WO2018028553A1 - 水稻条纹叶枯病抗性基因Stv-bi及其应用 - Google Patents

水稻条纹叶枯病抗性基因Stv-bi及其应用 Download PDF

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WO2018028553A1
WO2018028553A1 PCT/CN2017/096359 CN2017096359W WO2018028553A1 WO 2018028553 A1 WO2018028553 A1 WO 2018028553A1 CN 2017096359 W CN2017096359 W CN 2017096359W WO 2018028553 A1 WO2018028553 A1 WO 2018028553A1
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protein
rice
sequence
transgenic
gene
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French (fr)
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储成才
唐九友
左示敏
吴旭江
殷文超
潘学彪
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中国科学院遗传与发育生物学研究所
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    • 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)
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • C12N15/8283—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 virus resistance

Definitions

  • the invention relates to the field of genetic engineering, in particular to a rice stripe disease resistance gene Stv-b i and application thereof.
  • Rice (Oryza sativa L.) is the most important food crop in nearly half of the world's population. Its yield and quality have long been severely threatened by pathogens such as bacteria, fungi, and viruses. Years of production practice have shown that breeding resistant varieties by using the plant's own disease resistance gene (R gene) is the most economical, safe and effective response (Ronald PC.1997. The molecular basis of disease resistance in rice. Plant Mol .Biol. 35: 179-186). In the past ten years, several rice bacterial blight and rice blast resistance genes have been successfully cloned and effectively integrated into rice resistance improvement research and application (Helliwell EE, Yang Y.2013.Molecular strategies to Improve rice disease resistance. Methods Mol. Biol.
  • Rice stripe disease is one of the most important viral diseases in rice.
  • the disease is caused by rice stripe virus (RSV), the first reported plant virus with a double-sense coding strategy, which is mainly transmitted in a more persistent manner by Laodelphax striatellus Fallén ( Ramirez BC, Haenni AL. 1994. Molecular biology of tenuiviruses, a remarkable group of plant viruses. J. Gen. Virol. 75: 467-475).
  • the early heart of the plant has intermittent yellow-green or yellow-white streaks along the veins. Then the streaks merge into pieces, and the heart leaves are curled into a paper-like shape.
  • Stv-b allele Stv-b i gene with incomplete dominant disease resistance. Since 1960s, the Stv-b i gene derived from the indica variety 'Modan' has been introduced into indica rice varieties, and these intermediate varieties have been widely used in the breeding of high quality japonica rice varieties resistant to RSV. In the past half century, the Stv-b i gene is the only RSV resistance gene that provides sustained and high-efficiency resistance without affecting rice quality and yield. There have been no reports of pathogenic races (Hayano-Saito Y) , Tsuji T, Fuji i K, Saito K, Iwasaki M, Saito A. 1998.
  • Stv-b i Localization of the rice stripe disease resistance gene, Stv-b i , by graphical genotyping and linkage analysis with molecular markers. Theor.Appl.Genet.96 , 1044-1049).
  • Stv-b i Rice stripe disease resistance gene
  • Theor.Appl.Genet.96 1044-1049.
  • the cloning of the Stv-b i gene can help unravel the antiviral molecular mechanism of rice; provide a reference for the antiviral mechanism of monocotyledon; and also for further RSV resistant breeding, especially genetic engineering improved breeding and molecular Marker-assisted breeding provides the basis for better control and reduction of the risk of RSV to rice production.
  • the technical problem to be solved by the present invention is how to improve the resistance of rice stripe disease.
  • the present invention first provides a protein.
  • the protein provided by the present invention may be as follows: a1) or a2) or a3) or a4) or a5) or a6):
  • amino acid sequence is the protein shown in SEQ ID NO: 4 in the Sequence Listing;
  • A2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 4 in the Sequence Listing;
  • amino acid sequence is the protein shown in SEQ ID NO: 7 in the Sequence Listing;
  • A5) a protein associated with plant disease resistance obtained by subjecting a protein represented by a1) or a2) or a3) or a4) to substitution and/or deletion and/or addition of one or several amino acid residues;
  • A6 A protein having 70% or more identity with the protein represented by a1) or a2) or a3) or a4).
  • sequence 4 in the sequence listing can be composed of 1710 amino acid residues.
  • Sequence 7 in the Sequence Listing can consist of 1708 amino acid residues.
  • the label shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein shown in SEQ ID NO: 7 or Sequence 7 in the Sequence Listing.
  • substitution and/or deletion and/or addition of the one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 850 amino acid residues.
  • the protein in the above a5) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the protein in the above a5) can be obtained by SEQ ID NO: 2 in the sequence listing, sequence 5 in the sequence listing, sequence 128 in the sequence listing from the 5' end, 128 to 5260, or the DNA shown in SEQ ID NO:1 in the sequence listing. Codons lacking one or several amino acid residues in the sequence, and/or one or several base pair missense mutations, and/or at their 5' and/or 3' ends are shown in Table 1. The coding sequence of the tag is obtained.
  • identity refers to a similarity to a natural amino acid sequence. “Identity” includes an amino acid sequence having 70% or more, or 80% or more, or 90% or more identity with the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7 of the Sequence Listing of the present invention. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the disease resistance may be against stripe leaf blight.
  • the disease resistance may be a disease caused by rice stripe virus.
  • the plant may be any one of c1) to c14): c1) dicotyledon; c2) monocotyledon; c3) gramineous plant; c4) rice; c5) barley; c6) wheat; c7) tobacco; C8) tomato; c9) oats; c10) sorghum; c11) corn; c12) cotton; c13) soybean; c14) canola.
  • the rice may specifically be a rice variety Wuyujing No. 3 or a rice variety Wulingyu No. 1.
  • Active fragments of the protein are also within the scope of the invention.
  • Nucleic acid molecules encoding the proteins are also within the scope of the invention.
  • the nucleic acid molecule encoding the protein may specifically be a DNA molecule as shown in (b1) or (b2) or (b3) or (b4) or (b5) or (b6):
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing;
  • nucleotide sequence is the DNA molecule shown in position 128 to position 5260 of the sequence 2 from the 5' end of the sequence listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 5 in the Sequence Listing;
  • (b6) a DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by (b1) or (b2) or (b3) or (b4) and which encodes the protein.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • Sequence 1 in the sequence listing consists of 11390 nucleotides.
  • Sequence 2 in the sequence listing consists of 5260 nucleotides, and the nucleotide of SEQ ID NO: 2 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing.
  • the sequence 5 in the sequence listing consists of 5127 nucleotides, and the nucleotide of the sequence 5 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
  • nucleotide sequence encoding the protein of the present invention can readily mutate the nucleotide sequence encoding the protein of the present invention using known methods, such as directed evolution and point mutation methods. Those artificially modified nucleotides having 50% or greater identity to the nucleotide sequence of the protein isolated from the present invention, as long as the protein is encoded, are derived from the nucleotide sequence of the present invention. And equivalent to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes a nucleotide sequence of a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 of the coding sequence listing of the present invention, 50% or more, or 60% or more, or 70% or more, or A nucleotide sequence of 80% or higher, or 90% or higher identity. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • Molecular marker A developed based on the nucleic acid molecule, or molecular marker B which is tightly linked to the nucleic acid molecule and which can be used to identify or assist in the identification of plant disease resistance is also within the scope of the present invention.
  • Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing or partially containing the nucleic acid molecules are also within the scope of the invention.
  • the recombinant vector is a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or a cloning vector.
  • the expression vector may specifically be the vector pCAMBIA2300 mentioned in the examples or the derivative vector pCAMBIA2300-XmaI/AvrII or the like of the vector pCAMBIA2300.
  • the recombinant vector may specifically be 480C or 500ox mentioned in the examples.
  • the recombinant microorganism is a recombinant strain obtained by introducing the recombinant vector into a starting microorganism.
  • the recombinant microorganism may specifically be a recombinant strain obtained by introducing the recombinant plasmid 480C or 500ox into a starting microorganism.
  • the starting microorganism may be Agrobacterium tumefaciens.
  • the Agrobacterium tumefaciens may specifically be Agrobacterium tumefaciens AGL1.
  • the transgenic cell line can be obtained by transforming the recombinant vector into a recipient cell.
  • the transgenic cell line can be specifically obtained by transforming the recombinant plasmid 480C or 500ox into a recipient cell.
  • Antibodies that bind to the protein are also within the scope of the invention.
  • D1 the protein, or the nucleic acid molecule, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing or partially containing the nucleic acid molecule, for use in regulating plant disease resistance;
  • D3 the protein, or the nucleic acid molecule, or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism or a transgenic cell line, for use in preparing a disease-resistant drug;
  • the disease resistance may be resistance to stripe leaf blight.
  • the disease resistance may be a disease caused by rice stripe virus.
  • the plant may be any one of c1) to c14): c1) dicotyledon; c2) monocotyledon; c3) gramineous plant; c4) rice; c5) barley; c6) wheat; c7 ) tobacco; c8) tomato; c9) oats; c10) sorghum; c11) corn; c12) cotton; c13) soybean; c14) canola.
  • the rice may specifically be a rice variety Wuyujing No. 3 or a rice variety Wulingyu No. 1.
  • the present invention also provides a method of cultivating a transgenic plant.
  • the method for cultivating a transgenic plant may specifically be a method comprising the steps of: expressing or over-expressing the protein in a recipient plant, or increasing the activity of the protein in the recipient plant to obtain a transgenic plant; The disease resistance of the transgenic plants is enhanced compared to the recipient plants.
  • the "expression or overexpression of the protein in the recipient plant, or increase the activity of the protein in the recipient plant” may be through multiple copies, alteration of promoters, regulatory factors, transgenes, etc.
  • a well-known method achieves the effect of expressing or overexpressing the protein or increasing the activity of the protein.
  • the "expressing or overexpressing the protein in a recipient plant, or increasing the activity of the protein in the recipient plant” may specifically be introducing a nucleic acid molecule encoding the protein into the recipient plant.
  • the method for cultivating a transgenic plant provided by the present invention may specifically be a method 2, comprising the steps of inhibiting the expression level of the protein in the recipient plant or inhibiting the activity of the protein in the recipient plant to obtain a transgenic plant; The resistance of the transgenic plants is reduced compared to the recipient plants.
  • the "inhibiting the expression level of the protein in the recipient plant or inhibiting the activity of the protein in the recipient plant” may be by methods well known in the art such as RNA interference, homologous recombination, gene-site editing, and the like. The purpose of inhibiting the expression amount or activity of the protein is achieved.
  • the "inhibiting the expression level of the protein in the recipient plant or inhibiting the activity of the protein in the recipient plant” may specifically be introduced into the recipient plant to inhibit expression of the nucleic acid molecule encoding the protein. Substance.
  • the nucleic acid molecule encoding the protein may specifically be a DNA molecule represented by the following (b1) or (b2) or (b3) or (b4) or (b5) or (b6):
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing;
  • nucleotide sequence is the DNA molecule shown in position 128 to position 5260 of the sequence 2 from the 5' end of the sequence listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 5 in the Sequence Listing;
  • (b6) a DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by (b1) or (b2) or (b3) or (b4) and which encodes the protein.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • Sequence 1 in the sequence listing consists of 11390 nucleotides.
  • Sequence 2 in the sequence listing consists of 5260 nucleotides, and the nucleotide of SEQ ID NO: 2 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing.
  • the sequence 5 in the sequence listing consists of 5127 nucleotides, and the nucleotide of the sequence 5 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
  • the "introducing a nucleic acid molecule encoding the protein into a recipient plant” can be achieved by introducing a recombinant vector A into a recipient plant; the recombinant vector A can be inserted into an expression vector or a cloning vector.
  • the recombinant vector A may specifically be the 480C or the 500ox.
  • the "substance that inhibits expression of a nucleic acid molecule encoding the protein” can be achieved by introducing a recombinant vector B into a recipient plant.
  • the recombinant vector B may specifically be 480Ri mentioned in the examples.
  • the method for cultivating a transgenic plant provided by the present invention may specifically be the method 3, comprising the steps of inhibiting or increasing the expression level of the eEF1A protein in the receptor or the activity of the eEF1A protein in the receptor, and obtaining a transgenic organism; The disease resistance of the transgenic organism is enhanced.
