WO2010067801A1 - GÈNE DE RÉSISTANCE À LA PYRICULARIOSE CHEZ LE RIZ Pb1 ET SON UTILISATION - Google Patents

GÈNE DE RÉSISTANCE À LA PYRICULARIOSE CHEZ LE RIZ Pb1 ET SON UTILISATION Download PDF

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WO2010067801A1
WO2010067801A1 PCT/JP2009/070552 JP2009070552W WO2010067801A1 WO 2010067801 A1 WO2010067801 A1 WO 2010067801A1 JP 2009070552 W JP2009070552 W JP 2009070552W WO 2010067801 A1 WO2010067801 A1 WO 2010067801A1
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dna
seq
plant
present
set forth
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Japanese (ja)
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長生 林
博志 高辻
晴彦 井上
恭宏 加藤
岳人 船生
雅毅 城田
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独立行政法人農業生物資源研究所
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Priority to JP2010542109A priority Critical patent/JP5618301B2/ja
Publication of WO2010067801A1 publication Critical patent/WO2010067801A1/fr

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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
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8282—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 fungal resistance

Definitions

  • the present invention relates to a rice panicle field resistance gene.
  • the blast field resistance derived from the indica variety “Modan” is characterized by high resistance to panicle and long-term stable resistance.
  • the resistance gene Pb1 governing this was first identified as a panicle field resistance gene (Non-patent Document 1).
  • Pb1 has the effect of field resistance, such as the formation of diseased lesions, but it is controlled by a single main gene and is easy to introduce by normal breeding, so it is used as a practical resistance gene source for breeding. It has been.
  • An object of the present invention is to provide a novel DNA by isolating and identifying the Pb1 gene.
  • the Pb1 gene revealed by the present invention is a CC-NBS-LRR type resistance gene and has a novel sequence.
  • the gene has characteristics of adult resistance unlike the rice blast resistance gene reported so far, and it has been clarified that the rice blast field resistance with high practicality can be imparted to rice by modifying it.
  • the present invention relates to a novel rice panicle field resistance gene Pb1, more specifically, [1] DNA according to any one of the following (a) to (d), which encodes a protein having a blast resistance function, (A) DNA comprising the base sequence set forth in SEQ ID NO: 1 (B) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • the DNA according to [1], wherein the blast is rice blast [3] a protein encoded by the DNA according to [1] or [2], [4] A vector comprising the DNA of [1] or [2], [5] A host cell into which the vector according to [4] has been introduced, [6] A plant cell into which the vector according to [4] is introduced, [7] A transformed plant comprising the plant cell according to [6], [8] A transformed plant that is a descendant or clone of the transformed plant according to [7], [9] A propagation material for the transformed plant according to [7] or [8], [10]
  • FIG. 1 It is a figure which shows the genetic map of Pb1 area
  • the figure below schematically shows the structure of the protein encoded by the gene (Pb1) of the present invention. It is a figure (linkage map) which shows typically the position of the molecular marker which exists near the gene (Pb1) of the present invention. It is the figure which looked at the resistance with respect to blast and the expression level of Pb1 gene. Resistance to blast was low at 2L and 6L, but increased from around 10L. And it became strongest at the end. On the other hand, the expression level of Pb1 increased with the growth stage and increased to 0.2% of ubiquitin in the leaf. In the ear, it was even higher.
  • the present inventors identified a panicle field resistance gene (this gene may be referred to as “the gene of the present invention” in the present specification).
  • the gene of the present invention is a gene having a blast resistance function and may be referred to as “Pb1 gene” or “P15 gene”.
  • the DNA sequence of the gene of the present invention is described in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene (in this specification, sometimes referred to as “the protein of the present invention”) is described in SEQ ID NO: 2.
  • sequence of the promoter region located upstream of the gene of the present invention is described in SEQ ID NO: 3. Furthermore, 3′-UTR of the gene of the present invention is described in SEQ ID NO: 4.
  • the gene of the present invention is not necessarily limited to DNA consisting of sequences specifically described in the sequence listing.
  • the protein of the present invention is not necessarily limited to a protein having an amino acid sequence specifically described in the sequence listing.
  • proteins other than the above have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequences described in the sequence listing, for example.
  • the protein which has the function (for example, blast resistance function) which the protein of this invention has is contained in the protein of this invention.
  • Pb1 ′ which is one amino acid different from Pb1
  • the protein is, for example, a protein comprising an amino acid sequence in which one or more amino acids are added, deleted, substituted, or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and the number of normally changing amino acids is 30 amino acids. Within 10 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.
  • the gene of the present invention includes, for example, an endogenous gene in another plant (such as a homolog of a rice Pb1 gene of another plant) corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1.
  • the endogenous DNA of other plants corresponding to the DNA consisting of the base sequence described in SEQ ID NO: 1 generally has high homology with the DNA described in SEQ ID NO: 1.
  • High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, or 96%, 97%, 98% or 99% or more).
