EP1047783A1 - Gene associe a la resistance aux maladies chez les vegetaux - Google Patents

Gene associe a la resistance aux maladies chez les vegetaux

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Publication number
EP1047783A1
EP1047783A1 EP98904517A EP98904517A EP1047783A1 EP 1047783 A1 EP1047783 A1 EP 1047783A1 EP 98904517 A EP98904517 A EP 98904517A EP 98904517 A EP98904517 A EP 98904517A EP 1047783 A1 EP1047783 A1 EP 1047783A1
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European Patent Office
Prior art keywords
plant
rice
sequence
plants
seq
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EP98904517A
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German (de)
English (en)
Inventor
Chaozu He
Guo-Liang Wang
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Institute of Molecular Agrobiology
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Institute of Molecular Agrobiology
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically 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
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically 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
    • C12N15/8282Phenotypically 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • blast fungus is believed to infect rice plants in a manner typical of other foliar pathogens.
  • Infection by M. grisea is initiated when a conidium lands on a leaf surface.
  • a conidium produces a germ tube that grows and differentiates a specialized infection structure called an appressorium that adheres tightly to the plant surface (Bourett and Howard, 1990).
  • the specialized cell generates enormous turgor pressure that is used to penetrate the underlying plant surface (Howard, 1994). The penetration into plant cells by pathogen invasion may damage the cell structure and activate genes responsive to wounding.
  • MAP mitogen-activated protein
  • MAP kinase signaling cascade is one of the major pathways involved in transducing extracellular stimuli into intracellular responses in mammals and yeasts (Shyy and Chien, 1997, Gabay et al., 1997, Samejima et al., 1997).
  • MAP kinase is a specific class of serine/threonine protein kinases and has been implicated in a wide variety of physiological processes, such as cell growth, differentiation, oncogenesis and response to environmental stresses (Herskowitz, 1995, Cohen, 1997) .
  • MAP kinases or extracellular signal regulated kinases (“ERKs") were originally identified as transducers of mitogens (substances that induce proliferation).
  • MAP kinases were also shown to be involved with signaling hormones, neurotransmitters and signals for differentiation (Marshall, 1994). At present, MAP kinase pathways are best understood in yeast and animals and several distinct MAP kinase pathways have been identified (Ruis and Schuller, 1995) .
  • the basic module of a MAP kinase cascade is a specific set of three functionally interlinked kinases. The activation of MAP kinases is brought about by upstream (i.e.
  • MAPKKs dual-specificity MAP kinase kinases
  • MAKKKs upstream kinases that either belong to the class of MAPKK kinases (MAKKKs), or are raf and mos proteins (Marshall, 1994; Hirt, 1997).
  • MAP kinases In plants, several genes encoding MAP kinases have been identified from alfalfa (Jonak et al., 1993; 1995), Arabidopsis (Mizoguchi et al., 1994), pea (Stafstrom et al., 1993), petunia (Decroocq-Ferrant et al., 1995), tobacco (Wilson et al . , 1993) and parsley (Lcklerink et al., 1997). Similar to mammalian kinases, AtMAPKl and AtMAPK2 are shown to be involved in cell proliferation (Jonak et al., 1993, Mizoguchi et al., 1994).
  • MAP kinases have also been identified in plants which are responsive to cold, heat, wounding, drought and mechanical stresses (Bogre et al., 1997, Jonak et al., 1996; Seo et al., 1995, Lipporink et al., 1997; Zhang and Klessig, 1997).
  • the 48 kD MAP kinase, ERMK is rapidly activated upon high-affinity binding of a fungal elicitor to a plasma membrane receptor in parsley cells (Lcklerink et al., 1997).
  • the activated ERMK is translocated into the nucleus where it may be involved in the transcriptional activation of defense genes.
  • SA salicylic acid
  • MAP kinases are an important component in the signal transduction pathway of plant defense to pathogen infection. Lddlerink et al. (1997) and Zhang and Klessig, (1997) have found a elicitor-responsive MAP kinase in parsley suspension cells and a SA-activated MAP kinase in tobacco suspension cells respectively. However, no evidence was found that MAP kinase is activated by natural pathogen infection in plant species. Accordingly, a need exists for the identification of MAP kinase genes associated with such defense mechanisms and means for expressing such genes in host plants (or regulating their expression) to confer disease resistance.