  • the eEF1A protein may be as follows G1) or G2) or G3):
  • the G1) amino acid sequence is the protein shown in SEQ ID NO: 9 in the Sequence Listing;
  • G2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 9 in the Sequence Listing;
  • G3 A protein associated with resistance to RNA virus disease obtained by substitution and/or deletion and/or addition of a protein represented by G1) or G2) by one or several amino acid residues.
  • the "inhibition of the expression level of the eEF1A protein in the receptor or the activity of the eEF1A protein in the receptor” can be inhibited by methods well known in the art, such as RNA interference, homologous recombination, and gene-site editing.
  • the expression level of the eEF1A protein in the body or the purpose of inhibiting the activity of the eEF1A protein in the receptor can be inhibited by methods well known in the art, such as RNA interference, homologous recombination, and gene-site editing.
  • the "inhibition of the expression level of the eEF1A protein in the receptor or the activity of the eEF1A protein in the receptor” may specifically be a substance introduced into the receptor to inhibit expression of the nucleic acid molecule encoding the eEF1A protein.
  • the "increasing the expression level of the eEF1A protein in the receptor or the activity of the eEF1A protein in the receptor” can increase the receptor by a method well known in the art such as multiple copies, altered promoters, regulatory factors, transgenes, and the like.
  • the effect of the expression level of the eEF1A protein or the activity of the eEF1A protein in the receptor can increase the receptor by a method well known in the art such as multiple copies, altered promoters, regulatory factors, transgenes, and the like.
  • the "increasing the expression level of the eEF1A protein in the receptor or the activity of the eEF1A protein in the receptor” may specifically be the introduction of a nucleic acid molecule encoding the eEF1A protein into the receptor.
  • Any of the above eEF1A proteins may specifically be protein SIP1.
  • the protein SIP1 can be F1) or F2) or F3) as follows:
  • F2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 9 in the Sequence Listing;
  • F3 A protein associated with resistance to RNA viral disease obtained by substituting and/or deleting and/or adding a protein represented by F1) or F2) with one or several amino acid residues.
  • the nucleic acid molecule encoding the protein SIP1 may specifically be a DNA molecule represented by the following (B1) or (B2) or (B3) or (B4):
  • the coding region is a DNA molecule represented by SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13 in the Sequence Listing;
  • the nucleotide sequence is sequence 10, sequence 11, sequence 12 or sequence 13 in the sequence listing, respectively.
  • DNA molecule
  • (B4) A DNA sequence which hybridizes under stringent conditions to a nucleotide sequence defined by (B1) or (B2) and which encodes the protein SIP1.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • Sequence 10, Sequence 11, Sequence 12 and Sequence 13 in the Sequence Listing are all composed of 1344 nucleotides (four sequences are highly homologous) and both encode the amino acid sequence shown in SEQ ID NO: 9 in the Sequence Listing.
  • the organism may be a plant.
  • the receptor may specifically be a recipient plant.
  • the "substance which inhibits expression of the nucleic acid molecule encoding the protein SIP1" can be achieved by introducing a recombinant vector pentamine into a recipient plant.
  • the recombinant vector VIII may specifically be SIP1Ri or SIP1ox mentioned in the examples.
  • the method for cultivating a transgenic plant provided by the present invention may specifically be the method 4, comprising the step of crossing the transgenic plant obtained by the above method 1 or the above method 2 or the above method 3 with the plant to be modified to obtain a progeny transgenic plant;
  • the progeny transgenic plants have the same disease resistance as the transgenic plants (i.e., transgenic plants that are parents).
  • the disease resistance may be resistance to stripe disease.
  • the disease resistance may be a disease caused by rice stripe virus.
  • the recipient plant may be any one of c1) to c14): c1) dicotyledon; c2) monocotyledon; c3) gramineous plant; c4) rice; c5) Barley; c6) wheat; c7) tobacco; c8) tomato; c9) oats; c10) sorghum; c11) corn; c12) cotton; c13) soybean; c14) canola.
  • the rice may specifically be a rice variety Wuyujing No. 3 or a rice variety Wulingyu No. 1.
  • the transgenic plants are understood to include not only the first generation of transgenic plants obtained by transforming the nucleic acid molecules into recipient plants, but also their progeny.
  • the nucleic acid molecules can be propagated in the species, and the nucleic acid molecules can also be transferred to other varieties of the same species, particularly in commercial varieties, using conventional breeding techniques.
  • the transgenic plants include seeds, callus, whole plants, and cells.
  • the present invention also provides a nucleic acid molecule, which may be a DNA molecule represented by the following (e1) or (e2) or (e3):
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing;
  • (e2) a DNA molecule having 75% or more identity with the nucleotide sequence defined by (e1);
  • (e3) A DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by (e1) or (e2).
  • H1 interacts with the eEF1A protein
  • H4 is used to regulate the biological function of eEF1A protein in vivo.
  • the present invention also protects a method for regulating the biological function of an eEF1A protein in a living body, comprising regulating the expression or activity of said protein in an organism by introducing the protein or increasing the expression or activity of said protein in the living body, by regulating the interaction of said protein with eEF1A protein.
  • the organism of the method may be a plant, an animal, or a microorganism.
  • the protein and its coding gene provided by the present invention can regulate plant disease resistance. Therefore, the protein provided by the invention has a very important role in controlling the resistance of stripe disease, and has a broad prospect in breeding rice varieties resistant to stripe disease.
  • Figure 1 shows the genotypic and phenotypic data of the fine mapping map of the Stv-b i gene and the single chromosome fragment substitution line.
  • Figure 2 is a schematic diagram showing the genomic and transcript structure of Nipponbare, Lemont and Teqing in the localization interval. Among them, Nipponbare is Nipponbare, and Teqing is Teqing.
  • Example 3 is an experimental result of Example 2.
  • Fig. 5 is an experimental result of Example 4.
  • Fig. 6 is an experimental result of Example 6.
  • Fig. 7 is an experimental result of Example 7.
  • Figure 8 is a graph showing the results of the LCI protein interaction verification of Example 8.
  • FIG. 9 is a result of the verification of the pull-down interaction of the embodiment 8.
  • Figure 10 is a result of the Co-IP mutual verification of the embodiment 8.
  • FIG. 11 shows the experimental results of Example 9.
  • the vectors pCAMBIA2300 and pCAMBIA1300 are products of Cambia.
  • the cloning vector pMD18-T is a product of TaKaRa.
  • the vector pUC18 is a product of ThermoFisher.
  • Vector pUC-RNAi a fragment between the recognition sequences of the restriction endonucleases BamHI and BglII of the vector pUC18 (vector pUC18 was cut into a large fragment and a small fragment by restriction enzymes BamHI and BglII, the DNA being this small The fragment was replaced with the sequence of the intron of the potato GA20 oxidase gene, and a recombinant plasmid was obtained, which was designated as vector pUC-RNAi.
  • vector pCAMBIA2300-XmaI/AvrII cloning of the vector pCAMBIA2300 The point was replaced with a fragment containing the recognition sequence of restriction endonuclease XmaI and the recognition sequence of restriction endonuclease AvrII.
  • the rice susceptible variety Wuyujing No. 3 is a commercially available variety cultivated from Wujin Rice and Wheat Breeding Farm.
  • the rice susceptible variety Wuyujing No. 3 is hereinafter referred to as Wuyujing No. 3 or WYJ3.
  • the rice resistant variety Wulingyu No.1 is a commercial variety selected from the Agricultural College of Yangzhou University, the Seed Management Station of Jiangsuzhou and the Rice and Wheat Breeding Farm of Wujin District through multiple generations of backcross breeding.
  • the rice resistant variety Wulingyu No. 1 is hereinafter referred to as Wulingyu No. 1 or WLJ1.
  • the rice susceptible variety Lemont is a commercially available rice variety from the United States.
  • the rice susceptible variety Lemont is hereinafter referred to as Lemont.
  • the rice resistant variety Teqing is a commercially available variety cultivated by the Rice Research Institute of the Guangdong Academy of Agricultural Sciences.
  • the rice resistant variety Teqing is hereinafter referred to as Teqing or Teqing.
  • the near-isogenic line NIL39 was obtained by the inventor with Lemont as the recurrent parent, and the Teqing was obtained from the donor parent by molecular marker multi-generation assisted selection.
  • the near isogenic line NIL39 is hereinafter referred to as NIL39.
  • the Japanese susceptible variety Nipponbare is a commercially available rice variety from Japan.
  • the Japanese susceptible variety Nipponbare is hereinafter referred to as Nipponbare or Nipponbare.
  • the anti-strand leaf blight gene qSTV11 TQ derived from Teqing (the Stv-b i gene found in the present application is an allele, so in the present invention, if there is no other explanation, the gene is uniformly named Stv- The b i gene is located within a 55.7 kb interval between the 11th chromosome molecular marker CAPS2 and CAPS3 (Wu XJ, Zuo SM, Zhang YF, Zhu JK, Ma N, Tang JY, Chu CC, Pan XB.2011. Fine mapping of qSTV11 TQ , a major gene coferring resistance to rice strip disease. Theor. Appl. Genet. 122: 915-23).
  • STV11S gene Show, hereinafter referred to as the STV11S gene); in Teqing and Lemont, another alternative splicing of the first exon, Exon1A, was detected in front of the predicted first exon Exon1B, and two transcripts were detected, ie, transcription
  • STV11L nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing, hereinafter referred to as STV11L gene
  • transcript STV11S one transcript STV11-pa1 of LOC_Os11g31500 was detected in three varieties of Nipponbare, Teqing and Lemont
  • the nucleotide sequence is shown in SEQ ID NO: 5 in the Sequence Listing, hereinafter referred to as the STV11-pa1 gene).
  • the first intron can also function as a promoter, thus showing that the LOC_Os11g31480 gene contains two alternative splicing promoters and two alternatively spliced first exons.
  • vector pCAMBIA2300-XmaI/AvrII A fragment between the recognition sequences of the restriction endonucleases XmaI and AvrII of the vector pCAMBIA2300-XmaI/AvrII (vector pCAMBIA2300-XmaI/AvrII was cleaved into a large fragment and a small fragment by restriction endonucleases XmaI and AvrII
  • the DNA is replaced by the DNA molecule shown in positions 2621 to 11390 of the sequence 1 from the 5' end of the sequence listing to obtain a recombinant plasmid designated as T-480C.
  • T-480C was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, and was named AGL1/T-480C.
  • AGL1/500C was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, and was named AGL1/500C.
  • AGL1/480C was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, and was named AGL1/480C.
  • the vector pCAMBIA2300-XmaI/AvrII was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, and was named AGL1/pCAMBIA2300.
  • T 0 transgenic rice plants were harvested and screened by G418 to obtain T 1 transgenic rice. After T 1 generation transgenic rice was harvested, it was again screened by G418 and then sown to obtain T 2 generation transgenic homozygous rice.
  • Two T 2 generation transgenic rice lines (T-480C L1 and T-480C L2) were taken separately for the experiment of the third step.
  • AGL1/T-480C was replaced with AGL1/500C, and the other steps were the same, and a homozygous plant of the T 2 generation transgenic rice was obtained.
  • Two T 2 generation transgenic rice lines (500C L1 and 500C L2) were taken separately for the experiment of the third step.
  • AGL1/T-480C was replaced with AGL1/480C, and the other steps were the same, and a homozygous plant of T 2 generation transgenic rice was obtained.
  • Two T 2 generation transgenic rice lines (480C L1 and 480C L2) were taken separately for the experiment of the third step.
  • AGL1 / T-480C is replaced with AGL1 / pCAMBIA2300, other steps are the same, to give homozygous plants T 2
  • the total RNA of T-480C L1 grown to 4 weeks was extracted by Trizol method, and the total RNA of T-480C L1 was obtained.
  • the total RNA was reverse transcribed from the first strand cDNA by reverse transcriptase, referred to as T-480C.
  • T-480C L1 was replaced with T-480C L2, 480C L1, 480C L2, 500C L1 and 500C L2, respectively, to obtain T-480C L2 cDNA, 480C L1 cDNA, 480C L2 cDNA, 500C L1 cDNA and cDNA of 500C L2.
  • T-480C L1 was replaced with Wuyujing No. 3 to obtain a blank control cDNA.
  • T-480C L1 was replaced with the empty vector rice to obtain an empty control cDNA.
  • T-480C L1 was replaced with Wulingjing No. 1, and a positive control cDNA was obtained.
  • the primers for identifying the STV11L gene were 5'-AGGCCGTCATCTACCTCA-3' and 5'-ACATTCAGCACAAGGTTTCT-3'.