  • mBLAST algorithm Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7).
  • stringent conditions for example, “2 ⁇ SSC, 0.1% SDS, 50 ° C.”, “2 ⁇ SSC, 0.1% SDS, 42 ° C.”, “1 ⁇ SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 ⁇ SSC, 0.1% SDS, 65 ° C.”, “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.” and “0.2 ⁇ SSC, 0.1% SDS, 65 ° C.” Can do.
  • the protein of the present invention can be prepared not only as a natural protein but also as a recombinant protein using genetic recombination technology.
  • Natural proteins can be prepared, for example, by a method using affinity chromatography using an antibody against the protein of the present invention to an extract of a cell (tissue) that is considered to express the protein of the present invention. is there.
  • a recombinant protein can be prepared by culturing cells transformed with a DNA encoding the protein of the present invention.
  • a DNA according to any one of the following (a) to (d) encoding a protein having a blast resistance function.
  • A DNA comprising the base sequence set forth in SEQ ID NO: 1
  • B DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • C DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1
  • D DNA encoding 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: 2
  • a protein encoded by the above DNA is also included in the present invention.
  • blast resistance usually means that rice has an increased resistance to blast, making it less susceptible to blast.
  • the “rice blast” in the present invention is preferably rice blast.
  • by overexpressing the protein (Pb1) of the present invention it exhibits resistance to leaf blast. Therefore, “leaf blast” in the present invention includes “leaf blast”.
  • the protein in the present specification means a polymer composed of a plurality of amino acids, and the length of the amino acids is not particularly limited. Therefore, the protein of the present invention includes so-called “polypeptides” and “oligopeptides”.
  • the proteins of the present invention include both those that are not modified from the naturally occurring state and those that are modified.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond , Crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cystine formation, pyroglutamate formation, formylation, ⁇ -carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, Examples include methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, ubiquitination, and the like.
  • the polypeptide of the present invention can be produced by a general chemical synthesis method according to its amino acid sequence, and this method includes peptide synthesis methods by a normal liquid phase method and a solid phase method. More specifically, the peptide synthesis method includes a stepwise elongation method in which each amino acid is sequentially synthesized one by one based on the amino acid sequence information to extend the chain, and a fragment consisting of several amino acids is synthesized in advance. Then, a fragment condensation method in which each fragment is subjected to a coupling reaction is included, and any method may be used for the synthesis of the protein of the present invention.
  • the condensation method used in such a peptide synthesis method can also be performed according to various methods. Specific examples thereof include an azide method, a mixed acid anhydride method, a DCC method, an active ester method, an oxidation-reduction method, a DPPA (diphenylphosphoryl azide) method, and a Woodward method.
  • solvents that can be used in these various methods, commonly used solvents can be appropriately used. Examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and mixed solvents thereof.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ethyl acetate ethyl acetate and the like
  • amino acids that do not participate in the reaction and carboxyl groups in the peptide are generally esterified, for example, lower alkyl esters such as methyl ester, ethyl ester, tertiary butyl ester, such as benzyl ester, P- It can be protected as methoxybenzyl ester, P-nitrobenzyl ester aralkyl ester and the like.
  • amino acid having a functional group in the side chain for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like, but such protection is not necessarily essential.
  • the protein of the present invention that can be obtained as described above can be obtained by, for example, ion exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method according to a conventional method. Purification can be appropriately performed according to a method widely used in the field of peptide chemistry.
  • the protein of the present invention is a gene that is synthesized, for example, by synthesizing the polypeptide shown in SEQ ID NO: 2 or the DNA nucleic acid molecule shown in SEQ ID NO: 1, and then introducing it into an appropriate expression vector. It can also be obtained by engineering methods.
  • the protein of the present invention includes, for example, a protein (polypeptide) functionally equivalent to the Pb1 protein.
  • “functionally equivalent” means that the target protein has the same (equivalent) biological or biochemical function (activity) as the protein of the present invention. Examples of such a function include a function of imparting blast resistance to a plant body.
  • Whether the target protein (polypeptide) has a biological or biochemical function (activity) equivalent to that of the protein of the present invention is determined, for example, by blast in a plant in which the protein is expressed. It can be determined by evaluating the resistance. Specifically, it can be evaluated by inoculating a test plant with a blast fungus and observing whether or not it exhibits blast.
  • a polypeptide functionally equivalent to the polypeptide can be prepared.
  • amino acid mutations in the polypeptide may occur naturally.
  • the amino acid sequence of Pb1 protein (SEQ ID NO: 2) identified by the present inventors has an amino acid sequence in which one or more amino acid sequences are mutated.
  • a polypeptide functionally equivalent to the polypeptide is included in the protein (polypeptide) of the present invention.
  • the number of amino acids to be mutated in the mutant is not limited as long as the function of the protein of the present invention is maintained, but is usually within 15 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids. More preferably 1 to 4 amino acids.
  • amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain.
  • amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids with amino acids (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino groups with base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses indicate single letter amino acids).