  • MAP kinase gene and protein that it encodes have been discovered. Based on sequence analysis, this novel gene is a new member of the MAP kinase gene family which encodes a 519 amino acid 59 kD protein. It is designated as BIMKl for blast jLnduced MAP _kinase. BIMKl was strongly induced by rice blast fungus M. grisea and is postulated to be involved in the defense response of rice to blast infection.
  • the invention relates to the deoxyribonucleic acid ("DNA”) that comprises the novel MAP kinase gene, its messenger ribonucleic acid (“mRNA) transcript and the protein that it encodes.
  • DNA deoxyribonucleic acid
  • mRNA messenger ribonucleic acid
  • the invention involves expression vectors that contain the novel gene operably linked to a plant active promoter and to plant cells and plants that have been transformed with such vectors.
  • the invention concerns a method for conferring disease resistance in plants, particularly monocot plants such as rice, wheat, maize, barley and asparagus, which comprises genetically modifying the plant to effect expression of the novel MAP kinase gene.
  • Figure 1 is an autoradiogram of a Southern hybridization analysis of restriction enzyme digested rice genomic DNA using labeled BIMKl cDNA as a probe.
  • Figure 2 is an autoradiogram of a Northern analysis of total RNA (50 mg) isolated from rice leaf tissue at different time points after inoculation with M. grisea using labeled BIMKl cDNA as a probe.
  • the gene encoding a MAP kinase, identified as BIMKl has been identified for rice, cloned and sequenced.
  • the sequence of the full-length clone, including 5' and 3' untranslated regions, is provided in SEQ ID NO:l.
  • the region from nucleotide 13 through nucleotide 1569 encodes the 519 amino acid 59kD protein whose sequence is shown in SEQ ID NO: 2.
  • the BIMKl gene was isolated from rice infected with the rice blast pathogen, Magnaporthe grisea .
  • the invention provides an isolated DNA having substantially the sequence spanning nucleotides 13 through 1569 of SEQ ID NO:l.
  • the invention further provides isolated mRNA complementary to the deoxyribonucleic acid having substantially the sequence spanning nucleotides 13 through 1569 of SEQ ID N0:1.
  • the invention also provides an isolated protein having substantially the sequence shown in SEQ ID NO: 2.
  • isolated as used herein, means that the nucleic acid or protein is in an environment different from its natural environment. For example, it may be cloned in a cloning or expression vector, it may reside in a bacterial cell, it may be associated with other means for transformation of plants or plant cells or it may reside in a plant with which it is not naturally associated.
  • substantially the sequence means a sequence that is predominantly that of the identified sequence, provided that the nucleic acid or protein retains the kinase functions of the native molecule. Thus, conservative substitutions, deletions and additions that do not significantly reduce the function of the protein are contemplated.
  • Probes, primers, antisense molecules and other nucleic acid molecules that are complementary to regions of the BIMKl gene will be useful for its amplification and analysis, regulation of its expression and the like.
  • the invention provides DNA or RNA molecules that are capable of hybridizing to the DNA molecules described above (or their complements) under stringent hybridization conditions. Such conditions are well known in the art and include those conditions under which stable hybrids will form when there is at least about 75%, preferably at least about 80%, most preferably at least about 90%- 100% homology between the DNA or RNA molecule and the corresponding region of the target DNA.
  • the DNA can be incorporated in plant or bacterial cells using conventional recombinant DNA technologies. Generally, such techniques involve inserting the DNA into an expression vector which contains the necessary elements for the transcription and translation of the inserted protein coding sequences and one or more marker sequences to facilitate selection of transformed cells or plants.
  • Suitable promoters include, for example, the nos promotor, the small subunit chlorophyll A/B binding polypeptide, the 35S promotor of cauliflower mosaic virus, and promoters naturally associated with MAP kinase genes, such as BIMKl in plants.