  • the primers for identifying the STV11S gene are 5'-GCCTCTCAGCTCGATCTAC and 5'-ACATTCAGCACAAGGTTTCT-3'.
  • the primers for identifying the STV11-pa1 gene were 5'-TCTGCCACTGGATTGACTA-3' and 5'-GGGTCCAATCTTGTCTATGA-3'.
  • the primers for identifying the Actin1 gene were 5'-CAGGCCGTCCTCTCTCTGTA-3' and 5'-AAGGATAGCATGGGGGAGAG-3'.
  • FIG. 3, C the expression level of STV11L gene in Wulingjing 1 was taken as 1, and the expression levels of STV11L gene in transgenic vector rice, 480C L1 and 480C L2 were 0, 1.2 and 2.08, respectively.
  • the expression of STV11L gene was not detected in ⁇ 3 and transgenic rice.
  • the expression level of STV11S gene in transgenic vector rice was 1, and the expression of STV11S gene in T-480C L1 and T-480C L2 was 5.8.
  • STV11S gene There was no significant difference in the expression of STV11S gene in rice with 13.4, Wuyujing 3 and transgenic rice; the expression level of STV11-pa1 gene in transgenic rice was used as STV11-pa1 gene in 1,500C L1 and 500C L2 The expression levels of 4.5 and 2.9, respectively, were not significantly different between the expression of STV11-pa1 gene in Wuyujing 3 and transgenic rice.
  • Rice stripe disease resistance was determined by using Wuyujing No. 3, Wulingyu No. 1, transgenic rice, T-480C L1, T-480C L2, 480C L1, 480C L2, 500C L1 and 500C L2 as experimental materials. Sex.
  • each strain was planted with 40 plants in 2L glass beakers.
  • each rice seedling was inoculated with 4 2-3 years old rice stripe virus nymphal nymph (2-3
  • the age-bearing rice stripe virus, the nymphal nymph was provided by the Plant Protection Institute of Jiangsu Academy of Agricultural Sciences), and the gray planthopper was driven 4 times a day to facilitate uniform exposure of the tested rice seedlings.
  • all the gray planthoppers were removed with a straw, and the rice seedlings were transplanted into a rice greenhouse without the gray planthopper, and the incidence of the experimental materials was investigated about one month later.
  • the disease resistance of rice stripe disease in each test material was determined by two indicators: 1) the ratio of healthy plants, that is, the percentage of plants that were not infected after the infestation accounted for the total number of plants (Wu XJ, Zuo SM, Zhang YF, Zhu JK, Ma N, Tang JY, Chu CC, Pan XB.2011. Fine mapping of qSTV11 TQ , a major gene coferring resistance to rice stripdisease.
  • Theor.Appl.Genet.122:915-23); 2) The disease index, that is, according to the severity of the disease, according to Washio's resistance identification criteria, the incidence of each seedling is divided into six levels of A, B, Bt, Cr, C and D, and the number of different grades in each family is counted. Calculate the disease index according to the formula (100 ⁇ A + 80 ⁇ B + 60 ⁇ Bt + 40 ⁇ Cr + 20 ⁇ C + 5 ⁇ D) / total number of seedlings tested (Washio O, Ezuka A, Sakurai Y, Toriyama K. 1967. Studies on the breeding of rice varieties resistant to rice stripe disease. I. Varietaldifference in resistance to stripe disease. Jpn. J. Breed. 17: 91-98).
  • the LOC_Os11g31480 gene contains two alternative splicing promoters and two alternatively spliced first exons, ie, the transcript shown in SEQ ID NO: 2 in the Sequence Listing STV11L and the transcript STV11S shown in SEQ ID NO:3 in the Sequence Listing.
  • the T-480C L1 and T-480C L2 obtained in step 2 were identified for the stripe disease phenotype.
  • the results showed that T-480C L1 and T-480C L2 did not show significant resistance to stripe disease caused by Wuyujing No.3. This result indicates that the interaction of STV11L or STV11L with STV11S plays a key role in the resistance of stripe disease.
  • Example 3 Acquisition and identification of Stv-b i silenced strain
  • DNA molecule 1 a. PCR amplification of Teqing genomic DNA to obtain DNA molecule 1.
  • the nucleotide sequence of DNA molecule 1 is shown in the sequence 2 of the sequence listing from positions 4670 to 5031 (length 362 bp) from the 5' end.
  • DNA molecule 1 and the cloning vector pMD18-T were ligated to obtain a recombinant plasmid.
  • the sequencing results showed that the recombinant plasmid contained DNA molecule 1.
  • the recombinant plasmid constructed in step b was digested with restriction endonucleases SalI and BamHI to recover a DNA fragment of about 360 bp.
  • the vector pUC-RNAi was digested with the restriction enzymes SalI and BamHI, and the vector backbone 1 was recovered (the vector pUC-RNAi was cut into a large fragment and a small fragment, and the vector backbone 1 was a large fragment).
  • the vector backbone 1 and the DNA fragment A are ligated to obtain a recombinant plasmid.
  • the recombinant plasmid constructed in step e was digested with restriction endonucleases BglII and XhoI, and the vector backbone 2 was recovered (the recombinant plasmid constructed in step e was cut into a large fragment and a small fragment, and the vector backbone 2 was a large fragment).
  • the vector backbone 2 and the DNA fragment A are ligated to obtain a recombinant plasmid.
  • the recombinant plasmid constructed in step g was digested with restriction endonuclease PstI, and the DNA fragment C was recovered (the recombinant plasmid constructed in step g was cut into a large fragment and a small fragment, and the DNA fragment C was a small fragment).
  • the expression vector pCAMBIA2301A (recombined by the expression vector pCAMBIA2300 with the Actin1 promoter and OCS terminator) was digested with restriction endonuclease PstI, and the vector backbone 3 was recovered (the vector pCAMBIA2301A was cut into a large fragment and a small fragment, The carrier skeleton 3 is a large fragment).
  • 480Ri a recombinant plasmid designated as 480Ri.
  • 480Ri is based on the expression vector pCAMBIA2300, and the following expression cassette is inserted into the restriction site: from the upstream to the downstream, the Actin1 promoter, the DNA fragment C and the OCS terminator are sequentially composed.
  • DNA molecule 1 was replaced with the DNA molecule 2, and the other steps were unchanged, and a recombinant plasmid was obtained, which was named 500Ri.
  • the nucleotide sequence of DNA molecule 2 is shown in the sequence 5 of the sequence listing from positions 4469 to 4814 (length 346 bp) from the 5' end.
  • 480Ri was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, and was named AGL1/480Ri.
  • 500Ri was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, and was named AGL1/500Ri.
  • pCAMBIA2301A was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, designated as AGL1/pCAMBIA2301A, as a control.
  • the Wulingjing No. 1 seed is shelled and sterilized, and then subjected to Agrobacterium infection method (Liu, XQ, Bai, XQ, Wang, XJ & Chu, CC2007. OsWRKY71, a rice transcription factor, is involved in rice defense response. J. Plant Physiol. 164, 969-79)
  • AGL1/480Ri was transformed into Wulingjing No. 1, and a T 0 generation silent strain was obtained.
  • the seed produced by self-crossing of T 0 generation silenced strain was named T 1 generation silent seed, and the rice plant grown from T 1 generation silenced seed was named as T 1 generation silenced strain, and the T 1 generation transgenic rice was harvested again and then passed G418 again. Seeding after screening, T 2 generation transgenic homozygous rice was obtained.
  • Two T 2 generation transgenic rice lines were designated as 480Ri L1 and 480Ri L2, respectively, for the subsequent step 3.
  • AGL1/480Ri was replaced with AGL1/500Ri, and the other steps were the same.
  • T 2 generation homozygous silenced strain was obtained, and two silent strains were randomly selected and named as 500Ri L1 and 500Ri L2.
  • AGL1/480Ri was replaced with AGL1/pCAMBIA2301A, and the other steps were the same, and the T 2 generation homozygous empty vector rice plant was obtained, hereinafter referred to as the transgenic vector rice.
  • RNA of 480Ri L1 and 480Ri L2 leaf sheath tissues grown to 4 weeks and fully expanded by leaves was extracted by Trizol method to obtain total RNA of 480Ri L1 and 480Ri L2, and the total RNA was reverse transcribed by reverse transcriptase.
  • the strand cDNA abbreviated as 480Ri L1 cDNA and 480Ri L2 cDNA.
  • RNA of 500Ri L1 and 500Ri L2 spikes grown to 5 cm in young ears was extracted by Trizol method to obtain total RNA of 500Ri L1 and 500Ri L2, and the total RNA was reverse transcribed from the first strand cDNA by reverse transcriptase. , abbreviated as 500Ri L1 cDNA and 500Ri L2 cDNA.
  • the primers for identifying the STV11L gene were 5'-AGGCCGTCATCTACCTCA-3' and 5'-ACATTCAGCACAAGGTTTCT-3'.
  • Primers for identifying the STV11S gene were 5'-GCCTCTCAGCTCGATCTAC-3' and 5'-ACATTCAGCACAAGGTTTCT-3'.
  • the primers for identifying the STV11-pa1 gene were 5'-TCTGCCACTGGATTGACTA-3' and 5'-GGGTCCAATCTTGTCTATGA-3'.
  • the primers for identifying the Actin1 gene were 5'-CAGGCCGTCCTCTCTCTGTA-3' and 5'-AAGGATAGCATGGGGGAGAG-3'.
  • the expression of STV11S gene in transgenic rice was used as the expression of STV11S gene in 1,480Ri L1, 480Ri L2, 500Ri L1 and 500Ri L2.
  • STV11S gene in Wulingjing 1 and the empty-transferred rice were not significantly different in the quantities of 0.21, 0.18, 0.79 and 0.87, respectively.
  • the expression level of STV11-pa1 gene in transgenic rice was 1,480Ri L1, 480Ri.
  • the expression levels of STV11-pa1 gene in L2, 500Ri L1 and 500Ri L2 were 0.73, 0.91, 0.17 and 0.12, respectively. There was no significant difference in the expression of STV11-pa1 gene in Wulingjing 1 and transgenic rice.
  • the resistance of stripe leaf blight in rice was identified by using Wulingyu No.1, empty carrier rice, Wuyujing No.3, 480Ri L1, 480Ri L2, 500Ri L1 and 500Ri L2 as experimental materials.
  • the identification method is the same as that in the fourth step of the second embodiment.
  • Seedlings of different developmental stages of the near-isogenic line NIL39 containing the chromosome fragments of the Sterling Stv-b i resistance gene were collected in the background of Lemont and Lemont (including 2, 3, 4, and 5 weeks old seedlings, corresponding to the map). 2w, 3w, 4w, 5w Shoot) in 5, leaves (including 1 week before unfolding, 1 day before unfolding, 0 days after unfolding, and 1 week after unfolding), correspondingly labeled as -1w, 0d in Figure 5.
  • leaf sheath including the corresponding leaf sheath of the three age groups 1 week before deployment, 0 days after unfolding, and 1 week after unfolding, correspondingly labeled as -1w, 0d, 1w Leaf sheath in Figure 5
  • Spikes including the young ears of the 5 cm stage and the old ears of 3 weeks after flowering, corresponding to the Y and O Panicle in Figure 5
  • stems including the young stems of the jointing stage and the old stems of the three sections
  • Samples labeled as Y and O Culm in Figure 5 and roots including 2 weeks old roots and old roots at jointing stage, corresponding to Y and O Root in Figure 5 were placed in liquid nitrogen.
  • the total RNA of each sample in the step (1) was extracted using a Trizol kit (product of Invitrogen Life Technologies), and the total RNA was reverse transcribed out of the first strand cDNA using a reverse transcription kit (product of Toyobo Co., Ltd.), and then The cDNA was used as a template, and the relative expression level of the STV11L gene or the STV11S gene in each sample was quantitatively detected by a real-time quantitative PCR instrument (Bio-Rad) (Actin1 gene was used as an internal reference gene).
  • the primers for identifying the STV11L gene were 5'-AGGCCGTCATCTACCTCA-3' and 5'-ACATTCAGCACAAGGTTTCT-3'.
  • Primers for identifying the STV11S gene were 5'-GCCTCTCAGCTCGATCTAC-3' and 5'-ACATTCAGCACAAGGTTTCT-3'.
  • the primers 5'-CAGGCCGTCCTCTCTCTGTA-3' and 5'-AAGGATAGCATGGGGGAGAG-3' of the Actin1 gene were identified.
