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WO2004013331A1 - Genes pathogenes de xanthomonas oryzae pv. oryzae et leur utilisation - Google Patents

Genes pathogenes de xanthomonas oryzae pv. oryzae et leur utilisation Download PDF

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Publication number
WO2004013331A1
WO2004013331A1 PCT/JP2003/009922 JP0309922W WO2004013331A1 WO 2004013331 A1 WO2004013331 A1 WO 2004013331A1 JP 0309922 W JP0309922 W JP 0309922W WO 2004013331 A1 WO2004013331 A1 WO 2004013331A1
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Prior art keywords
dna
rice
plant
seq
present
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Japanese (ja)
Inventor
Hisatoshi Kaku
Hirokazu Ochiai
Masaru Takeya
Yasuhiro Inoue
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National Institute of Agrobiological Sciences
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National Institute of Agrobiological Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention relates to crop disease diagnosis and crop breeding.
  • Bacteria have a genome size much smaller than animals and plants, and their genome size is about 1/10 or less than that of filamentous fungi, which is the same microorganism. Genome analysis is progressing.
  • Genome analysis analyzes all the genes present in a genome by revealing the genome that is the source of genetic information at the nucleotide sequence level, and understands the whole life phenomenon such as gene networks and interactions. It is expected to provide valuable information, and is widely used worldwide with a wide variety of organisms. In particular, in the field of animal pathogenic bacteria, elucidation of the pathogenic mechanism of human infection and disease, and the knowledge obtained therefrom, various research developments and applications such as genomic drug discovery are expected. Currently, tuberculosis bacteria ( Cole, ST et al. (1998) .Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.Nature 393: 537-44) and E. coli 0-157 (Hayashi, T.
  • Bacterial leaf blight of rice is a globally important disease of rice that occurs not only in Japan, but also in rice cultivation areas in Asia, Africa, Australia, Latin America and the United States. It is.
  • the pathogenic bacterium that causes this disease is Xanthom as oryzae pv. Oryzae, a rice cultivar that is known to have a large number of races that differ in pathogenicity to rice varieties, and that is highly differentiated. .
  • the genus to which this bacterium belongs is composed of bacteria that cause disease in various plants, and is known to be of a very large variety, and is a group of bacteria important in agriculture.
  • rice the infection partner (host)
  • This is a model plant that has been studied.
  • the process by which phytopathogenic bacteria cause disease in plants involves two processes, host recognition / infection establishment and pathogen release / pathogenesis, and many genes are thought to be involved in these processes. .
  • details are still unknown.
  • rice blight disease bacterium has no effective materials such as pesticides.
  • rice blast blight resistance genes are mainly introduced by breeding to control rice blast blight resistant varieties. It has been raised and cultivated. However, it has been reported that cultivation of disease-resistant varieties into which a resistance gene has been introduced becomes ill with the emergence of pathogenic bacteria exhibiting a new pathogenic type (race), so-called resistance crushing.
  • the present invention has been made in view of such a situation, and an object of the present invention is to identify a gene involved in the pathogenicity of a rice bacterial wilt disease which is an important pathogenic bacterium of rice. More specifically, it is an object of the present invention to provide a method for easily diagnosing rice leaf blight on a test rice by setting the presence or absence of a gene related to the virulence of rice leaf blight fungus as an indicator. .
  • pathogenic bacteria both plants and animals, exist in the form of large clusters (pathogenic islands) on genomes or plasmids, causing genes that cause pathogens. It was predicted that the bacterial pathogen of the present invention may form such a virulence gene cluster. Therefore, the present invention
  • the hrp gene class encoding a pathogenic secretory device (type III secretion device) that is important for expressing pathogenicity in plant pathogenic bacteria such as Bacterial Blight Fungus of Rice, and race differentiation ⁇ Pathogenic genes involved in diversification of pathogenicity such as
  • the present inventors set out to establish a molecular biological basis for elucidating the mechanism of pathogenicity expression and response mechanism on the microorganism side in plant-microbe interaction.