  • a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by another amino acid of one or more amino acid residues to a certain amino acid sequence can maintain its biological function (activity).
  • It is possible that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by another amino acid of one or more amino acid residues to a certain amino acid sequence can maintain its biological function (activity).
  • already known Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. & Smith, M. Nucleic Acids Research (1982 10, 6487-650087, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413) .
  • polypeptide comprising a sequence in which amino acids are appropriately modified based on these amino acid sequences, It is possible to evaluate whether or not the polypeptide has the above-described function and to select the protein (polypeptide) of the present invention as appropriate.
  • the protein having a plurality of amino acid residues added to the amino acid sequence of the protein of the present invention includes a fusion polypeptide containing these proteins.
  • a fusion polypeptide is a fusion of these polypeptides with other peptides or polypeptides.
  • a method for producing a fusion polypeptide is such that the DNA (eg, SEQ ID NO: 1) encoding the protein of the present invention (eg, SEQ ID NO: 2) and the DNA encoding another peptide or polypeptide match in frame.
  • SEQ ID NO: 1 encoding the protein of the present invention
  • SEQ ID NO: 2 DNA encoding another peptide or polypeptide match in frame.
  • Other peptides or polypeptides that are subjected to fusion with the protein of the present invention are not particularly limited.
  • Examples of other peptides subjected to fusion with the polypeptide of the present invention include GST (glutathione-S-transferase), immunoglobulin constant region, ⁇ -galactosidase, MBP (maltose binding protein) and the like.
  • GST glutthione-S-transferase
  • immunoglobulin constant region ⁇ -galactosidase
  • MBP maltose binding protein
  • the present invention includes proteins encoded by DNA that hybridizes with DNA encoding the protein of the present invention and functionally equivalent to the protein of the present invention.
  • proteins include rice and other plant homologues.
  • Hybridization conditions for isolating a DNA encoding a protein functionally equivalent to the protein of the present invention can be appropriately selected by those skilled in the art.
  • Examples of hybridization conditions include low stringency conditions.
  • Low stringent conditions are, for example, conditions of 42 ° C., 0.1 ⁇ SSC, and 0.1% SDS, and preferably 50 ° C., 0.1 ⁇ SSC, and 0.1% SDS in washing after hybridization.
  • More preferable hybridization conditions include highly stringent conditions. Highly stringent conditions are, for example, conditions of 65 ° C., 5 ⁇ SSC and 0.1% SDS. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. .
  • the protein of the present invention is encoded using gene amplification technology (PCR) (CurrentPCRprotocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4) instead of hybridization.
  • PCR gene amplification technology
  • a primer is designed based on a part of the DNA to be isolated (for example, SEQ ID NO: 1), a DNA fragment highly homologous to the DNA encoding the protein identified by the present inventors is isolated, and the DNA It is also possible to obtain a protein functionally equivalent to the protein identified by the present inventors.
  • the protein of the present invention may be in the form of a “mature” polypeptide or may be part of a larger polypeptide, such as a fusion polypeptide.
  • the protein of the present invention may contain a leader sequence, a pro sequence, a sequence useful for purification, such as multiple histidine residues, or an additional sequence ensuring stability during recombinant production.
  • a protein functionally equivalent to the protein of the present invention encoded by DNA isolated by these hybridization techniques and gene amplification techniques is usually higher in amino acid sequence than the protein of the present invention (for example, SEQ ID NO: 2).
  • the protein of the present invention includes a protein that is functionally equivalent to the Pb1 protein and has high homology with the amino acid sequence of the protein.
  • High homology is usually at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% (eg 96%) at the amino acid level. Above, 97% or higher, 98% or higher, 99% or higher).
  • To determine protein homology follow the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730). Good.
  • the protein of the present invention can be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art.
  • a DNA encoding the protein of the present invention for example, the DNA described in SEQ ID NO: 1 is incorporated into an appropriate expression vector and introduced into an appropriate host cell. After recovering the transformant and obtaining the extract, it is subjected to chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against the protein of the present invention is immobilized on the column, or further It is possible to purify and prepare by combining a plurality of these columns.
  • the protein of the present invention when expressed in a host cell (for example, plant cell or microbial cell) as a fusion polypeptide with glutathione S-transferase protein or as a recombinant polypeptide to which a plurality of histidines are added.
  • the expressed recombinant polypeptide can be purified using a glutathione column or a nickel column. After purification of the fusion polypeptide, if necessary, a region other than the target polypeptide in the fusion polypeptide can be cleaved with thrombin or factor Xa and removed.
  • an affinity column in which an antibody having an affinity for the protein of the present invention is bound to an extract of a tissue or a cell expressing the protein of the present invention is applied to a method well known to those skilled in the art. And can be isolated by purification.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the protein of the present invention can be used, for example, for production of an antibody that recognizes the protein of the present invention.
  • the DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA. Moreover, as long as it can code the protein of this invention, DNA which has the arbitrary base sequences based on the degeneracy of a genetic code is contained.