  • the results showed that the expression of STV11L gene and STV11S gene were detected in tissues at different stages, and the relative expression of these two genes in the old leaf sheath was high.
  • the experimental results were related to the stripe leaf virus vector insect ash.
  • the feeding habit of the locust is that the phenotype of the outer layer of the outer leaf sheath is completely consistent.
  • the cDNA sequence of the rice Stv-b i gene was used as a probe and compared in a public database ( www.ncbi.nih.nlm.gov ). The alignment showed that there was Stv-b i on chromosome 12 of rice.
  • the third homologous gene of the gene, LOC_Os12g29350 has a nucleotide sequence of transcript STV11-pa2 as shown in SEQ ID NO:6 in the sequence listing, and expresses the protein STV11-pa2 shown in SEQ ID NO:8 in the sequence listing.
  • transcript STV11-pa2 At the nucleotide level, there is 58.5% sequence identity between transcript STV11-pa2 and transcript STV11L, and 57.3% sequence identity between transcript STV11-pa2 and transcript STV11-pa1, transcript STV11L and The transcript STV11-pa1 has 74% sequence identity; at the amino acid level, there is 50.2% sequence identity between protein STV11-pa2 and protein STV11L, and 48.5% between protein STV11-pa2 and protein STV11-pa1. Sequence identity, protein STV11L has a sequence identity of 67.9% with protein STV11-pa1.
  • amino acid sequence alignment indicated that the protein STV11-pa1 has higher sequence identity with the protein STV11L, suggesting that the protein STV11-pa1 and protein STV11L There may be functional conservatism.
  • the constitutive high level of CaMV 35S promoter of cauliflower mosaic virus was used to drive the expression of STV11-pa1 gene in Wuyujing 3 to detect this. The function of the protein.
  • Recombinant plasmid 500ox The expression vector pCAMBIA2300 was used as a starting vector, and the following expression cassette was inserted into the restriction site: from the upstream to the downstream, the cDNA molecule represented by the CaMV 35S promoter, the sequence 5 in the sequence listing, and the polyA termination sequence were sequentially composed.
  • the recombinant plasmid 500ox is hereinafter referred to as 500ox.
  • AGL1/500ox was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, which was named AGL1/500ox.
  • the vector pCAMBIA2300-35S was introduced into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium, designated as AGL1/pCAMBIA2300-35S, as a control.
  • the Wuyujing No. 3 seed is shelled and sterilized, and then subjected to Agrobacterium infection method (Liu, XQ, Bai, XQ, Wang, XJ & Chu, CC2007. OsWRKY71, a rice transcription factor, is involved in rice defense response. J. Plant Physiol. 164, 969-79)
  • AGL1/500ox was transformed into Wuyujing No. 3 to obtain a T 0 transgenic line.
  • T 0 transgenic rice plants were harvested and screened by G418 to obtain T 1 transgenic rice. After T 1 generation transgenic rice was harvested, it was again screened by G418 and then sown to obtain T 2 generation transgenic homozygous rice.
  • Two T 2 generation transgenic rice lines (500ox L1 and 500ox L2) were taken separately for the next step of the experiment.
  • AGL1 / 500ox replaced AGL1 / pCAMBIA2300-35S, other steps are the same, to give homozygous plants T 2
  • RNA of 500ox L1 and 500ox L2 aboveground tissues grown to 4 weeks and fully expanded by leaves was extracted by Trizol method.
  • the total RNA was reverse transcribed from the first strand cDNA by reverse transcriptase, abbreviated as 500ox L1 cDNA and cDNA of 500ox L2.
  • 500 ox L1 was replaced with the empty vector rice to obtain an empty control cDNA.
  • the relative expression level of the STV11-pa1 gene was quantitatively detected in real time (with the Actin1 gene as an internal reference).
  • the primers for identifying the STV11-pa1 gene were 5'-TCTGCCACTGGATTGACTA-3' and 5'-GGGTCCAATCTTGTCTATGA-3'.
  • the primers for identifying the Actin1 gene were 5'-CAGGCCGTCCTCTCTCTGTA-3' and 5'-AAGGATAGCATGGGGGAGAG-3'.
  • the expression level of STV11L gene in transgenic vector rice was 1629 and 1756 as the expression levels of STV11L gene in 1,500ox L1 and 500ox L2, respectively, Wuyujing 3 and empty vector There was no significant difference in the expression level of STV11-pa1 gene in rice.
  • DNA molecule 3 is as shown in the sequence of Table 2 from the 5' end at positions 128 to 727 (length 600 bp).
  • the DNA molecule 3 was ligated to the restriction enzyme cleavage site NdeI and XhoI on the cloning vector pGBKT7 by the InFusion cloning system (Clontech) to obtain a recombinant plasmid which was designated as a bait vector and designated as BD-STV11 N .
  • the sequencing results showed that the recombinant plasmid contained DNA molecule 3.
  • DNA molecules 4, 5, 6, and 7 are shown as positions 1 to 690 (length 690 bp) and 688 to 1341 of sequence 10 from the 5' end of the sequence table, respectively (length 654 bp) ), shown in bits 688 to 960 (length 273 bp), 961 to 1341 (length 381 bp).
  • the DNA molecules 4, 5, 6, and 7 were ligated to the restriction enzyme sites NdeI and PstI on the cloning vector pGADT7 by the InFusion cloning system (Clontech) to obtain the recombinant plasmid, which is the SIP1 verification vector.
  • the recombinant plasmid which is the SIP1 verification vector.
  • AD-STV11 D1 , AD-STV11 D23 , AD-STV11 D2 , and AD-STV11 D3 The sequencing results showed that the recombinant plasmid contained DNA molecules 4, 5, 6, and 7, respectively.
  • the BD-STV11 N decoy vector and the pGBKT7 empty vector were separately transformed into the yeast strain Gold Yeast (Clontech). After the culture of the bacterium, the transformed colonies were placed on the medium containing SD/-Trp/Xa-Gal and cultured at 30 °C. 3 to 4 days. Observations showed that the colonies transformed with the bait vector BD-STV11 N grew normally on SD/-Trp/Xa-Gal medium and showed no blue color, indicating that the bait protein was in yeast cells. It is non-toxic and has no self-activating activity and can be used in yeast two-hybrid library screening experiments.
  • yeast two-hybrid screening we identified the interaction protein SIP1 of STV11L.
  • yeast two-hybrid validation assay we found that the two domains of the carboxy terminus of SIP1 (corresponding vector number AD-STV11 D23 ) play an irreplaceable role in the interaction between STV11L and SIP1 (Fig. 7).
  • the expression vector pCAMBIA1300-nLuc (hereinafter abbreviated as nLuc) was used as the starting vector, and the full-length cDNA of STV11L was inserted into the SacI/SalI at the restriction site, that is, the sequence shown in the sequence table from the 5' end from the 5' end to the sequence of the sequence 2 (length) It is 5130bp).
  • the recombinant plasmid obtained is hereinafter referred to as STV11L-nLuc.
  • the expression vector pCAMBIA1300-cLuc (hereinafter abbreviated as cLuc) was used as a starting vector, and the sequence shown in SEQ ID NO: 10 (length: 1344 bp) was inserted into the full length cDNA of SIP1 at the restriction site KpnI/PstI.
  • the recombinant plasmid obtained is hereinafter referred to as cLuc-SIP1.
  • the following expression cassette was inserted into the restriction enzyme site: from upstream to downstream, the CaMV 35S promoter, the cDNA molecule shown by the sequence 10 in the sequence listing, and the polyA termination sequence were sequentially composed.
  • the recombinant plasmid obtained is hereinafter referred to as SIP1ox.
  • the expression vector pGEX-6P-1 (GE Healthcare) was used as the starting vector, and the full-length cDNA of STV11L was inserted into the restriction enzyme site, that is, the sequence shown in the sequence list from the 5' end of the sequence 2 to the position of 128 to 5260 (length 5133 bp) .
  • the recombinant plasmid obtained is hereinafter referred to as GST-STV11L.
  • the following expression cassette was inserted into the restriction enzyme site: from the upstream to the downstream, the CaMV 35S promoter, the sequence 2 in the sequence listing from the 5' end, from the 128th to the 5260th position, 3 ⁇ Flag tag sequence molecule and polyA termination sequence composition.
  • the recombinant plasmid obtained is hereinafter referred to as STV11L-Flag.
  • Agrobacterium containing recombinant plasmid combinations STV11L-nLUC/cLuc-SIP1, STV11L-nLUC/cLuc, nLUC/cLuc-SIP1, nLuc/cLuc were injected into corresponding regions of tobacco leaves, respectively. After 3 days, tobacco leaves injected with Agrobacterium were sprayed with 1 mM Luciferin (Promega) on the surface of the leaves, left to stand for 5 minutes, and then placed in a low-light cooled CCD imaging apparatus (CHEMIPROHT1300B/LND, 16bits, Roper Scientific).
  • the SIP1ox recombinant plasmid was expressed in tobacco leaves by Agrobacterium infection. Use liquid nitrogen to express The tobacco leaves of the SIP1 protein were ground uniformly, followed by the addition of an appropriate amount of protein extract (0.01 M PBS [Ph7.3], 0.1% Triton X-100, EDTA-free protease inhibitor cocktail [Roche], 1 mM PMSF). After centrifugation at 15,000 rpm for 30 minutes, the supernatant was taken to obtain a crude SIP1 protein extract. The pGEX-6P-1 empty vector and the GST-STV11L recombinant plasmid were transformed into E. coli BL21 (DE3) pLysS (Promega) competent cells, respectively.
  • protein expression was induced overnight at 16 °C using 0.6 mM isopropyl ⁇ -D-1-thiogalactopyranoside (Pierce).
  • the GST and GST-STV11L proteins were subsequently purified by Glutathione Sepharose 4FF beads (GE Healthcare).
  • Glutathione Sepharose 4FF beads bound to GST or GST-STV11L were co-cultured with the crude SIP1 protein extract at 4 ° C for 1 hour, and then the above-mentioned beads were washed with protein extract and appropriate amount of washed beads were dissolved in 1 ⁇ SDS.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Agrobacterium containing recombinant plasmid combination STV11L-Flag/SIP1ox and Flag (empty vector after STV11L full-length cDNA removal in STV11L-Flag recombinant plasmid)/SIP1ox was injected into tobacco leaves, respectively. After 3 days, total protein was extracted from tobacco leaves injected with Agrobacterium. The two protein extracts were incubated with appropriate amounts of anti-Flag magnetic beads (Sigma) for two hours at 4 °C. The above beads were then washed at least five times using 4 ° C pre-cooled IP buffer. An appropriate amount of beads was used for immunoblot analysis using anti-Flag (Sigma) and anti-SIP1 antibodies, respectively. The results show that STV11L can co-precipitate SIP1 (Fig. 10), again demonstrating the interaction between STV11L and SIP1.
  • DNA molecule 1 was replaced with the DNA molecule 8, and the other steps were unchanged, and a recombinant plasmid was obtained, which was named SIP1Ri.
  • SIP1Ri The nucleotide sequence of DNA molecule 8 is shown in SEQ ID NO: 228 to 545 from the 5' end of the sequence 9 in the sequence listing (length 318 bp).
  • SIP1 silence inhibits RSV virus replication
  • SIP1 is an eEF1A (eukaryotic translation elongation factor 1alpha) protein. Encoded by four highly homologous genes on the genome. The gene coding region sequences are sequence 10, sequence 11, sequence 12, and sequence 13 in the sequence listing, respectively. The protein sequence corresponds to sequence 9 in the sequence listing.
  • eEF1A eukaryotic translation elongation factor 1alpha
  • RNA was sampled at 0, 24, and 48 hours after transfer and the corresponding cDNA was prepared.
  • the relative expression levels of the SIP1 gene and the RSV gene RSVCP were quantitatively detected in real time (with the Actin1 gene as an internal reference).
  • the primer for identifying the SIP1 gene is 5'-GGAGAAGACGCACATCAACA-3' and 5'-CTCTTGGGCTCGTTGATCTG-3'.
  • the primers for identifying the RSVCP gene were 5'-TTTGCAGAAGGCAATCAATG-3' and 5'-CTGGCATGGTCTTTTTGGTT-3'.
  • the primers for identifying the Actin1 gene were 5'-CAGGCCGTCCTCTCTCTGTA-3' and 5'-AAGGATAGCATGGGGGAGAG-3'.