  • the present inventors have analyzed the genome of rice white leaf blight fungus.
  • the strain used for genome analysis was Xal (Yoshimura, S. et al. (1998), a rice leaf blight resistance gene in rice genome research.
  • Xal a bacterial blight-resistance gene in rice, is induced by bacterial inoculat ion.Pro Natl.Acad.Sci. USA 95: 1663-8) and its structural analysis was performed. In other words, in elucidating the interaction, it is considered that strains having a non-pathogenic gene (avrXal) with respect to the resistance gene (Xal) may have more non-pathogenic genes (pathogenicity). As a result of considering the narrow spectrum, we tested MAFF311018 (T7174), a representative strain of Race I in Japan.
  • PCR-based detection that amplifies a gene region specific to Bacterial Blight Leaf Blight is effective.
  • it is effective to introduce a virulence gene derived from a pathogenic bacterium into a plant body so that the plant can always induce resistance to the plant side.
  • the present inventors cloned the DNA of the virulence gene group and the DNA in the vicinity thereof from Japanese rice leaf blight fungus stored at the National Institute for Agricultural Resources, and determined the nucleotide sequence. We succeeded in identifying 14 novel virulence genes unique to Bacterial Blight Pathogen that do not show significant homosexuality.
  • the novel gene discovered by the present inventors can be said to be extremely useful.
  • the present inventors have actually hybridized to the novel virulence gene and succeeded in specifically detecting rice Bacterial Blight on test rice using a PCR primer set capable of spreading the gene.
  • the information on the genome analysis of Bacterial Leaf blight obtained by the present invention will be a powerful 'I blueprint' for elucidating the pathogenic mechanism, as well as genetic information including the diversity of pathogenicity of XanthomoMS bacteria. It is expected to be useful knowledge in the analysis of genetic diversity and classification research.
  • the present invention relates to a novel virulence gene group of rice bacterial wilt and its use.
  • SEQ ID NO: Encodes a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the even-numbered amino acid sequence of any one of SEQ ID NOS: 2 to 28 DNA.
  • [4] A transformed cell carrying the DNA of [1] or [2] or the vector of [3].
  • [6] A transformed plant comprising the transformed cell according to [5].
  • a transformed plant which is a progeny or clone of the transformed plant of [6].
  • An oligonucleotide comprising at least 15 consecutive nucleotide sequences in the arrangement (J number: any one of the odd-numbered numbers from 1 to 27, or its complementary arrangement [J].
  • SEQ ID NO: 1 An oligonucleotide which specifically hybridizes with DNA consisting of the nucleotide sequence of any one of odd numbers of SEQ ID NO: 27 and has a chain length of at least 15 bases.
  • a method for determining that the rice to be diagnosed is infected with Bacterial Leaf blight, if the presence of the DNA or its expression product according to [1] is detected in step (b). Things.
  • the present inventors have identified 14 novel virulence genes of the bacterial leaf blight fungus.
  • the present invention provides DNA derived from the bacterial wilt of rice.
  • the nucleotide sequence of the virulence gene of Bacterial Leaf blight of Rice isolated by the present inventors, which is included in the present invention, is encoded by an odd number of SEQ ID NO: 1 and the like by each of the nucleotide sequences.
  • the amino acid sequence of the protein is shown in SEQ ID NO: 2 Shown in the numbers. That is, the present invention relates to a DNA encoding a protein consisting of the amino acid sequence described in any one of SEQ ID NOS: 2 to 28 derived from Bacterial Leaf blight of rice, and any one of SEQ ID NOs: 1 to 27.
  • a DNA comprising the coding region of the nucleotide sequence described in the odd number.
  • the present invention also provides a DNA encoding a protein structurally similar to the protein described in any of SEQ ID NOs: 2 to 28.
  • Examples of such MA include a DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted or inserted in the protein.