  • SEQ ID NO: 6 provides the sequence of the 5' untranslated region upstream of the BIMKl coding sequence. This region contains the putative promoter for this gene. SEQ ID NO: 6 overlaps the 5' end of the BIMKl coding region, the ATG start codon appearing at position 1378-80.
  • TATA box appears at positions 1302-1306 of the sequence.
  • this promoter has general utility as a plant-active promoter, particularly for effecting expression of transgenes in monocotylodonous plants, such as rice.
  • plant cell is intended to encompass any cell derived from a plant including undifferentiated tissues such as callus and suspension cultures, as well as plant seeds, pollen or plant embryos.
  • Plant tissues suitable transformation include leaf tissues, root tissues, meristems, protoplasts, hypocotyls, cotyledons, scutellum, shoot apex, root, immature embryo, pollen, and anther.
  • One technique for transforming plants is by contacting tissue of such plants with an inoculum of a bacterium transformed with a vector comprising DNA in accordance with the present invention. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Bacteria from the genus Agroba cterium can be utilized advantageously to transform plant cells. Suitable species of such bacteria include Agrobacterium tumefaciens and Agrobacterium rhizogens . Agrobacterium tumefaciens ( e . g. , strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Another approach to transforming plant cells with the nucleic acid of this invention involves propelling inert or biologically active particles into plant cells. This technique is disclosed in U.S. Pat. Nos . 4,945,050, 5,036,006 and 5,100,792 all to Sanford et. al., which are hereby incorporated by reference.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector comprising the isolated DNA of this invention.
  • Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
  • Another method of transforming plant cells is the electroporation method.
  • This method involves mixing the protoplasts and the desired DNA and forming holes in the cell membranes by electric pulse so as to introduce the DNA into the cells, thereby transforming the cells.
  • This method currently has high reproducibility and various genes have been introduced into monocotyledons, especially rice plants by this method (Toriyama et . al., 1988, Shimamoto et al., 1989 and Rhodes et al . , 1988).
  • Similar to the electroporation method is a method in which the desired gene and protoplasts are mixed and the mixture is treated with polyethylene glycol (“PEG”), thereby introducing the gene into the protoplasts.
  • PEG polyethylene glycol
  • This method is different from the electroporation method in that PEG is used instead of an electric pulse (Zhang W. et. al., 1988, Datta et al., 1990 and Christou et al., 1991).
  • transgenic plants of the present invention may be used in preparing transgenic plants of the present invention.
  • explants, callus tissues or suspension cultures can be exposed to the appropriate chemical environment (e.g. , cytokinin and auxin) so the newly grown cells can differentiate and give rise to embryos which then regenerate into roots and shoots.
  • the appropriate chemical environment e.g. , cytokinin and auxin
  • the isolated DNA of the present invention is believed to be useful in enhancing resistance to disease-causing pathogens in both monocotyledonous plants (“monocots”), and dicotyledonous plants
  • BIMKl a gene that influences expression of the endogenous gene, rather than transforming the plant with a vector containing the gene.
  • control may be achieved, for example, by modifying or replacing endogenous promoters, enhancers or other control signals that regulate expression of the gene, for example, to achieve enhanced expression or programmed expression.
  • the predicted protein sequence of BIMKl carries all 11 conserved domains for the catalytic function of serine/threonine protein kinase. The expression of BIMKl was rapidly induced as early as 4 hours after inoculation with M.
  • BIMKl only has about 50% identity with these two stress-related MAP kinases isolated from dicot plants. This suggests the divergence of MAP kinases in onocot and dicot plant species. In addition to sequence differences, BIMKl is about 500 bp longer than all cloned MAP kinase genes. The 3' region of the gene contains a domain similar with ADH genes in animals. The function of this domain in the defense response to blast infection is unknown. The invention is further illustrated by the following examples, which are not intended to be limiting . EXAMPLES
  • the resistant isogenic line C101A51 carrying the Pi-2 gene and the susceptible cultivar C039 were used in the experiment.
  • Three week-old rice plants were inoculated with a Philippine isolate P06-6 of M. grisea . After inoculation, plants were kept in dark in a dew chamber for 24 hours at 26° C. Then, inoculated plants were move into a growth chamber in 10 hours light with 14 hours dark at 25-26° C for 7 days.