  • the DNA of the present invention can be prepared by methods known to those skilled in the art. For example, it is prepared by preparing a cDNA library from cells expressing the protein of the present invention and performing hybridization using a part of the DNA of the present invention (for example, the nucleotide sequence described in SEQ ID NO: 1) as a probe. it can.
  • the cDNA library may be prepared by the method described in the literature (Sambrook, J. et al., Molecular Cloning, Cold Harbor Laboratory Press (1989)), or a commercially available DNA library may be used. Good.
  • RNA is prepared from cells expressing the protein of the present invention
  • cDNA is synthesized by reverse transcriptase
  • oligo DNA is prepared based on the DNA of the present invention (for example, the base sequence described in SEQ ID NO: 1). It can also be prepared by synthesizing and performing a PCR reaction using this as a primer to amplify a cDNA encoding the protein of the present invention.
  • genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.
  • mRNA is isolated from cells, tissues, and organs that express the protein of the present invention.
  • known methods such as guanidine ultracentrifugation (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. and Sacchi, N. , Anal. Biochem. (1987) 162, 156-159), etc.
  • mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia).
  • mRNA can be directly prepared by using QuickPrep® mRNA® Purification® Kit® (Pharmacia) ®.
  • CDNA is synthesized from the obtained mRNA using reverse transcriptase.
  • Synthesis of cDNA can also be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation).
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation).
  • 5'-Ampli IN FINDER RACE Kit ⁇ (manufactured by Clontech)
  • 5'-RACE method polymerase chain reaction; PCR) (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) And amplification can be performed.
  • a target DNA fragment is prepared from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector.
  • the base sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.
  • a base sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucelic Acids Research ( 1981) 9, r43-74).
  • the DNA of the present invention can be modified by a commercially available kit or a known method. Examples of modifications include digestion with restriction enzymes, insertion of synthetic oligonucleotides and appropriate DNA fragments, addition of linkers, insertion of start codon (ATG) and / or stop codon (TAA, TGA, or TAG). .
  • the present invention also provides a vector into which the DNA of the present invention has been inserted.
  • the vector of the present invention includes a vector for expressing the DNA of the present invention in a plant cell in addition to the above-mentioned vector used for production of a recombinant protein in order to produce a transformed plant body.
  • a vector preferably contains a promoter sequence that can be transcribed in plant cells and a terminator sequence including a polyadenylation site necessary for stabilization of the transcript.
  • the vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells.
  • plant cell includes various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, callus and the like.
  • the vector of the present invention may contain a promoter for constitutively or inducibly expressing the protein of the present invention.
  • promoters for constant expression include cauliflower mosaic virus 35S promoter, rice actin promoter, corn ubiquitin promoter, and the like.
  • the promoter preferably includes a promoter consisting of the DNA represented by SEQ ID NO: 3 existing upstream of the Pb1 gene.
  • SEQ ID NO: 3 existing upstream of the Pb1 gene.
  • a vector having a structure in which the gene (Pb1) of the present invention is operably linked to a promoter capable of overexpression can be exemplified.
  • a corn ubiquitin promoter and the like can be preferably shown.
  • the present invention includes the DNA described in any of (a) and (b) below and having promoter activity.
  • the vector of the present invention is also useful for retaining the DNA of the present invention in a host cell or for expressing the protein of the present invention.
  • the DNA in the present invention is usually carried (inserted) in an appropriate vector and introduced into a host cell.
  • the vector is not particularly limited as long as it can stably hold the inserted DNA.
  • Escherichia coli is used as a host, a pBluescript vector (Stratagene) is preferred as a cloning vector, but commercially available.
  • a pBluescript vector (Stratagene) is preferred as a cloning vector, but commercially available.
  • An expression vector is particularly useful when a vector is used for the purpose of producing the protein of the present invention.
  • the expression vector is not particularly limited as long as it is a vector that expresses the polypeptide in vitro, in E. coli, in cultured cells, or in individual plants.
  • pBEST vector manufactured by Promega
  • E. coli PET vector manufactured by Invitrogen
  • pME18S-FL3 vector GenBank Accession No. AB009864
  • pME18S vector Mol Cell Biol. 8: 466-472 (1988)
  • the nucleic acid of the present invention can be inserted into the vector by a conventional method, for example, by a ligase reaction using a restriction enzyme site.
  • the host cell is not particularly limited, and various host cells can be used depending on the purpose.
  • Examples of cells for expressing the protein of the present invention include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), insect cells (eg, Drosophila S2, Spodoptera SF9), animal cells ( Examples: CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells.
  • Vector introduction into host cells can be performed by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons.Section 9.1-9.9), lipofection method (GIBCO -BRL) and a known method such as a microinjection method.
  • an appropriate secretion signal can be incorporated into the target protein.
  • These signals may be endogenous to the protein of interest or they may be heterologous signals.