  • the DNA of the present invention includes natural or isolated / purified genomic DNA, cDNA, and chemically synthesized DNA.
  • Preparation of genomic DNA and cDNA can be performed by a person skilled in the art using conventional means.
  • genomic DNA is extracted from an organism having a gene encoding a protein represented by any one of SEQ ID NOs: 2 to 28, and a genomic library (plasmid, phage , Cosmids, BACs, PACs, etc. can be used), developed and developed using a probe prepared based on the DNA encoding the protein. It can be prepared by performing knee hybridization or plaque hybridization.
  • cDNA for example, a cDNA is synthesized based on mRNA extracted from an organism having a gene encoding the protein, inserted into a vector such as ⁇ ZAP to prepare a cDNA library, and developed. Then, it can be prepared by performing colony hybridization or plaque hybridization as described above, or by performing PCR.
  • DNA that can be isolated by the hybridization technique or the PCR technique and that hybridizes with the DNA consisting of the base sequence described in any of the odd-numbered SEQ ID NO: 27 or 27 is also used. It is included in the DNA of the present invention.
  • a hybridization reaction is preferably performed under stringent conditions.
  • the stringent hybridization conditions refer to conditions of 6 M urea, 0.4% SDS, 0.5 XSSC or a hybridization condition of a stringency equivalent thereto. Under conditions of higher stringency, for example, 6M urea, 0.4% SDS, and 0.1 ⁇ SSC, it is expected that more homologous DNA can be isolated.
  • the DNA thus isolated is considered to have high homology at the amino acid level to the amino acid sequence described in any of the even-numbered SEQ ID NOs: 2 to 28.
  • High homology refers to sequence identity of at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the entire amino acid sequence.
  • the amino acid sequence and nucleotide sequence identity can be determined by the algorithm BLAST (Proc. Natl. Acad. Sei. USA, 1990, 87, 2264-2268. Karl in, S. & Al tscliul, SF. Natl. Acad. Sei. USA, 1993, 90, 5873.).
  • a program called BLASTN or BLASTX based on the BLAST algorithm.
  • a program has been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.).
  • the present invention also provides a DNA encoding an antisense RNA complementary to a transcript of the DNA of the present invention, and a DNA encoding an RNA having a lipozyme activity that specifically cleaves the transcript of the DNA of the present invention. .
  • the above-mentioned DNA can be used, for example, to produce a plant resistant to bacterial blight of rice with suppressed expression of the DNA of the present invention.
  • a DNA for suppressing the expression of the DNA of the present invention is inserted into an appropriate vector and introduced into a plant cell. Then, the transformed plant cells thus obtained are regenerated.
  • "Suppression of expression of the DNA of the present invention” includes suppression of gene transcription and suppression of translation into Z or protein. It also includes a decrease in expression as well as a complete cessation of DNA expression.
  • the action of the antisense nucleic acid to suppress the expression of the target gene has several factors as follows. In other words, inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally formed by RNA polymerase, inhibition of transcription by formation of a hybrid with RNA that is undergoing synthesis Inhibition of splicing by hybridization at the junction of intron and exon; inhibition of splicing by hybridization with spliceosome-forming sites; inhibition of nuclear-to-cytoplasmic translocation by hybridization with mRNA; capping; (A) Splicing inhibition by the formation of a hybrid with the additional site, translation initiation inhibition by the formation of a hybrid with the translation initiation factor binding site, translation inhibition by the formation of a hybrid with the liposomal binding site near the start codon, translation region of mRNA And hybrids with polysome binding sites Outgrowth inhibitory peptide chain by forming, and the like gene expression inhibition by Haipuriddo forming the interaction site with our and
  • antisense nucleic acids suppress target gene expression by inhibiting various processes such as transcription, splicing, and translation.
  • Hirashima and Inoue Shinsei Kagaku Kenkyusho Lecture Nucleic acid IV gene replication and expression (Japan Biochemical Society, Tokyo Chemical Dojin) p.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.