  • Leaf tissue was harvested from both cultivars at 0, 4, 8, 12, 24, 48. 72 hours after inoculation.
  • RNeasy mini kit (Qiagen, Germeny) was used to isolate total RNA from 150-200 mg rice leaf tissue.
  • RT-PCR reverse transcriptase-mediated polymerase chain reaction
  • Two primers, CF9-RT and CF9-Rev were designed based on the DNA sequence of the cloned gene Cf-9, a tomato resistance gene to the leaf mould fungus Cladosporium fulvum (Jones et al., 1994).
  • the primer sequence of CF9-RT is 5 ' -AAAAGCACAAGTTGGTGC-3 ' (SEQ ID NO: 3) which is the DNA sequence 217-235 bp after the start codon.
  • the sequence of CF9-Rev is 5'TAACGTCTATCGACTTCT-3' (SEQ. ID NO : 4 ) which is the reverse strand sequence of Cf-9 from 1408 to 1426 bp after the start codon.
  • RT-PCR was conducted following protocols provided by the manufacturer (GIBCO-BRL, Life-Technology, USA) . The amplified cDNAs were then separated in 1.2% agarose gel.
  • Rice genomic DNA was isolated as described by Dellporta et al. (1984) . DNA was digested with restriction enzymes and separated in 0.8% agarose gel, and then transferred onto Hybond-N+ membrane (Amersham, UK) . Probes were labeled using megaprimer labelling kit (Amersham, UK) . Rapid hybridization solution (Clonetech, USA) was used.
  • RNA used in the Northern blot analysis was isolated using a Trizol total RNA isolation reagent (GIBCO-BRL, Life-Technology, USA) . Fifty micrograms of total RNA per lane was separated in 1.0% agarose gel and transferred onto Hybond-N+ membrane (Amersham, UK) using NorthernMax kit (A bion, USA) following the manufacturer's instruction. Northern hybridization was carried out same as Southern hybridization described above.
  • a rice BAC library of cultivar IR64 (Yang et al., 1997) was screened using the 350 bp cDNA fragment described in Example 1 as a probe.
  • Four positive BAC clones (3-07, 17-H21, 43-H15 and 43-F5) were identified from the whole BAC library.
  • the miniprepared DNA of the three BAC clones was digested with 3 different enzymes to check if they are overlapping clones in a chromosomal region. Based on the restriction patterns, it was found that these three clones were overlapping clones.
  • BAC clone (3-07) was chosen and subcloned into pBluescript-SK (Strategene, USA) .
  • a primer containing sequence spanning the start codon ATG(5'-AACACAGTGGAAATGGAGTTCTTCA-3' ) SEQ ID NO: 5 was designed based on the genomic DNA sequence. RT-PCR was performed using this primer and a oligo-dT primer (Life-Technologies, USA) . From the cDNA prepared from the infected leaves of C101A51 (8 hours after inoculation), a 2.0 kb PCR product was obtained. This PCR product was cloned into pGEM-T vector and sequenced. The sequence is shown in SEQ ID N0:1.
  • BIMKl for blast induced MAP kinase.
  • This amino acid sequence was compared to the sequence of several MAP kinases isolated from a variety of organisms. As shown in Table 1, the sequences are significantly homologous. In section A of the Table, multiple alignment of the deduced amino acid sequence (N-terminal) of BIMKl with other members of MAP kinases from other organisms is shown. The amino acid sequence of BIMKl is compared to that of MsERK (Duerr et al .
  • the 11 MAP kinase subdomains are labeled in Roman numerals (Hanks et al., 1988).
  • the M. grisea BIMKl gene contains all 11 highly conserved subdomains which are present in all known MAP kinases in mammals and plants.
  • BIMKl also contains 50 amino acids homologous to mammalian alcohol dehydrogenase (ADH) in its C-terminal.
  • Section B of the Table shows multiple alignment of the deduced amino acid sequence (C-terminal) of BIMKl with other ADH genes in animals and plants.
  • ADH is present in many organisms that metabolize ethanol, including human, in an oxidoreductase reaction with NAD+/NADH as an essential co-factor .