  • the protein is collected when the protein of the present invention is secreted into the medium.
  • the protein of the present invention is produced intracellularly, the cell is first lysed and then the protein is recovered.
  • ammonium sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl
  • acid extraction anion or cation exchange chromatography
  • phosphocellulose chromatography phosphocellulose chromatography
  • hydrophobic interaction chromatography phosphocellulose chromatography
  • affinity chromatography hydroxyl
  • Known methods including apatite chromatography and lectin chromatography can be used.
  • the DNA of the present invention is incorporated into an appropriate vector, for example, by electroporation method, Agrobacterium method, liposome method, cationic liposome method, etc. Examples thereof include a method of introducing into a living body.
  • General genetic manipulations such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Molecular Cloning, 5.61-5.63).
  • Administration into a plant body may be an ex-vivo method or an in-vivo method.
  • the method for introducing the DNA of the present invention into a plant body preferably includes a method for introducing a gene via Agrobacterium.
  • an antibody that binds to this can be prepared.
  • a polyclonal antibody can be prepared by immunizing an immunized animal such as a rabbit with the purified protein of the present invention or a partial peptide thereof, collecting blood after a certain period, and removing the blood clot. is there.
  • the monoclonal antibody is obtained by fusing an antibody-producing cell of an animal immunized with the protein or peptide and a bone tumor cell, and isolating a single clone cell (hybridoma) that produces the target antibody.
  • 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. By using these antibodies, it is possible to determine the expression site of the protein of the present invention in a plant body, or to determine whether the plant species express the protein of the present invention.
  • the DNA encoding the protein of the present invention is inserted into an appropriate vector, which is introduced into plant cells, The transformed plant cell thus obtained is regenerated.
  • the proteins isolated by the present inventors have a function of imparting blast resistance to plants. By introducing and expressing the protein of the present invention in any plant species (such as rice varieties), those plants are expressed. It is possible to impart blast resistance to the body. The period required for this transformation is extremely short compared to conventional gene transfer by crossing, and is advantageous in that it does not involve other phenotypic changes.
  • the present invention also includes a method for imparting blast resistance to a plant comprising the step of expressing the DNA of the present invention in cells of the plant.
  • a plant body resistant to blast or its seed is produced. Therefore, the present invention provides a method for producing a rice blast resistant plant or its seed, which comprises the step of expressing the DNA of the present invention in the cells of the plant.
  • the present invention also provides a transformed cell into which the vector of the present invention has been introduced.
  • the cells into which the vector of the present invention is introduced include plant cells for the production of transformed plants in addition to the cells used for the production of recombinant proteins.
  • a plant cell For example, the rice of various varieties is mentioned.
  • the plant cells of the present invention include cultured cells as well as cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.
  • various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used.
  • Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells.
  • methods for producing transformed plants include a method for regenerating plants by introducing genes into protoplasts using polyethylene glycol, a method for regenerating plants by introducing genes into protoplasts using electric pulses, and a particle gun method.
  • Some techniques have already been established, such as a method for directly introducing a gene into a cell and regenerating a plant, and a method for regenerating a plant by introducing a gene via Agrobacterium. Widely used. In the present invention, these methods can be suitably used.
  • the above recombinant vector contains an appropriate selection marker gene or is introduced into a plant cell together with a plasmid vector containing a selection marker gene. It is preferable to do.
  • Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene resistant to the antibiotic hygromycin, the neomycin phosphotransferase gene resistant to kanamycin or gentamicin, and the acetyl resistant to the herbicide phosphinothricin. Examples include transferase genes.
  • Plant 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 transformed plant cell can regenerate the plant body by redifferentiation.
  • the method of redifferentiation differs depending on the type of plant cell. For example, in the case of rice, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be mentioned.
  • the present invention includes a plant cell into which the DNA of the present invention has been introduced, a plant containing the cell, progeny and clones of the plant, and propagation material of the plant, its progeny and clones.
  • the plant thus produced or its seed is expected to be resistant to blast.
  • a method for producing a transformed plant comprising the steps of introducing the DNA or vector of the present invention into a plant cell and regenerating the plant from the plant cell is also included in the present invention.
  • the blast-resistant plant of the present invention or its seeds can be produced by a breeding method.
  • the breeding method include a general breeding method (cross breeding method or the like) characterized by crossing with a variety having the DNA of the present invention. By this method, a plant body resistant to blast or its seed can be produced.
  • a preferred embodiment of the breeding method of the present invention is a method including the following steps (a) and (b). (A) including the step of crossing with a plant having the DNA of the present invention, (B) A step of selecting a plant variant having the DNA
  • the plant B having the DNA of the present invention and the plant to which blast resistance is to be imparted are crossed, and the DNA of the present invention possessed by B is inherited.
  • an individual close to the plant A is selected, and a “backcross” in which crossing with the plant A is repeated is intentionally introduced into the DNA trait of the present invention possessed by B.