  • designing an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of a gene is considered to be effective in inhibiting translation of the gene.
  • a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
  • the antisense DNA used in the present invention includes not only the translated region of the gene but also the DNA containing the antisense sequence of the untranslated region.
  • the antisense DNA to be used is ligated downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is ligated on the 3 'side.
  • the DNA thus prepared can be transformed into a desired plant by using a known method. Wear.
  • the sequence of the antisense DNA is preferably a sequence that is complementary to the pathogenic gene of rice leaf blight of the present invention or a part thereof, but is completely complementary as long as gene expression can be effectively suppressed. It does not have to be.
  • the transcribed RNA has preferably 90% or more, and most preferably 95% or more complementarity to the transcript of the target gene.
  • the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more.
  • the length of commonly used antisense DNA is shorter than 5 kb, preferably shorter than 2.5 kb.
  • Liposomes refer to RNA molecules that have catalytic activity. There are various types of liposomes that have various activities.Research focused on lipozymes as enzymes that cleave RNA has made it possible to design liposomes that cleave RNA in a site-specific manner. Was.
  • Lipozymes include those with a size of 400 nucleotides or more, such as Daphlepe I intron type and Ml RNA contained in RNase P.Hammerhead type ⁇ Hairpin type has an active domain of about 40 nucleotides There are also others (Makoto Koizumi and Eiko Otsuka: Protein Nucleic Acid Enzyme, 35: 2191, 1990).
  • the self-cleaving domain of the hammerhead lipozyme cleaves the 3 'side of C15 in the sequence G13U14C15, but its activity requires base pairing between U14 and A9, and A15 instead of C15.
  • U15 can also be cleaved (Koizumi M, et al: FEBS Lett 228: 228, 1988).
  • Hairpin type liposomes are also useful for the purpose of the present invention.
  • This lipozyme is found, for example, in the minus strand of satellite RNA of evening bacillus spot virus (Buzayan JM: Nature 323: 349, 1986). It has been shown that a hairpin-type lipozyme can also produce a target-specific NA-cleaved lipozyme (Kikuchi Y & Sasaki N: Nucl Acids Res 19: 6751, 1991; Kikuchi, Hiroshi: Chemistry and biology 30: 112, 1992) .
  • the lipozyme designed to cleave the target is ligated to a promoter and transcription termination sequence, such as the cauliflower mosaic virus 35S promoter, so that it is transcribed in plant cells.
  • a promoter and transcription termination sequence such as the cauliflower mosaic virus 35S promoter
  • lipozyme activity may be lost.
  • the transcripted ribozyme contains
  • another trimming ribozyme that acts on cis on the 5 'or 3' side of the lipozyme portion (Taira K, et al: Protein Eng 3: 733) , 1990, Dzianott AM & Bujarski JJ: Proc Natl Acad Sc i USA 86: 4823, 1989, Gross ans CA & Cech T: Nucl Acids Res 19: 3875, 1991, Taira K, et al: Nucl Acids Res 19: 5125, 1991).
  • the DNA of the present invention can be used, for example, for preparing a recombinant protein.
  • the DNA of the present invention is inserted into an appropriate expression vector, the vector is introduced into appropriate cells, and the expressed protein is purified by culturing the transformed cells. I do.
  • the recombinant protein can be expressed as a fusion protein with another protein, for example, to facilitate purification. For example, a method for preparing a fusion protein with maltose binding protein using E.
  • the host cell is not particularly limited as long as it is a cell suitable for the expression of the recombinant protein.
  • yeast various animal and plant cells, insect cells, and the like can be used.
  • Various methods known to those skilled in the art can be used for introducing a vector into a host cell.
  • the recombinant protein expressed in the host cell can be purified and recovered from the host cell or a culture supernatant thereof by a method known to those skilled in the art.
  • affinity purification can be easily performed.