  • eEF1A plays a key role in the conservation of RNA viruses in other plants and animals (Sasikumar, AN, Perez WB, Kinzy TG.2012. The many roles of the eukaryotic Elongation factor 1complex.Wiley Interdiscip.Rev.RNA.3: 543-555; Li D, Wei T, Abbott CM, Harrich D. 2013. The unexpected roles of eukaryotic translation in the form of RNA virus replication and pathogenesis. Microbiol.Mol. Biol. Rev. 77: 253-266).
  • the present patent also provides a method for regulating the function of the eEF1A protein by manipulating the expression of the STV11L protein in different species, thereby improving the resistance of the corresponding species to the RNA virus.
  • the protein provided by the present invention and the gene encoding the same can regulate the disease resistance of plant RNA virus disease. Therefore, the protein provided by the present invention has broad prospects for breeding crop varieties resistant to RNA virus diseases. At the same time, the protein and the coding gene thereof provided by the invention have great application potential in the field of human and poultry RNA virus disease prevention and treatment.

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Abstract

一种水稻条纹叶枯病抗性基因Stv-bi及其应用。Stv-bi的氨基酸序列如序列表中序列4或序列表中序列7所示。Stv-bi的蛋白质通过与生物体内功能保守的eEF1A蛋白质直接互作来调控eEF1A蛋白质的生物学功能。实验证明，提供的Stv-bi蛋白质对调控条纹叶枯病的抗性具有重要作用，在培育抗条纹叶枯病的水稻品种中具有广阔前景。

Description

水稻条纹叶枯病抗性基因Stv-bi及其应用 技术领域
本发明涉及基因工程领域，具体涉及水稻条纹叶枯病抗性基因Stv-bi及其应用。
背景技术
水稻(Oryza sativa L.)作为全球近半数人口最主要的粮食作物，其产量及品质长期经受各类细菌、真菌、以及病毒等病原菌的严重威胁。多年的生产实践表明，通过利用植物自身的抗病基因(R基因)培育抗性品种是最为经济、安全而又有效的应对措施(Ronald PC.1997.The molecular basis of disease resistance in rice.Plant Mol.Biol.35:179-186)。在过去的十几年中，多个水稻白叶枯病及稻瘟病抗病基因被成功克隆并有效的整合到水稻的抗性改良研究和应用中(Helliwell EE,Yang Y.2013.Molecular strategies to improve rice disease resistance.Methods Mol.Biol.956:285-309.)，但有关水稻抗病毒病基因的分离和应用鲜有报道(Mandadi KK,Scholthof KB.2013.Plant immune responses against viruses:how does a virus cause disease？Plant Cell 25:1489-1505)。
条纹叶枯病(rice stripe disease)是水稻最为主要的病毒病之一。这一病害由水稻条纹病毒(rice stripe virus，RSV)引起，后者是第一个报道的具有双义编码策略的植物病毒，主要通过灰飞虱(Laodelphax striatellus Fallén)以较为持久的方式传播(Ramirez BC,Haenni AL.1994.Molecular biology of tenuiviruses,a remarkable group of plant viruses.J.Gen.Virol.75:467-475)。该病毒侵染水稻后，发病较早的植株心叶沿叶脉呈现断续的黄绿色或黄白色条斑，随后这些条斑融合成片，心叶卷曲成纸捻状，弯曲下垂呈“假枯心”；发病迟的植株只在剑叶或叶鞘上有褪色斑，抽穗不良或畸形不结实。此外，发病植株一般还表现为分蘖少和矮化(Fargette D，Ghesquiere A，Albar L，Thresh JM.2006.Virus resistance in rice.G.Loebenstein and J.P.Carr(eds.).Natural Resistance Mechanisms of Plants to Virus.431-446)。不同于细菌或真菌病害的是，条纹叶枯病一旦发病，就无法通过现有的理化方法逆转，因此也是水稻种植上最难防治的病害之一。
条纹叶枯病频繁爆发于温带及亚热带地区，包括中国、朝鲜、日本以及远东。早在上世纪60年代，日本每年超过600，000公顷的水稻种植面积(Shimizu T,Nakazono-Nagaoka E，Uehara-Ichiki T，Sasaya T，Omura T.2011.Targeting specific genes for RNA interference is crucial to the development of strong resistance to Rice stripe virus.Plant Biotechnol.J.9:503-512)，韩国40％的水稻种植面积(Jonson MG，Choi HS，Kim JS，Choi IR，Kim KH.2009.Complete genome dequence of the RNAs 3and 4segments of rice  stripe virus isolates in Korea and their phylogenetic relationshipswith Japan and China isolates.Plant Pathol.J.25:142-150)，以及中国20多个省大约2，660，000公顷的水稻种植面积受到RSV爆发的影响(Wang HD，Chen JP，Zhang HM，Sun XL，Zhu JL，Wang AG，Sheng WX，Adams MJ.2008.Recent Rice stripe virus epidemics in Zhejiang province，China，and experiments on sowing date，disease–yield loss relationships，and seedling susceptibility.Plant Dis.92:1190-1196)。此后，RSV多次爆发于上述粳稻种植区，其中从2000年开始的最近一轮大规模条纹叶枯病爆发引起了人们的严重关切。这轮RSV爆发在中国多个省份，普遍性的导致了20％～30％的产量损失，个别重病区引起了30～40％的产量损失，甚至颗粒无收(Zhang S，Li L，Wang X，Zhou G.2007.Transmission of rice stripe virus acquired from frozen infected leaves by the small brown planthopper(Laodelphax striatellus Fallén).J.Virol.Methods 146:359-362)；而在韩国同样影响了84％的水稻种植区，造成了巨大的经济损失(Jonson MG，Choi HS，Kim JS，Choi IR，Kim KH.2009.Complete genome sequence of the RNAs 3and 4segments of rice stripe virus isolates in Korea and their phylogenetic relationshipswith Japan and China isolates.Plant Pathol.J.25:142-150)。
通过对大量品种的表型筛选，日本育种家们在上世纪60年代发现一些籼稻、热带粳稻以及一些粳型旱稻中存在RSV的有效抗源，而其它温带粳稻品种普遍感条纹叶枯病(Washio O，Ezuka A，Sakurai Y，Toriyama K.1967.Studies on the breeding of rice varieties resistant to rice stripe disease.I.Varietal difference in resistance to stripe disease.Jpn.J.Breed.17:91-98)。其中，来自旱稻品种的两个抗性基因Stv-a和Stv-b虽然具有显著且稳定的RSV抗性，但是由于其与其它产量和品质副效基因紧密连锁，一直没有在RSV抗性育种中得到推广利用。一些籼稻品种中含有Stv-b的等位基因Stv-bi基因具有不完全显性的抗病性。从1960s起，来源于籼稻品种‘Modan’的Stv-bi基因被导入粳稻品种，随后这些中间品种被广泛地应用到抗RSV的优质粳稻品种的选育中。在过去的近半个世纪中，Stv-bi基因是唯一一个提供持续高效抗性同时不影响水稻品质和产量的RSV抗性基因，迄今还没有致病小种产生的报道(Hayano-Saito Y，Tsuji T，Fuji i K，Saito K，Iwasaki M，Saito A.1998.Localization of the rice stripe disease resistance gene，Stv-bi，by graphical genotyping and linkage analysis with molecular markers.Theor.Appl.Genet.96，1044-1049)。由此，克隆Stv-bi基因，有助于揭开水稻抗病毒分子机制；为单子叶植物的抗病毒机制研究提供参考；同时也为进一步的RSV抗性育种尤其是基因工程改良育种以及分子标记辅助育种提供依据，以更好地控制和减低RSV对水稻生产的危害。
发明公开
本发明所要解决的技术问题是如何提高水稻的条纹叶枯病抗性。
为解决上述技术问题，本发明首先提供了蛋白质。
本发明所提供的蛋白质，可为如下a1)或a2)或a3)或a4)或a5)或a6)：
a1)氨基酸序列是序列表中序列4所示的蛋白质；
a2)在序列表中序列4所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
a3)氨基酸序列是序列表中序列7所示的蛋白质；
a4)在序列表中序列7所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
a5)将a1)或a2)或a3)或a4)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与植物抗病性相关的蛋白质；
a6)与a1)或a2)或a3)或a4)所示的蛋白质具有70％或70％以上同一性的蛋白质。
其中，序列表中序列4可由1710个氨基酸残基组成。序列表中序列7可由1708个氨基酸残基组成。
为了使a1)或a3)中的蛋白质便于纯化，可在序列表中序列4或序列表中序列7所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1.标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述a5)中的蛋白质，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过850个氨基酸残基的取代和/或缺失和/或添加。
上述a5)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述a5)中的蛋白质的编码基因可通过将序列表中序列2、序列表中序列5、序列表中序列2自5′末端起第128至5260位、或序列表中序列1所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
这里使用的术语“同一性”指与天然氨基酸序列相似性。“同一性”包括与本发明序列表的序列4或序列7所示的氨基酸序列具有70％或更高，或80％或更高，或90％或更高同一性的氨基酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
所述抗病性可为抗条纹叶枯病。
所述抗病性可为抗水稻条纹病毒引起的病害。
所述植物可为c1)至c14)中的任一种：c1)双子叶植物；c2)单子叶植物；c3)禾本科植物；c4)水稻；c5)大麦；c6)小麦；c7)烟草；c8)番茄；c9)燕麦；c10)高粱；c11)玉米；c12)棉花；c13)大豆；c14)油菜。所述水稻具体可为水稻品种武育粳3号或水稻品种武陵粳1号。
所述蛋白质的活性片段也属于本发明的保护范围。