  • a DNA marker generally used for genome breeding to select a plant having the DNA of the present invention, it is possible to efficiently perform the replacement by the “backcross”. As a result, the breeding period can be shortened, and extra genomic region contamination can be accurately removed.
  • the above method if necessary, it can be repeated until the entire genome other than the DNA of the present invention is homo-fixed with the target genetic trait. That is, in a preferred embodiment of the present invention, a plant having the DNA of the present invention and having a genomic structure close to the plant A using a general DNA marker for the individuals crossed in the step (d). Individuals can be selected. Furthermore, the selected plant individual can be “backcrossed” (crossed with rice cultivar A) as necessary.
  • selecting using a DNA marker means selecting based on information on the base type of a base sequence (for example, polymorphism) that characterizes the DNA marker. For example, when a polymorphic mutation exists in the vicinity of the DNA of the present invention, it refers to selecting an individual having the same polymorphic mutation as the polymorphic mutation.
  • a base sequence for example, polymorphism
  • the breeding method of the present invention is preferably a “genomic breeding” method using a DNA marker.
  • the “genome breeding” is also called “marker breeding”.
  • “Genome breeding” uses DNA markers that exist in high density throughout the entire genome, and depicts the situation of chromosome replacement for the entire genome of a progeny individual at each crossing. This is a breeding method in which an individual having sucrose is selected at the seedling stage and used for the next crossing. In conventional breeding methods, trait evaluation was used to select individuals to be used for the next cross, so it was necessary to grow to a large number of individual harvest periods, and crosses once a year or once every two years However, it took a great deal of labor and experience to cultivate a large number of individuals uniformly and to evaluate their characteristics correctly.
  • a preferred embodiment of the breeding method of the present invention is a method characterized by selecting using the DNA sequence of the present invention or a DNA marker present in the peripheral sequence.
  • the DNA marker that can be used in the breeding method of the present invention is not particularly limited, and various generally known DNA markers can be suitably used.
  • RFLP restriction fragment length polymorphism
  • SSR simple repetitive sequence
  • SNP single nucleotide polymorphism
  • the present invention provides a molecular marker present in the Pb1 gene region or in the vicinity thereof.
  • a preferred embodiment of the molecular marker is a molecular marker linked to the rice blast resistance gene Pb1, which is shown in the linkage map of FIG.
  • molecular markers of the present invention include molecular markers composed of at least one primer set shown in any of the following (A) to (G).
  • D DNA consisting of the base sequence set forth in SEQ ID NO: 15 and DNA consisting of the base sequence set forth in SEQ ID NO: 16
  • the blast-resistant plant can be identified or selected using at least one of the molecular markers of the present invention as an index.
  • a method for identifying or selecting a blast-resistant plant using the molecular marker of the present invention (which may be described as “the method of the present invention” in the present specification) is also included in the present invention.
  • the blast resistance of a test rice can be specifically or efficiently identified or selected by examining whether or not the test rice has the blast resistance gene Pb1.
  • the desired plant to be identified for the presence or absence of blast resistance (hereinafter referred to as “test plant” in some cases) has a Pb1 gene, the test plant has blast disease.
  • test plant has a Pb1 gene, the test plant has blast disease.
  • a molecular marker linked to Pb1 is used.
  • “Molecular marker” in the present invention refers to a DNA region genetically linked to Pb1, which can be distinguished from other DNA regions.
  • the molecular markers described in FIG. 3 or Table 1 can be exemplified.
  • molecular markers are more useful because the shorter the map distance in units of cM, the closer to the gene and the inheritance of the gene is.
  • One embodiment of the method of the present invention is a method in which a test rice is determined to be resistant to blast when it shows a genotype similar to that of rice having a blast resistance trait.
  • the above-mentioned “rice having a blast resistance trait” refers to rice that has been confirmed to have the blast resistance gene Pb1.
  • the test plant is determined to have blast resistance.
  • Comparison of the molecular marker in the test plant with the molecular marker in “rice having a rice blast resistance trait” is performed not only by comparing the DNA sequence of the molecular marker but also by comparing the information characterized by the DNA sequence. can do.
  • Information characterized by the DNA sequence of a molecular marker includes, but is not limited to, information about the presence or absence of a molecular marker and information about the presence or absence of a mutation site or polymorphic site contained in a molecular marker. It is not something.
  • the molecular weight of the amplified product is determined by electrophoresis using the molecular weight of the "rice having a rice blast resistance trait” amplified product.
  • the molecular weight is the same, it can be said that “shows the same genotype”. Therefore, “showing the same genotype” includes not only the case where the DNA sequences of the molecular markers are completely identical, but also the case where the information characterized by the DNA sequence is the same.
  • “use a molecular marker” means that the molecular marker is used as an index for identifying or selecting rice blast resistance. That is, in a preferred embodiment of the present invention, when the molecular marker of the test plant shows a genotype similar to that of rice having a trait with resistance to blast, the test plant is judged to have trait with blast resistance. Is done.