  • the protein encoded by the DNA of the present invention thus produced is also included in the present invention.
  • an antibody that binds to the protein can be prepared.
  • the antibody can be used for a method for diagnosing whether or not the rice is infected with the bacterial leaf blight described below.
  • the polyclonal antibody is, for example, a purified protein of the present invention or one thereof.
  • a part of the peptide can be immunized to immunized animals such as egrets, and after a certain period of time, blood can be collected and prepared from serum from which blood bladders have been removed.
  • the monoclonal antibody is obtained by fusing antibody-producing cells of the animal immunized with the above-mentioned protein or peptide with bone tumor cells to produce a single-clonal cell (hybrid-ma) that produces an antibody that is used as an eye-opening stump. It can be prepared by isolating and obtaining an antibody from the cells. The antibody thus obtained can be used for purification and detection of the protein of the present invention.
  • the antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and the fragmentability of these antibodies.
  • the present invention provides a vector comprising the above DNA, a transformed meniscus which carries the vector, a transformed cell which is a plant cell, a transformed plant comprising the transformed cell, a transformed plant comprising the transformed cell, Provided are a transformed plant that is a progeny or a clone, and a propagation material of the transformed plant.
  • the introduction of the DNA or nucleic acid of the present invention into plant cells can be carried out by those skilled in the art by known methods, for example, the agrobacterium method, the electroporation method (elect-portation method), and the particle gun method. it can.
  • the agrobacterium method for example, the method of Nagel et al. (Microbiol. Lett., 1990, 67, 325.) is used.
  • the recombinant vector is transformed into bacterium agrobacterium, and the transformed agrobacterium is then introduced into plant cells by a known method such as a leaf disk method.
  • the vector contains an expression promoter so that the DNA of the present invention is expressed in the plant, for example, after introduction into the plant.
  • the DNA of the present invention is located downstream of the promoter, and furthermore, one minute and one minute is located downstream of the DNA.
  • the recombinant vector used for this purpose is appropriately selected by those skilled in the art according to the method of introduction into the plant or the type of the plant.
  • Examples of the above promoter include CaMV35S derived from cauliflower mosaic virus, corn ubiquitin promoter (Japanese Patent Publication No. 2-79983), and the like.
  • the above-mentioned one-minute-one-one-one-one can be exemplified by a terminator derived from a cauliflower mosaic virus or a terminator-derived one derived from a nopaline synthase gene. If it is one, it is not limited to these.
  • the plant into which the DNA or nucleic acid of the present invention is introduced may be an explant, or cultured cells may be prepared from these plants and introduced into the obtained cultured cells.
  • the "plant cell” of the present invention includes, for example, plant cells such as leaves, roots, stems, flowers, and scutellum in seeds, virulent, suspension cultured cells, and the like.
  • the above-mentioned recombinant vector contains an appropriate selection marker gene and a selection marker marker gene. It is preferably introduced into a plant cell together with the containing plasmid vector.
  • Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene, which is resistant to the antibiotic hygromycin, the neomycin phosphotransferase, which is resistant to kinamicin or genyumycin, and the herbicide phosphinothricin And an acetyltransferase gene that is resistant to acetyl.
  • the plant cells into which the recombinant vector has been introduced are placed on a known selection medium containing an appropriate selection agent and cultured according to the type of the introduced selection marker gene. As a result, transformed plant culture cells can be obtained.
  • a plant is regenerated from the transformed cell into which the DNA or nucleic acid of the present invention has been introduced.
  • Plant regeneration 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, 1995, 100, 1503-1507.).
  • a method for producing a transformed plant is to introduce a gene into protoplasts using polyethylene glycol to regenerate the plant (Indian rice varieties are suitable) (Dat ta, S K. et al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.), 1995, 66-74. Gene transfer into toplasts to regenerate plants (suitable for Japanese rice varieties) (Mi.
