed plant cell according to claim 6;
(B) contacting the transformed plant with a blast fungus and a test compound;
(C) The process of selecting the test compound which suppresses the blast disease of this transformed plant body compared with the case where the test compound is not made to contact.   The kit for using for the screening method in any one of Claims 31-34.