编码所述蛋白质的核酸分子也属于本发明的保护范围。
编码所述蛋白质的核酸分子，具体可为如下(b1)或(b2)或(b3)或(b4)或(b5)或(b6)所示的DNA分子：
(b1)核苷酸序列是序列表中序列2所示的DNA分子；
(b2)核苷酸序列是序列表中序列2自5′末端起第128至5260位所示的DNA分子；
(b3)核苷酸序列是序列表中序列1所示的DNA分子；
(b4)核苷酸序列是序列表中序列5所示的DNA分子；
(b5)与(b1)或(b2)或(b3)或(b4)限定的核苷酸序列具有50％或50％以上同一性，且编码所述蛋白质的DNA分子；
(b6)在严格条件下与(b1)或(b2)或(b3)或(b4)限定的核苷酸序列杂交，且编码所述蛋白质的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
序列表中序列1由11390个核苷酸组成。序列表中序列2由5260个核苷酸组成，序列表中序列2的核苷酸编码序列表中序列4所示的氨基酸序列。序列表中序列5由5127个核苷酸组成，序列表中序列5的核苷酸编码序列表中序列7所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码所述蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的所述蛋白质的核苷酸序列50％或者更高同一性的核苷酸，只要编码所述蛋白质，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列表的序列4所示的氨基酸序列组成的蛋白质的核苷酸序列具有50％或更高，或60％或更高，或70％或更高，或80％或更高，或90％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
基于所述核酸分子开发的分子标记甲，或，与所述核酸分子紧密连锁并可用于鉴定或辅助鉴定植物抗病性的分子标记乙也属于本发明的保护范围。
含有或部分含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系也属于本发明的保护范围。
所述重组载体为向表达载体或克隆载体插入所述核酸分子得到的重组质粒。
所述表达载体具体可为实施例中提及的载体pCAMBIA2300或载体pCAMBIA2300的衍生载体pCAMBIA2300-XmaI/AvrII等。
所述重组载体具体可为实施例中提及的480C或500ox。
所述重组微生物为将所述重组载体导入出发微生物得到的重组菌。
所述重组微生物具体可为将所述重组质粒480C或500ox导入出发微生物得到的重组菌。
所述出发微生物可为根癌农杆菌。
所述根癌农杆菌具体可为根癌农杆菌AGL1。
所述转基因细胞系可为将所述重组载体转化受体细胞获得的。
所述转基因细胞系具体可为将所述重组质粒480C或500ox转化受体细胞获得的。
与所述蛋白质结合的抗体也属于本发明的保护范围。
d1)或d2)或d3)或d4)的应用也属于本发明的保护范围：
d1)所述蛋白质，或，所述核酸分子，或，含有或部分含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在调控植物抗病性中的应用；
d2)所述蛋白质，或，所述核酸分子，或，含有或部分含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在培育抗病性提高或降低的转基因植物中的应用；
d3)所述蛋白质，或，所述核酸分子，或，含有或部分含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在制备抗病性药物中的应用；
d4)所述核酸分子产生的分子标记在选育抗病性植物中的应用。
上文中，所述抗病性可为抗条纹叶枯病。
上文中，所述抗病性可为抗水稻条纹病毒引起的病害。
上文中，所述植物可为c1)至c14)中的任一种：c1)双子叶植物；c2)单子叶植物；c3)禾本科植物；c4)水稻；c5)大麦；c6)小麦；c7)烟草；c8)番茄；c9)燕麦；c10)高粱；c11)玉米；c12)棉花；c13)大豆；c14)油菜。所述水稻具体可为水稻品种武育粳3号或水稻品种武陵粳1号。
为解决上述技术问题，本发明还提供了培育转基因植物的方法。
本发明所提供的培育转基因植物的方法，具体可为方法一，包括使所述蛋白质在受体植物中表达或过表达、或提高受体植物中所述蛋白质的活性，得到转基因植物的步骤；与所述受体植物相比，所述转基因植物的抗病性增强。
上述方法一中，所述“使所述蛋白质在受体植物中表达或过表达、或提高受体植物中所述蛋白质的活性”可通过多拷贝、改变启动子、调控因子、转基因等本领域熟知的方法，达到表达或过表达所述蛋白质、或提高所述蛋白质的活性的效果。
上述方法一中，所述“使所述蛋白质在受体植物中表达或过表达、或提高受体植物中所述蛋白质的活性”具体可为向受体植物中导入编码所述蛋白质的核酸分子。
本发明所提供的培育转基因植物的方法，具体可为方法二，包括抑制受体植物中所述蛋白质的表达量或抑制受体植物中所述蛋白质的活性，得到转基因植物的步骤；与所述受体植物相比，所述转基因植物的抗病性降低。
上述方法二中，所述“抑制受体植物中所述蛋白质的表达量或抑制受体植物中所述蛋白质的活性”可通过、RNA干扰、同源重组、基因定点编辑等本领域熟知的方法，达到抑制所述蛋白质的表达量或活性的目的。
上述方法二中，所述“抑制受体植物中所述蛋白质的表达量或抑制受体植物中所述蛋白质的活性”具体可为向受体植物中导入抑制编码所述蛋白质的核酸分子的表达的物质。
上述方法中，编码所述蛋白质的核酸分子，具体可为如下(b1)或(b2)或(b3)或(b4)或(b5)或(b6)所示的DNA分子：
(b1)核苷酸序列是序列表中序列2所示的DNA分子；
(b2)核苷酸序列是序列表中序列2自5′末端起第128至5260位所示的DNA分子；
(b3)核苷酸序列是序列表中序列1所示的DNA分子；
(b4)核苷酸序列是序列表中序列5所示的DNA分子；
(b5)与(b1)或(b2)或(b3)或(b4)限定的核苷酸序列具有50％或50％以上同一性，且编码所述蛋白质的DNA分子；
(b6)在严格条件下与(b1)或(b2)或(b3)或(b4)限定的核苷酸序列杂交，且编码所述蛋白质的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
序列表中序列1由11390个核苷酸组成。序列表中序列2由5260个核苷酸组成，序列表中序列2的核苷酸编码序列表中序列4所示的氨基酸序列。序列表中序列5由5127个核苷酸组成，序列表中序列5的核苷酸编码序列表中序列7所示的氨基酸序列。
上述方法中，所述“向受体植物中导入编码所述蛋白质的核酸分子”可通过向受体植物中导入重组载体甲实现；所述重组载体甲可为向表达载体或克隆载体插入编码所述蛋白质的核酸分子得到的重组质粒。
所述重组载体甲具体可为所述480C或所述500ox。
上述方法中，所述“抑制编码所述蛋白质的核酸分子的表达的物质”可通过向受体植物中导入重组载体乙实现。所述重组载体乙具体可为实施例中提及的480Ri。
本发明所提供的培育转基因植物的方法，具体可为方法三，包括抑制或提高受体中eEF1A蛋白质的表达量或受体中eEF1A蛋白质的活性，得到转基因生物的步骤；与所述受体相比，所述转基因生物的抗病性增强。
上述方法三中，所述eEF1A蛋白质可为如下G1)或G2)或G3)：
G1)氨基酸序列是序列表中序列9所示的蛋白质；
G2)在序列表中序列9所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
G3)将G1)或G2)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与RNA病毒病抗病性相关的蛋白质。
上述方法三中，所述“抑制受体中eEF1A蛋白质的表达量或抑制受体中eEF1A蛋白质的活性”可通过、RNA干扰、同源重组、基因定点编辑等本领域熟知的方法，达到抑制受体中eEF1A蛋白质的表达量或抑制受体中eEF1A蛋白质的活性的目的。
上述方法三中，所述“抑制受体中eEF1A蛋白质的表达量或抑制受体中eEF1A蛋白质的活性”具体可为向受体中导入抑制编码eEF1A蛋白质的核酸分子的表达的物质。
上述方法三中，所述“提高受体中eEF1A蛋白质的表达量或受体中eEF1A蛋白质的活性”可通过多拷贝、改变启动子、调控因子、转基因等本领域熟知的方法，达到提高受体中eEF1A蛋白质的表达量或受体中eEF1A蛋白质的活性的效果。
上述方法三中，所述“提高受体中eEF1A蛋白质的表达量或受体中eEF1A蛋白质的活性”具体可为向受体中导入编码eEF1A蛋白质的核酸分子。
上述任一所述eEF1A蛋白质具体可为蛋白质SIP1。
所述蛋白质SIP1可为如下F1)或F2)或F3)：
F1)氨基酸序列是序列表中序列9所示的蛋白质；
F2)在序列表中序列9所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
F3)将F1)或F2)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与RNA病毒病抗病性相关的蛋白质。
编码所述蛋白质SIP1的核酸分子具体可为如下(B1)或(B2)或(B3)或(B4)所示的DNA分子：
(B1)编码区为序列表中序列10、序列11、序列12或序列13所示的DNA分子；
(B2)核苷酸序列分别为序列表中序列10、序列11、序列12或序列13所 示的DNA分子；
(B3)与(B1)或(B2)或限定的核苷酸序列具有50％或50％以上同一性，且编码所述蛋白质SIP1的DNA分子；
(B4)在严格条件下与(B1)或(B2)限定的核苷酸序列杂交，且编码所述蛋白质SIP1的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
序列表中序列10、序列11、序列12和序列13均由1344个核苷酸组成(四个序列高度同源)，且均编码序列表中序列9所示的氨基酸序列。
上述方法三中，所述生物可为植物。所述受体具体可为受体植物。
上述方法三中，所述“抑制编码所述蛋白质SIP1的核酸分子的表达的物质”可通过向受体植物中导入重组载体戊实现。所述重组载体戊具体可为实施例中提及的SIP1Ri或SIP1ox。
本发明所提供的培育转基因植物的方法，具体可为方法四，包括将通过上述方法一或上述方法二或上述方法三获得的转基因植物与待改良植物杂交，得到后代转基因植物的步骤；所述后代转基因植物与所述转基因植物(即作为亲本的转基因植物)具有相同的抗病性。
上述任一所述方法中，所述抗病性可为抗条纹叶枯病。
上述任一所述方法中，所述抗病性可为抗水稻条纹病毒引起的病害。
上述任一所述方法中，所述受体植物可为c1)至c14)中的任一种：c1)双子叶植物；c2)单子叶植物；c3)禾本科植物；c4)水稻；c5)大麦；c6)小麦；c7)烟草；c8)番茄；c9)燕麦；c10)高粱；c11)玉米；c12)棉花；c13)大豆；c14)油菜。所述水稻具体可为水稻品种武育粳3号或水稻品种武陵粳1号。
上文中，所述转基因植物理解为不仅包含将所述核酸分子转化受体植物得到的第一代转基因植物，也包括其子代。对于转基因植物，可以在该物种中繁殖所述核酸分子，也可用常规育种技术将所述核酸分子转移进入相同物种的其它品种，特别包括商业品种中。所述转基因植物包括种子、愈伤组织、完整植株和细胞。
本发明还提供了一种核酸分子甲，所述核酸分子甲可为如下(e1)或(e2)或(e3)所示的DNA分子：
(e1)核苷酸序列是序列表中序列3所示的DNA分子；
(e2)与(e1)限定的核苷酸序列具有75％或75％以上同一性的DNA分子；
(e3)在严格条件下与(e1)或(e2)限定的核苷酸序列杂交的DNA分子。
所述蛋白质的应用也属于本发明的保护范围；所述蛋白质的应用可为如下H1)至H4)中的至少一种：
H1)与eEF1A蛋白质互作；
H2)能够检测eEF1A蛋白质；
H3)用于富集eEF1A蛋白质；
H4)用于调控生物体内eEF1A蛋白质的生物学功能。
本发明还保护一种调控生物体内源eEF1A蛋白质生物学功能的方法，包括通过导入生物体内所述蛋白质或提高生物体内所述蛋白质的表达或活性，通过所述蛋白质与eEF1A蛋白质的互作来调控生物体内eEF1A蛋白质生物学功能。所述互作可为直接互作。鉴于eEF1A蛋白质在生物体内功能保守，本方法所述生物体可为植物、动物、或微生物。
实验证明，利用本发明提供的蛋白质及其编码基因能调控植物抗病性。因此，本发明提供的蛋白质对调控条纹叶枯病的抗性具有非常重要的作用，在培育抗条纹叶枯病的水稻品种中具有广阔前景。
附图说明
图1为Stv-bi基因的精细定位图谱和单染色体片段代换系的基因型和表型数据。
图2为日本晴、Lemont和特青在定位区间内的基因组和转录本结构示意图。其中Nipponbare为日本晴，Teqing为特青。
图3为实施例2的实验结果。
图4为实施例3的实验结果。
图5为实施例4的实验结果。
图6为实施例6的实验结果。
图7为实施例7的实验结果。
图8为实施例8LCI蛋白互作验证结果。
图9为实施例8pull-down互作验证结果。
图10为实施例8Co-IP互作验证结果。
图11为实施例9的实验结果。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
载体pCAMBIA2300和pCAMBIA1300为Cambia公司产品。克隆载体pMD18-T为TaKaRa公司的产品。载体pUC18为ThermoFisher公司的产品。
载体pUC-RNAi：将载体pUC18的限制性内切酶BamHI和BglII的识别序列间的片段(载体pUC18被限制性内切酶BamHI和BglII切成一个大片段和一个小片段，该DNA为该小片段)替换为马铃薯GA20氧化酶基因的内含子的序列，得到重组质粒，命名为载体pUC-RNAi。
载体pCAMBIA2300-XmaI/AvrII的构建方法：将载体pCAMBIA2300的多克隆位 点替换成含有限制性内切酶XmaI的识别序列和限制性内切酶AvrII的识别序列的片段。