  • the “test rice” is not particularly limited as long as it is rice, and may be an Indica species or a Japonica species, or a hybrid variety / line of an Indica species and a Japonica species or a wild species. It may be rice, or a hybrid / crossbred variety of cultivar and wild rice.
  • “rice having a blast resistance trait” may be any rice having Pb1, and examples thereof include “Modan”. The above identification method can be carried out by using as a control the molecular marker type in rice varieties / lines already known to have Pb1.
  • the “test plant” preferably refers to a growing line whose parent is known. That is, in the test plant, a plant having the same genotype as the parent of “blast resistance” is determined to have a blast resistance trait (having a Pb1 gene) with a high probability.
  • the probability in this case can be represented by 1-0.01xP when the recombination value is P (%).
  • 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 ears, leaves, roots, stems, seeds, endosperm parts, bran, embryos and the like.
  • the method for preparing (extracting) the DNA sample of the present invention can be carried out by those skilled in the art by a known method.
  • a preferable preparation method for example, a method of extracting DNA using the CTAB method can be mentioned.
  • electrophoretic analysis of the present invention may be performed according to a conventional method.
  • electrophoresis is performed by applying voltage in an agarose or polyacrylamide gel, and the separated DNA pattern is analyzed.
  • the present invention also provides an oligonucleotide having a chain length of at least 15 nucleotides complementary to DNA consisting of the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, and 4 or a complementary strand thereof.
  • complementary strand refers to the other strand of one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs.
  • 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 specifically hybridizes to DNA consisting of the base sequence described in any of SEQ ID NOs: 1, 3, and 4 or a complementary strand thereof.
  • “specifically hybridizes” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular® Cloning, Cold® Spring® Harbor® Laboratory® Press, New® York, USA, In the second edition 1989) means that no significant cross-hybridization with other DNA occurs.
  • 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 any one of SEQ ID NOs: 1, 3, and 4. Moreover, the oligonucleotide of the present invention can be used in the form of a substrate of a DNA array.
  • the oligonucleotide When 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 part of the DNA of the present invention or its complementary strand.
  • the 3 ′ region can be complementary, and a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ side.
  • a primer set used for detection and amplification of DNA consisting of the base sequences described in SEQ ID NOs: 1, 3, and 4, for example, at least one primer shown in any of (a) to (g) above A set can be illustrated.
  • the probe When the oligonucleotide is used as a probe, the probe specifically hybridizes to at least a part of DNA consisting of the base sequence described in any one of SEQ ID NOs: 1, 3, and 4 or a complementary strand thereof. If it is a thing, it will not restrict
  • the probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp.
  • the oligonucleotide of the present invention is used as a probe, it is preferably used after being appropriately labeled.
  • 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 thereof include a method (such as a random prime method) in which a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin is incorporated as a primer.
  • the oligonucleotide of the present invention can be produced by, for example, 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.
  • oligonucleotide for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffering agent
  • a preservative or the like may be mixed as necessary.
  • rice identified as resistant to blast can be selected early.
  • the present invention also provides a method for early selection of rice identified as resistant to blast disease.
  • “early” refers to the state before the heading of rice, preferably the state immediately after germination.
  • the present invention also provides a reagent (for example, a plant breeding reagent) containing an oligonucleotide for use in the plant of the present invention or a method for producing the seed thereof, or a breeding method thereof.
  • a reagent for example, a plant breeding reagent
  • the reagent of the present invention is a reagent containing the following oligonucleotides (a) and (b).
  • (b) DNA of the present invention Oligonucleotide probe that hybridizes to a region under stringent conditions and has a chain length of at least 15 nucleotides
  • the reagent of the present invention includes a reagent containing a molecular marker consisting of at least one primer set, as shown in any of (A) to (G) above.
  • the primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it.
  • the length of the base sequence complementary to the genomic DNA of the primer or probe of the present invention is usually 15 to 100, generally 15 to 50, preferably 15 to 30.
  • a technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known.
  • any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin.
  • a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.
  • the present invention also provides a kit for use in the various methods of the present invention, for example, a breeding kit for use in the plant breeding method of the present invention.
  • the kit contains at least one kind of oligonucleotide (a) or (b).
  • there is a blast disease resistance identification kit containing a molecular marker comprising at least one primer set as shown in any of (A) to (G) above.
  • the kit of the present invention can be appropriately packaged with positive and negative standard samples, instructions describing the method of use, and the like.
  • the DNA or vector of the present invention can be used, for example, for production of a plant body to which blast disease resistance is imparted, or seeds thereof.
  • a blast resistance imparting agent comprising the DNA of the present invention, the protein encoded by the DNA, or the vector of the present invention as an active ingredient.
  • the “blast resistance imparting agent” in the present invention is the active ingredient of the DNA of the present invention, the protein encoded by the DNA, or the vector of the present invention for use in imparting blast resistance to plants.
  • the drug of the present invention for example, blast resistance imparting agent
  • in addition to the active ingredient DNA or vector for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffering agent.