  • the plant regenerated from the transformed cells is then cultured in a conditioned medium. Thereafter, when the regenerated acclimated plant is cultivated under normal cultivation conditions, a plant is obtained, which can be matured and ripened to obtain a seed.
  • the presence of the introduced foreign DNA or nucleic acid in the transformed and cultivated transformed plant is determined by a known PCR method or Southern hybridization method, or by the base of the nucleic acid in the plant. It can be confirmed by analyzing the sequence. In this case, extraction of DNA or nucleic acid from the transformed plant can be performed according to the known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
  • an amplification reaction is performed using the nucleic acid extracted from the regenerated plant as type III as described above.
  • the nucleic acid of the present invention is DNA
  • a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA is used as a primer, and the resulting mixture is mixed in a reaction solution.
  • DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the MA sequence of the present invention.
  • the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, each amplified DNA fragment is fractionated, and it is confirmed that the DNA fragments correspond to the DNA of the present invention. It is possible to do.
  • progeny can be obtained from the plant by sexual or asexual reproduction.
  • a propagation material eg, seeds, fruits, cuttings, tubers, tubers, strains, calli, protoplasts, etc.
  • the present invention also provides an oligonucleotide comprising at least 15 contiguous base sequences in any of the odd-numbered base sequences of SEQ ID NOs: 1 to 27 or a complementary sequence thereof.
  • acquisition sequence refers to the other sequence for one strand of a double-stranded DNA consisting of A: T, G: C base pairs.
  • the nucleotide sequence is not limited to a completely complementary sequence in at least 15 contiguous nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95% or more.
  • DNA is useful as a probe for detecting or isolating the DNA of the present invention, and as a primer for performing amplification.
  • the present invention provides an oligonucleotide having a chain length of at least 15 bases, which specifically hybridizes with a DNA consisting of the base sequence represented by any of the odd-numbered SEQ ID NOs: 1 to 27.
  • the U-nucleotide specifically hybridizes to the DNA of the present invention.
  • “specifically hybridizes” means under ordinary hybridization conditions, preferably under stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York). , USA, 2nd edition 1989), does not significantly cause cross-hybridization with DNA encoding other proteins.
  • the oligonucleotide need not be completely complementary to the DNA of the present invention, if specific hybridization is possible.
  • Oligonucleotides that hybridize to the DNA of the present invention and have a chain length of at least 15 nucleotides can be used as probes and primers for detecting the DNA of the present invention.
  • the oligonucleotide is used as a primer, its 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 a part of the DNA of the present invention.
  • the probe is not particularly limited as long as it specifically hybridizes to the MA of the present invention.
  • the probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp or more.
  • the oligonucleotide of the present invention is used as a probe, it is preferable that the oligonucleotide is appropriately labeled before use. Labeling can be performed by using T4 polynucleotide kinase to phosphorylate the 5 'end of the oligonucleotide with 32 P, or by random DNA oligonucleotide using a DNA polymerase such as Klenow enzyme. A method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin or the like using the primers as a primer (random prime method, etc.) can be exemplified.
  • the present invention provides a method for diagnosing whether or not rice is infected with the bacterial wilt of rice.
  • a nucleic acid sample is prepared from a rice plant or a part thereof.
  • detecting the presence of MA of the present invention or its expression product in the nucleic acid sample is detected, it is determined that the rice to be diagnosed is infected with the bacterial wilt of rice.
  • the “part thereof” refers to, for example, a plant tissue, a cell, a cell extract, or the like.
  • One embodiment of the diagnostic method of the present invention uses a primer or a probe to detect a DNA encoding a rice bacterial wilt disease protein.
  • a probe group As the primer, the above oligonucleotide of the present invention can be used.
  • the present inventors have actually hybridized to the gene of the present invention, and succeeded in detecting the bacterial leaf blight of a test rice in a specific white stump using a PCR primer set capable of amplifying the gene. Therefore, the above-mentioned oligonucleotide of the present invention can be suitably used for the diagnostic method of the present invention.