  • Example 4 BIMKl is conserved in rice genome and mapped to a region clustering blast resistance genes
  • BIMKl was induced bv rice blast fungus Total RNA was isolated from rice leaf tissue collected at different timepoints after inoculation. The blot was hybridized using BIMKl cDNA fragment as probe labelled with 32p. It was found that BIMKl was highly induced as early as 4 hours after inoculation. The expression of the gene BIMKl was reduced 24 hours after inoculation ( Figure 2). The induction level of BIMKl in both resistant (C101A51) and susceptible (C039) lines was very similar ( Figure 2). Since C101A51 and Co39 have the same genetic background except C101A51 carries a rice blast resistance gene, Pi-2, it is suggested that BIMKl was induced independently from Pi-2 and is involved in a general defense pathway to blast. Table 1
  • ATMPK1 DLKPGNLLVNANCDL KICDFGLARASNTKG QFMTEYWTRWY RAPELL-LCCDNYGT SIDVWSVGCIFAELL GRKPIFQGTECLNQL 243
  • MsERKl a mitogen-activatedprotem kinase from a flowering plant. Plant Cell, 5(1): 87-96. Fukuda, M.G.Y., Nishida, E. (1997). Interaction of MAP kinase with MAP kinase: its possible role in the control of nucleocytoplasmictransport of MAP kinase. EMBO J. 16(8): 1901-1908.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ATCTTCGTGG CACCTGAATT ACTTGCATAC TGTACAATAT CATTATTTCT TTTTTTCTAT 660 GATCTGTGCA AAGTGCAATA CAGCTCAAGT GCAGGTAAAG CTTGCGTGTT CTATCCAATC 720
  • CTATGTACTG AAAATGTCAA TTTTTGTTAG GTTGGTCATG ATATTCCCAG GGAAAAGATC 1260 ATGGTTTTGT TTATAGGGCT ATTCTACTAC TGAAGAAGTT TTATAACCAG CCACTCTGTA 1320

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Abstract

Cette invention concerne un gène codant une nouvelle protéine-kinase activée par mitogène (MAP), lequel gène a été identifié dans le riz puis isolé et cloné. L'expression de ce gène est induite en réponse à une infection par le pathogène d'échaudage M. grisea. Ce gène permet de conférer une résistance aux maladies aux plantes, notamment aux plantes monocotylédones telles que le riz, le blé, le maïs, l'orge et les asperges. Cette invention concerne également des vecteurs contenant ce nouveau gène, ainsi que des cellules de plantes, des plantes et des graines transformées.
EP98904517A 1998-01-16 1998-01-16 Gene associe a la resistance aux maladies chez les vegetaux Withdrawn EP1047783A1 (fr)

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CN100366743C (zh) * 2004-07-16 2008-02-06 广西大学 一种植物抗病相关蛋白及其编码基因与应用
CN100348724C (zh) * 2005-02-02 2007-11-14 华中农业大学 水稻抗病相关基因OsDR8
CN101265294B (zh) * 2008-03-10 2010-08-04 中国农业科学院作物科学研究所 一种抗病相关的小麦myb蛋白及其编码基因与应用
CN101935660B (zh) * 2010-09-02 2012-07-04 北京大学 受稻瘟菌诱导的启动子及其应用
CN102154236B (zh) * 2011-03-24 2012-12-26 北京市农林科学院 一种小麦早熟相关蛋白TaMAPK1及其编码基因和应用
CN109423494B (zh) * 2017-08-26 2021-11-02 复旦大学 水稻tMAPKKK5基因在改良水稻产量性状方面的应用

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CN1079114C (zh) * 1993-07-29 2002-02-13 农林水产省农业生物资源研究所 稻瘟病抗性基因的核酸标记物以及通过使用这些标记物分离到的稻瘟病抗性基因
US6333151B2 (en) * 1993-07-29 2001-12-25 Research Development Corporation Of Japan Nucleic acid markers for rice blast resistance genes and rice blast resistance genes isolated by the use of these markers
JP2945953B2 (ja) * 1995-08-29 1999-09-06 農林水産省農業生物資源研究所長 傷ストレス誘導性mapキナーゼおよびその遺伝子

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