水稻感病品种武育粳3号为武进稻麦育种场培育而成的市售品种。水稻感病品种武育粳3号以下简称武育粳3号或WYJ3。
水稻抗病品种武陵粳1号为由扬州大学农学院、江苏省种子管理站和武进区稻麦育种场通过多代回交选育而成的市售品种。水稻抗病品种武陵粳1号以下简称武陵粳1号或WLJ1。
水稻感病品种Lemont为来源于美国的市售水稻品种。水稻感病品种Lemont以下简称Lemont。
水稻抗病品种特青为广东省农业科学院水稻研究所培育的市售品种。水稻抗病品种特青以下简称特青或Teqing。
近等基因系NIL39由发明人以Lemont为轮回亲本，特青为供体亲本通过分子标记多代辅助选择获得。近等基因系NIL39以下简称NIL39。
水稻感病品种日本晴为来源于日本的市售水稻品种。水稻感病品种日本晴以下简称日本晴或Nipponbare。
实施例1、Stv-bi基因的精细定位
研究结果表明来源于特青的抗条纹叶枯病基因qSTV11TQ(与本申请后续发现的Stv-bi基因是等位基因，故本发明中如无其他说明则统一将该基因命名为Stv-bi基因)定位于水稻第11条染色体分子标记CAPS2和CAPS3之间55.7kb的区间内(Wu XJ，Zuo SM，Zhang YF，Zhu JK，Ma N，Tang JY，Chu CC，Pan XB.2011.Fine mapping of qSTV11TQ,a major gene coferring resistance to rice strip disease.Theor.Appl.Genet.122:915-23)。为进一步精细定位Stv-bi基因，在上述55.7kb区间内开发多态性分子标记，并依据分子标记鉴定结果构建了以Lemont为遗传背景、包含更小特青染色体单片段的染色体单片段代换系(Chromosome single segment substitution lines，CSSSLs)，依据CSSSLs条纹叶枯病抗病性的鉴定(Wu XJ,Zuo SM,Zhang YF,Zhu JK,Ma N,Tang JY,Chu CC,Pan XB.2011.Fine mapping of qSTV11TQ,a major gene coferring resistance to rice stripdisease.Theor.Appl.Genet.122:915-23)，最终将Stv-bi基因定位于分子标记CAPS5(CAPS5F：5’-AAGTATGTCGCAAACTCGAT-3’和CAPS5R：5’-TCTAAAGCTGACTGCTCTGC-3’)和InDel11(InDel11F：5’-CAAATTTGGAAAGGTGGTAT-3’和InDel11R：5’-ACGTTAAATAAAAAGTCAACAGC-3’)之间29.6kb的区域(见图1)。参考日本晴基因组(http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml)的注释结果，在该29.6kb的区域检测到5个开放读码框(见图2)，包括2个转座子编码基因、一个反转座子编码基因以及一对同源基因LOC_Os11g31480(核苷酸序列如序列表中序列1所示)和LOC_Os11g31500，可见LOC_Os11g31480或 LOC_Os11g31500中的一个或两个即是抗条纹叶枯病基因。
采用cDNA末端快速扩增技术(Rapid amplification of cDNA ends，RACE)得到如下实验结果(见图2)：日本晴中仅检测到LOC_Os11g31480的一个转录本STV11S(核苷酸序列如序列表中的序列3所示，下文简称STV11S基因)；特青和Lemont中，均在预测的第一外显子Exon1B前面检测到另一个可变剪接第一外显子Exon1A，相应的检测到两个转录本，即转录本STV11L(核苷酸序列如序列表中序列2所示，下文简称STV11L基因)和转录本STV11S；在日本晴、特青和Lemont三个品种中，均检测到LOC_Os11g31500的一个转录本STV11-pa1(核苷酸序列如序列表中序列5所示,下文简称STV11-pa1基因)。上述结果表明，特青和日本晴在目的基因所在区域可能存在很大的序列差异。
采用基于PCR的基因组步移技术(PCR-based genome walking methods)得到如下实验结果(图2)：对特青相应基因组的序列进行了扩增和测序分析，在特青中分子标记CAPS5和InDel11界定的区域内，仅存在LOC_Os11g31480和LOC_Os11g31500两个同源基因，而且覆盖特青LOC_Os11g31480的第一内含子、第一外显子Exon1A以及第一个启动子的片段在日本晴中完全缺失或被其他片段替代；对Lemont的相应区域进行了序列分析，发现所在区域同样仅存在LOC_Os11g31480和LOC_Os11g31500两个同源基因。
上述结果表明：日本晴中，LOC_Os11g31480基因尽管缺失了第一外显子Exon1A和启动子，但仍然能检测到高水平的转录本STV11S，可见该转录本前面的区域即对应于特青等位基因的第一内含子同时也能行使启动子的功能，由此可见LOC_Os11g31480基因含有两个可变剪接启动子和两个可变剪接的第一外显子。
实施例2、Stv-bi基因的功能验证
一、重组质粒和重组农杆菌的构建
1、重组质粒的构建
(1)将载体pCAMBIA2300-XmaI/AvrII的限制性内切酶XmaI和AvrII的识别序列间的片段(载体pCAMBIA2300-XmaI/AvrII被限制性内切酶XmaI和AvrII切成一个大片段和一个小片段，该DNA为该小片段)替换为序列表中序列1自5′末端起第2621到11390位所示的DNA分子，得到重组质粒，命名为T-480C。
(2)将载体pCAMBIA2300-XmaI/AvrII的限制性内切酶XmaI和AvrII的识别序列间的片段(载体pCAMBIA2300-XmaI/AvrII被限制性内切酶XmaI和AvrII切成一个大片段和一个小片段，该DNA为该小片段)替换为序列表中序列5所示的cDNA分子对应的包括所有调控区段在内的基因组序列，得到重组质粒，命名为500C。500C表达序列表中序列7所示的蛋白质STV11-pa1。
(3)将载体pCAMBIA2300-XmaI/AvrII的限制性内切酶XmaI和AvrII的识别序列间的片段(载体pCAMBIA2300-XmaI/AvrII被限制性内切酶XmaI和AvrII切成一个大片段和一个小片段，该DNA为该小片段)替换为包含特青基因组的 LOC_Os11g31480第一可变启动子和序列表中序列1所示的双链DNA分子，得到重组质粒，命名为480C。480C表达序列表中序列4所示的蛋白质STV11L。蛋白质STV11L在下文中简称STV11L或STV11L蛋白。
2、重组农杆菌的构建
将T-480C导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/T-480C。
将500C导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/500C。
将480C导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/480C。
将载体pCAMBIA2300-XmaI/AvrII导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/pCAMBIA2300。
二、水稻转基因植株的再生
将武育粳3号种子脱壳灭菌，然后采用农杆菌侵染法将AGL1/T-480C转化武育粳3号，获得T0代转基因水稻植株。T0代转基因水稻植株收种后，经G418筛选后播种，得到T1代转基因水稻。T1代转基因水稻收种后，再次经G418筛选后播种，得到T2代转基因纯合水稻。分别取2个T2代转基因水稻株系(T-480C L1和T-480C L2)进行后续步骤三的实验。
按照上述方法，将AGL1/T-480C替换为AGL1/500C，其它步骤均相同，得到T2代转基因水稻的纯合植株。分别取2个T2代转基因水稻株系(500C L1和500C L2)进行后续步骤三的实验。
按照上述方法，将AGL1/T-480C替换为AGL1/480C，其它步骤均相同，得到T2代转基因水稻的纯合植株。分别取2个T2代转基因水稻株系(480C L1和480C L2)进行后续步骤三的实验。
按照上述方法，将AGL1/T-480C替换为AGL1/pCAMBIA2300，其它步骤均相同，得到T2代转空载体水稻的纯合植株，以下简称转空载体水稻。
三、转基因水稻的鉴定
采用Trizol法提取生长至4周的T-480C L1的地上组织总RNA，得到T-480C L1的总RNA，将该总RNA用逆转录酶反转录出第一链cDNA，简称为T-480C L1的cDNA。
按照上述方法，将T-480C L1分别替换为T-480C L2、480C L1、480C L2、500C L1和500C L2，得到T-480C L2的cDNA、480C L1的cDNA、480C L2的cDNA、500C L1的cDNA和500C L2的cDNA。
按照上述方法，将T-480C L1替换为武育粳3号，得到空白对照cDNA。
按照上述方法，将T-480C L1替换为转空载体水稻，得到空载对照cDNA。
按照上述方法，将T-480C L1替换为武陵粳1号，得到阳性对照cDNA。
以上述cDNA为模板，实时定量检测STV11L基因、STV11S基因或STV11-pa1基因的相对表达量(以Actin1基因为内参)。
鉴定STV11L基因的引物为5’-AGGCCGTCATCTACCTCA-3’和5’-ACATTCAGCACAAGGTTTCT-3’。鉴定STV11S基因的引物为 5’-GCCTCTCAGCTCGATCTAC和5’-ACATTCAGCACAAGGTTTCT-3’。鉴定STV11-pa1基因的引物为5’-TCTGCCACTGGATTGACTA-3’和5’-GGGTCCAATCTTGTCTATGA-3’。鉴定Actin1基因的引物为5’-CAGGCCGTCCTCTCTCTGTA-3’和5’-AAGGATAGCATGGGGGAGAG-3’。
实验结果表明(图3中C)：将武陵粳1号中STV11L基因的表达量作为1，转空载体水稻、480C L1和480C L2中STV11L基因的表达量分别为0、1.2和2.08，武育粳3号和转空载体水稻中均没有检测到STV11L基因的表达；将转空载体水稻中STV11S基因的表达量作为1，T-480C L1和T-480C L2中STV11S基因的表达量分别为5.8和13.4，武育粳3号和转空载体水稻中STV11S基因的表达量无显著差异；将转空载体水稻中STV11-pa1基因的表达量作为1，500C L1和500C L2中STV11-pa1基因的表达量分别为4.5和2.9，武育粳3号和转空载体水稻中STV11-pa1基因的表达量无显著差异。
四、转基因水稻的条纹叶枯病抗性鉴定
以武育粳3号、武陵粳1号、转空载体水稻、T-480C L1、T-480C L2、480C L1、480C L2、500C L1和500C L2为实验材料，测定水稻的条纹叶枯病抗性。
实验重复三次，每个株系每次种植40株于2L的玻璃烧杯中，在水稻生长至10天时，每株水稻苗接种4头2-3龄带水稻条纹病毒灰飞虱若虫(2-3龄带水稻条纹病毒灰飞虱若虫由江苏省农业科学院植物保护所提供)，每天驱赶灰飞虱4次以便于被测水稻苗均匀受毒。48小时后，用吸管将灰飞虱全部移走，并将水稻苗移栽到没有灰飞虱的水稻温室中，约一个月后调查实验材料的发病情况。
各供试材料的水稻条纹叶枯病抗病性通过两个指标进行测定：1)健康植株比率，即计算接虫后未发病植株数目占总接虫植株数目的百分比(Wu XJ,Zuo SM,Zhang YF,Zhu JK,Ma N,Tang JY,Chu CC,Pan XB.2011.Fine mapping of qSTV11TQ,a major gene coferring resistance to rice stripdisease.Theor.Appl.Genet.122:915-23)；2)病情指数，即按病害发生严重程度，根据Washio的抗性鉴定标准，将各苗的发病情况分成A、B、Bt、Cr、C和D六个级别，统计各家系中不同级别的株数，按公式计算病情指数＝(100×A+80×B+60×Bt+40×Cr+20×C+5×D)/被测总苗数(Washio O，Ezuka A，Sakurai Y，Toriyama K.1967.Studies on the breeding of rice varieties resistant to rice stripe disease.I.Varietaldifference in resistance to stripe disease.Jpn.J.Breed.17:91-98)。
健康植株比率的实验结果见图3中A，病情指数实验结果见图3中B。结果表明，转化LOC_Os11g31480能达到与抗病对照一致的抗病性。而相对应的是即使选用LOC_Os11g31500表达量高的转基因纯合株系进行表型鉴定，转基因也没有提高条纹叶枯病的抗病性。这些结果证实条纹叶枯病抗病基因对应于LOC_Os11g31480而不是LOC_Os11g31500。LOC_Os11g31480基因含有两个可变剪接启动子和两个可变剪接的第一外显子，即含有序列表中序列2所示的转录本 STV11L和序列表中序列3所示的转录本STV11S。为了进一步验证这两个转录本对条纹叶枯病抗病性的贡献，将步骤二获得的T-480C L1和T-480C L2进行条纹叶枯病表型鉴定。结果表明，T-480C L1和T-480C L2并没有出现比武育粳3号明显增强的条纹叶枯病抗病性。这一结果表明STV11L或STV11L与STV11S的互作在条纹叶枯病抗病性中发挥了关键作用。
实施例3、Stv-bi沉默株的获得和鉴定