  • a set of primers described in Example 5 described below can be shown, but is not limited thereto.
  • Those skilled in the art can appropriately design a set of primers capable of amplifying the DNA of the present invention based on the base sequence described in any one of the odd numbers of SEQ ID NOS: 1 to 28.
  • a probe capable of detecting the DNA of the present invention based on the base sequence described in any of the odd numbers of SEQ ID NO: 1 and 28. That power S is possible.
  • Primers and probes may be labeled as necessary. Examples of the label include a radiolabel. Primers or probes that can be used in the diagnostic method of the present invention are also included in the present invention.
  • the diagnostic method of the present invention includes, for example, a method of preparing a nucleic acid sample from a rice plant or a part thereof suspected of being infected with the bacterial leaf blight fungus, and a polymerase chain reaction (PCR) method using the above primer, or It can be carried out by a Northern plotting method or the like using the above-mentioned probe.
  • PCR polymerase chain reaction
  • Another aspect of the diagnostic method of the present invention is a method for diagnosing a test rice using an antibody as an index based on the presence or absence of a protein encoded by the pathogenic gene of rice leaf blight of the present invention. It is.
  • the antibody used for this diagnosis can be prepared, for example, as described above.
  • the antibody may be labeled if necessary. Examples of the label include an enzyme label.
  • the target protein may be detected by labeling with a substance that binds to the antibody, such as protein A.
  • a test sample is prepared from a rice plant or a part thereof suspected of having been infected with the bacterial leaf blight fungus, It can be performed by ELISA or Western blotting using antibodies.
  • FIG. 1 is a diagram showing a gene map of hrp and peripheral regions.
  • FIG. 2 shows the sequences of SEQ ID NO: 19 and SEQ ID NO: 21 used for preparing a set of PCR primers.
  • the portion where the sequence is indicated by a black frame indicates the sequence of each primer.
  • FIG. 3 is a photograph showing the results of PCR detection of various ⁇ 7 ⁇ iM3 ⁇ 47 bacteria using a primer set designed based on the nucleotide sequence of SEQ ID NO: 19. Bacterial names in each lane are shown below the photograph.
  • FIG. 3 is a diagram comparing the structures of the hpaB-hrpF regions between X. oryzae pv. Oryzae, I. axo lord odis pv. Citri, and yohi campestris pv. BEST MODE FOR CARRYING OUT THE INVENTION
  • BAC bacterial art if icial chromosome
  • the present inventors prepared a BAG library for about 16 genomes with an average insert length of about 100 to 120 kb (Ocliiai, H., Inoue, ⁇ ., Hasebe, A. and Kaku, H. (2001). Cons compilt ion and character izat ion of a Xan thomonas oryzae pv. oryzae bacterial artificial chromosome library. FEMS Microbiol. Lett. 200: 59-65).
  • phytopathogenic bacteria can cause disease in plants: the establishment of host infection by Shinkanzin Z and the release / development of pathogens.
  • the genes included in the hrp gene class are not only those encoding the pathogenic factor secretory mechanism (typelll), which plays an important role in the process of establishing host recognition infection, but also those that are not. It is also known to be the most important pathogenic genes in plant pathogenic bacteria (Bonas, U., Schulte, R., Fenselau, S., Minsavage, GV , Staskawicz, BJ, and Stall, RE (1991). Isolation of a gene cluster from Xanthomonas campestris pv.vesicatoria that determines pathogenicty and the hypersensitive response on pepper and tomato. Mol. Plant-Microbe Interact.
  • nucleotide sequence of the BAC clone having the hrp gene cluster isolated by PCR screening was determined by the shotgun method.
  • the determined region is about 220 kb including about 30 kb of the hrp gene cluster.
  • Figure 1 shows the results of gene prediction in this region. A total of 171 genes were predicted, including all 24 genes that make up the known hrp gene cluster in the XantJw genus.