  • a preservative or the like may be mixed as necessary.
  • Example 1 (1) Method (a) Genetic identification of Pb1 region “Noribayashi No. 8” / “St. No. 1” F 2 segregated population, and “St. No. 1” expected to improve recombination frequency / Kasalath Chromosome fragment replacement strains ⁇ SL234 '', ⁇ SL235 '', ⁇ SL51 '' F 2 segregated populations were trained, and the strains that had undergone recombination within this region by DNA markers located at both ends of Pb1 region 278 kb Selected, self-bred and fixed.
  • the candidate gene was incorporated into the binary vector pPZP2H-lac and introduced into ear glutinous resistant cultivar Norin No. 8 to produce a recombinant.
  • T 0 and T 1 Complementarity test Transformants were inoculated with rice blast fungus and examined whether the resistance was complemented.
  • the transformant (T 0 ) was spray-inoculated with a spore suspension at the panning stage.
  • the transformant (T 1 ) was inoculated by spraying or injuring the spore suspension into rice at the 10th leaf stage, the stationary leaf stage, or the panicle stage.
  • the region was located within 25.9 kb.
  • Five genes (P14, P15, P16, P17, P18) were predicted in this region (FIG. 1). Among these, the expression of P15 and P18 was confirmed by RT-PCR.
  • P15 has a base number of 3,897 bp, an amino acid number of 1,296, no intron, and has a CC-NBS-LRR structure with a coiled coil repeated seven times, a nucleotide-binding site, and 13 LRR domains (FIG. 2).
  • Example 2 (1) Examination of Pb1 expression and rice blast resistance associated with rice growth
  • Stage) 6L, 10L, leaf-leaved stage, panicle stage expression level of Pb1 gene and blast disease resistance at each stage were examined by real-time PCR and spray inoculation of blast fungus spore suspension, respectively.
  • the expression level of the Pb1 gene was low in 2L (leaf stage) and 6L, but increased with the transition of the growth stage and reached the maximum in the ear (Fig. 4).
  • the expression level and resistance were correlated. From this, it was considered that the characteristics of the field resistance of Pb1 due to the field resistance were affected by the expression level.
  • the Pb1 gene is a main gene, and is desirable as a resistant variety, such as adult resistance that exhibits resistance from the latter half of the growth stage, and long-term resistance lasting for more than 20 years after dissemination to general farmers. Moreover, although it has the feature which is easy to introduce
  • the change in the expression level of the Pb1 gene is considered to indicate adult resistance and sustained resistance because it coincides with the change in adult resistance observed in rice. Moreover, it became possible to cultivate varieties that had both resistance and high practicality by changing the tissue expression specificity and expression level of genes involved in field resistance in panicle.

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Abstract

L'invention porte sur un gène Pb1 de résistance à la pyriculariose chez le riz qui est isolé et identifié avec succès pour la première fois. Le gène Pb1 est un gène de résistance de type CC-NBS-LRR et a une nouvelle séquence. Le gène a de façon caractéristique une résistance chez le plant adulte, contrairement aux gènes de résistance à la pyriculariose classiques. Il a été trouvé qu'une résistance à la pyriculariose hautement utile en pratique peut être conférée à un plant de riz par modification du gène dans le plant de riz.
PCT/JP2009/070552 2008-12-09 2009-12-08 GÈNE DE RÉSISTANCE À LA PYRICULARIOSE CHEZ LE RIZ Pb1 ET SON UTILISATION WO2010067801A1 (fr)

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CN103026959A (zh) * 2011-03-18 2013-04-10 本田技研工业株式会社 水稻f1种子的生产方法、水稻f1种子及水稻雄性不育系
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Publication number Priority date Publication date Assignee Title
CN103026959A (zh) * 2011-03-18 2013-04-10 本田技研工业株式会社 水稻f1种子的生产方法、水稻f1种子及水稻雄性不育系
US9072230B2 (en) 2011-03-18 2015-07-07 Honda Motor Co., Ltd. Method for producing rice F1 seed, rice F1 seed, and rice male sterile line
US9943048B2 (en) 2011-03-18 2018-04-17 Honda Motor Co., Ltd. Method for producing rice F1 seed, rice F1 seed, and rice male sterile line
CN102703443A (zh) * 2012-05-23 2012-10-03 华南农业大学 稻瘟病抗性基因Pia功能特异性分子标记及其方法与应用
CN102703443B (zh) * 2012-05-23 2014-09-24 华南农业大学 稻瘟病抗性基因Pia功能特异性分子标记及其方法与应用
JP2015128385A (ja) * 2014-01-07 2015-07-16 国立研究開発法人農業生物資源研究所 いもち病抵抗性を付与する真性抵抗性遺伝子Ｐｉｔａ−２及びその対立遺伝子Ｐｉ１９の利用
CN112322633A (zh) * 2020-11-12 2021-02-05 华南农业大学 一种水稻根结线虫抗性基因OsBetvI及其应用

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