  • transposase homologue insert sequence: IS
  • IS transposase homologue
  • PCR was carried out using the primer set shown in FIG. 2A on the rice ⁇ and various Xantho nasae bacteria belonging to the same genus as the rice wilt fungus under the following conditions.
  • primer sets of 20 sense nucleotides and antisense nucleotides were prepared from SEQ ID NOs: 19 and 21, respectively.
  • Sense primer 5'-ATGATCTTGGAATCGCACAA (SEQ ID NO: 29)
  • Antisense primer 5'-TCATGATGCCACCTCCTGCG (SEQ ID NO: 30) PCR primer in SEQ ID NO: 21
  • Sense primer 5'-ATGAAACTCTCCGGCGGTAT (SEQ ID NO: 31)
  • Antisense primer 5'-TCATGCTCGCCCGCTTTGCC (SEQ ID NO: 32)
  • the PCR reaction conditions were as follows: initial denaturation 94 ° C for 2 minutes, denaturation 94 15 seconds, annealing 55 ° C 30 seconds, extension reaction 72 2 minutes 30 cycles, and final extension reaction 72 ° C 7 minutes .
  • the PCR amplification product was detected by agarose dull electrophoresis and detected after ethidium bromide staining. As a result, it was detected only in lanes A13, A16, B15, B16 and C15 on which the PCR amplification product of rice bacterial blight fungus DNA type II was placed (FIG. 3).
  • the pathogenic gene of Bacterial blight of the rice plant identified by the present invention has no significant homology with the known nucleotide sequence and amino acid sequence, a specific probe or PCR primer set using the nucleotide sequence is prepared. Then, it can be used for simple detection of rice leaf blight by PCR.

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Abstract

La présente invention concerne des gènes pathogènes et des ADN relatifs qui sont clonés à partir de Xanthomonas oryzae pv. oryzae, ainsi que 14 nouveaux gènes relatifs au pouvoir pathogène de x. oryzae pv. oryzae. L'utilisation de la présence/absence des ADN de ces gènes permet de diagnostiquer de manière pratique et précise une infection à x. oryzae pv. oryzae d'un échantillon de riz.
PCT/JP2003/009922 2002-08-06 2003-08-05 Genes pathogenes de xanthomonas oryzae pv. oryzae et leur utilisation Ceased WO2004013331A1 (fr)

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US8373021B2 (en) 2005-05-26 2013-02-12 National Institute Of Agrobiological Sciences Improving disease resistance in plants by introducing transcription factor gene
CN119881307A (zh) * 2024-12-31 2025-04-25 上海凌恩生物科技有限公司 一种稻黄单胞菌抗体检测试剂盒及其应用

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CN100348724C (zh) * 2005-02-02 2007-11-14 华中农业大学 水稻抗病相关基因OsDR8

Non-Patent Citations (3)

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Title
DA SILVA A.C. ET AL.: "Comparison of the genomes of two xanthomonas pathogens with differing host specificities", NATURE, vol. 417, no. 6887, May 2002 (2002-05-01), pages 459 - 463, XP002261557 *
OCHIAI H. ET AL.: "Construction and characterization of a xanthomonas oryzae pv. oryzae bacterial artificial chromosome library", FEMS MICROBIOL. LETT., vol. 200, no. 1, 2001, pages 59 - 65, XP002974827 *
ZHU W. ET AL.: "Identification of two novel hrp-associated genes in the hrp gene cluster of xanthomonas oryzae pv. oryzae", J. BACTERIOL., vol. 182, no. 7, 2000, pages 1844 - 1853, XP002974828 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8373021B2 (en) 2005-05-26 2013-02-12 National Institute Of Agrobiological Sciences Improving disease resistance in plants by introducing transcription factor gene
CN119881307A (zh) * 2024-12-31 2025-04-25 上海凌恩生物科技有限公司 一种稻黄单胞菌抗体检测试剂盒及其应用

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