系谱分析表明粳稻品种武陵粳1号的条纹叶枯病抗性基因来源于籼稻品种‘Modan’(PanXB，Chen ZX，Zuo SM，Zhang YF，Wu XJ，Ma N，Jiang QX，Que JH，Zhou CH.2009.A new rice cultivar'Wulingjing 1'with resistance to rice stripe virus bred by markerassisted selection.Acta Agron.Sin.35:1851-1857；Wu XJ，Zuo SM，Zhang YF，Zhu JK，Ma N，Tang JY，Chu CC，Pan XB.2011.Fine mapping of qSTV11TQ，a major gene coferring resistance to rice strip disease.Theor.Appl.Genet.122:915-23)。为证实在特青中克隆的条纹叶枯病抗病基因LOC_Os11g31480与来源于籼稻品种‘Modan’的抗病基因即Stv-bi是等位基因，我们选用武陵粳1号进行LOC_Os11g31480基因的RNAi敲除实验。
一、重组质粒和重组农杆菌的构建
1、重组质粒的构建
(1)480Ri的构建
a、PCR扩增特青基因组DNA获得DNA分子1。DNA分子1的核苷酸序列如序列表中序列2自5′末端起第4670到5031位所示(长度为362bp)。
b、将DNA分子1和克隆载体pMD18-T连接，得到重组质粒。测序结果表明，该重组质粒中含有DNA分子1。
c、用限制性内切酶SalI和BamHI酶切步骤b构建的重组质粒，回收约360bp的DNA片段甲。
d、用限制性内切酶SalI和BamHI酶切载体pUC-RNAi，回收载体骨架1(载体pUC-RNAi被切成一个大片段和一个小片段，载体骨架1为大片段)。
e、将载体骨架1和DNA片段甲连接，得到重组质粒。
f、用限制性内切酶BglII和XhoI酶切步骤e构建的重组质粒，回收载体骨架2(步骤e构建的重组质粒被切成一个大片段和一个小片段，载体骨架2为大片段)。
g、将载体骨架2和DNA片段甲连接，得到重组质粒。
h、用限制性内切酶PstI酶切步骤g构建的重组质粒，回收DNA片段丙(步骤g构建的重组质粒被切成一个大片段和一个小片段，DNA片段丙为小片段)。
i、用限制性内切酶PstI酶切表达载体pCAMBIA2301A(由表达载体pCAMBIA2300改造得到，具有Actin1启动子和OCS终止子)，回收载体骨架3(载体pCAMBIA2301A被切成一个大片段和一个小片段，载体骨架3为大片段)。
j、将载体骨架3和DNA片段丙连接，得到重组质粒，命名为480Ri。480Ri为以表达载体pCAMBIA2300为出发载体，在酶切位点插入如下表达盒：自上游至下游依次由Actin1启动子、DNA片段丙和OCS终止子组成。
(2)500Ri的构建
按照上述(1)的方法，将DNA分子1替换为DNA分子2，其它步骤均不变，得到重组质粒，命名为500Ri。DNA分子2的核苷酸序列如序列表中序列5自5′末端起第4469到4814位所示(长度为346bp)。
2、重组农杆菌的构建
将480Ri导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/480Ri。
将500Ri导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/500Ri。
将pCAMBIA2301A导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/pCAMBIA2301A，作为对照。
二、沉默株的获得
将武陵粳1号种子脱壳灭菌，然后采用农杆菌侵染法(Liu,X.Q.,Bai,X.Q.,Wang,X.J.&Chu,C.C.2007.OsWRKY71,a rice transcription factor,is involved in rice defense response.J.Plant Physiol.164,969-79)将AGL1/480Ri转化武陵粳1号，获得T0代沉默株。将T0代沉默株自交产生的种子命名为T1代沉默种子，由T1代沉默种子长成的水稻植株命名为T1代沉默株，T1代转基因水稻收种后，再次经G418筛选后播种，得到T2代转基因纯合水稻。分别取2个T2代转基因水稻株系将其命名为480Ri L1和480Ri L2进行后续步骤三的实验。
按照上述方法，将AGL1/480Ri替换为AGL1/500Ri，其它步骤均相同，得到T2代纯合沉默株，随机选择两个沉默株系，将其命名为500Ri L1和500Ri L2。
按照上述方法，将AGL1/480Ri替换为AGL1/pCAMBIA2301A，其它步骤均相同，得到T2代纯合转空载体水稻的植株，以下简称转空载体水稻。
三、沉默株的鉴定
采用Trizol法分别提取生长至4周且叶片全展开的480Ri L1和480Ri L2的叶鞘组织的总RNA，得到480Ri L1和480Ri L2的总RNA，将该总RNA用逆转录酶反转录出第一链cDNA，简称为480Ri L1的cDNA和480Ri L2的cDNA。
采用Trizol法分别提取幼穗生长至5厘米的500Ri L1和500Ri L2的幼穗的总RNA，得到500Ri L1和500Ri L2的总RNA，将该总RNA用逆转录酶反转录出第一链cDNA，简称为500Ri L1的cDNA和500Ri L2的cDNA。
按照上述方法，将480Ri L1或500Ri L1替换为武陵粳1号，得到对应空白对照cDNA。
按照上述方法，将480Ri L1或500Ri L1替换为转空载体水稻，得到对应空载对照cDNA。
以上述cDNA为模板，实时定量检测STV11L基因、STV11S基因和STV11-pa1基因的相对表达量(以Actin1基因为内参)。
鉴定STV11L基因的引物为5’-AGGCCGTCATCTACCTCA-3’和5’-ACATTCAGCACAAGGTTTCT-3’。鉴定STV11S基因的引物为5’-GCCTCTCAGCTCGATCTAC-3’和5’-ACATTCAGCACAAGGTTTCT-3’。鉴定STV11-pa1基因的引物为5’-TCTGCCACTGGATTGACTA-3’和5’-GGGTCCAATCTTGTCTATGA-3’。鉴定Actin1基因的引物为5’-CAGGCCGTCCTCTCTCTGTA-3’和5’-AAGGATAGCATGGGGGAGAG-3’。
实验结果表明(图4中C)：将转空载体水稻中STV11L基因的表达量作为1，480Ri L1、480Ri L2、500Ri L1和500Ri L2中STV11L基因的表达量分别为0.04、0.06、0.60和0.67，武陵粳1号和转空载体水稻中STV11L基因的表达量无显著差异；将转空载体水稻中STV11S基因的表达量作为1，480Ri L1、480Ri L2、500Ri L1和500Ri L2中STV11S基因的表达量分别为0.21、0.18、0.79和0.87，武陵粳1号和转空载体水稻中STV11S基因的表达量无显著差异；将转空载体水稻中STV11-pa1基因的表达量作为1，480Ri L1、480Ri L2、500Ri L1和500Ri L2中STV11-pa1基因的表达量分别为0.73、0.91、0.17和0.12，武陵粳1号和转空载体水稻中STV11-pa1基因的表达量无显著差异。
四、沉默株的条纹叶枯病抗性鉴定
以武陵粳1号、转空载体水稻、武育粳3号、480Ri L1、480Ri L2、500Ri L1和500Ri L2为实验材料，鉴定水稻的条纹叶枯病抗性。鉴定方法同实施例2步骤四。
结果表明(图4中A和B)，在武陵粳1号背景下特异敲除LOC_Os11g31480能完全破坏Stv-bi介导的条纹叶枯病抗病性，相应地在武陵粳1号背景下特异性敲除LOC_Os11g31500，并没有影响Stv-bi介导的条纹叶枯病抗病性，由此可见LOC_Os11g31480与Stv-bi系等位基因。在水稻中沉默Stv-bi基因可降低水稻对条纹叶枯病的抗性。
实施例4、STV11L基因的表达模式分析
实时定量检测不同组织的水稻样本中STV11L基因或STV11S基因的相对表达量
进行三次重复试验，每次重复试验的步骤均如下：
(1)获得样本
采集Lemont及Lemont背景下含有来自特青Stv-bi抗病基因染色体片段的近等基因系NIL39不同发育时期的幼苗(包括2周、3周、4周、5周大幼苗，对应标注为图5中的2w、3w、4w、5w Shoot)、叶片(包括拔节期叶片展开前1周、展开0天及展开1周后三个年龄段的叶片，对应标注为图5中的－1w、0d、1w Leaf blade)、叶鞘(包括拔节期叶片展开前1周、展开0天及展开1周后三个年龄段的相应叶鞘，对应标注为图5中的－1w、0d、1w Leaf sheath)、穗(包括5厘米阶段的幼穗以及开花后3周大的老穗，对应标注为图5中的Y和O Panicle)、茎秆(包括拔节期倒一节幼茎和倒三节老茎，对应标注为图5 中的Y和O Culm)和根(包括2周大幼根和拔节期的老根，对应标注为图5中的Y和O Root)的样本，放入液氮保存。
(2)STV11L基因或STV11S基因的表达模式分析
采用Trizol试剂盒(Invitrogen Life Technologies公司的产品)提取步骤(1)中各样本的总RNA，采用反转录试剂盒(Toyobo公司产品)将该总RNA反转录出第一链cDNA，然后以该cDNA为模板，采用荧光定量PCR仪(Bio-Rad公司产品)实时定量检测各样本中STV11L基因或STV11S基因的相对表达量(以Actin1基因作为内参基因)。鉴定STV11L基因的引物为5’-AGGCCGTCATCTACCTCA-3’和5’-ACATTCAGCACAAGGTTTCT-3’。鉴定STV11S基因的引物为5’-GCCTCTCAGCTCGATCTAC-3’和5’-ACATTCAGCACAAGGTTTCT-3’。鉴定Actin1基因的引物5’-CAGGCCGTCCTCTCTCTGTA-3’和5’-AAGGATAGCATGGGGGAGAG-3’。
各样本中STV11L基因的相对表达量见图5中A，各样本中STV11S基因的相对表达量见图5中B。
实验结果表明，在不同时期的组织中均能检测STV11L基因和STV11S基因表达，且这两个基因在老叶鞘中的相对表达量均较高，这一实验结果与条纹叶枯病毒因载体昆虫灰飞虱的取食习惯即首先大量侵染外层老叶鞘的表型完全一致。
实施例5、Stv-bi基因同源基因的鉴定
以水稻Stv-bi基因的cDNA序列为探针，在公共数据库(www.ncbi.nih.nlm.gov)上进行比对，比对结果显示在水稻的第12染色体上还有Stv-bi基因的第三个同源基因LOC_Os12g29350，其转录本STV11-pa2的核苷酸序列如序列表中序列6所示，表达序列表中序列8所示的蛋白质STV11-pa2。
通过cDNA扩增、测序及比对，得到如下结果：
核苷酸水平上，转录本STV11-pa2与转录本STV11L之间具有58.5％的序列一致性，转录本STV11-pa2与转录本STV11-pa1之间具有57.3％的序列一致性，转录本STV11L与转录本STV11-pa1之间具有74％的序列一致性；氨基酸水平上，蛋白质STV11-pa2与蛋白质STV11L之间具有50.2％的序列一致性，蛋白质STV11-pa2与蛋白质STV11-pa1之间具有48.5％的序列一致性，蛋白质STV11L与蛋白质STV11-pa1之间具有67.9％的序列一致性。
实施例6、组成性过表达STV11-pa1提高了水稻条纹叶枯病抗病性
实时定量PCR分析STV11-pa1基因的表达水平发现，在幼苗中STV11-pa1基因的表达处于极低的水平，近乎实时定量PCR的检测下限(图6中A)，这也解释了在实施例2中，通过转入STV11-pa1基因的全长基因组序列(即在对应转基因植株中，STV11-pa1转基因由其自身启动子驱动)并不能改变感病品种武育粳3号的条纹叶枯病抗病性。另一方面，氨基酸序列比对表明蛋白质STV11-pa1与蛋白质STV11L有较高的序列一致性，暗示蛋白质STV11-pa1与蛋白质STV11L 可能存在功能上的保守性。为了进一步明确蛋白质STV11-pa1在条纹叶枯病抗病性上的功能，采用花椰菜花叶病毒的CaMV 35S启动子组成性高水平驱动STV11-pa1基因在武育粳3号中的表达以检测该蛋白的功能。
一、重组质粒和重组农杆菌的构建
1、重组质粒的构建
重组质粒500ox：以表达载体pCAMBIA2300为出发载体，在酶切位点插入如下表达盒：自上游至下游依次由CaMV 35S启动子、序列表中序列5所示的cDNA分子和polyA终止序列组成。重组质粒500ox在下文中简称500ox。
2、重组农杆菌的构建
将500ox导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/500ox。
将载体pCAMBIA2300-35S导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/pCAMBIA2300-35S，作为对照。
二、STV11-pa1过表达株系的获得
将武育粳3号种子脱壳灭菌，然后采用农杆菌侵染法(Liu,X.Q.,Bai,X.Q.,Wang,X.J.&Chu,C.C.2007.OsWRKY71,a rice transcription factor,is involved in rice defense response.J.Plant Physiol.164,969-79)将AGL1/500ox转化武育粳3号，获得T0代转基因株系。T0代转基因水稻植株收种后，经G418筛选后播种，得到T1代转基因水稻。T1代转基因水稻收种后，再次经G418筛选后播种，得到T2代转基因纯合水稻。分别取2个T2代转基因水稻株系(500ox L1和500ox L2)行后续步骤三的实验。
按照上述方法，将AGL1/500ox替换为AGL1/pCAMBIA2300-35S，其它步骤均相同，得到T2代转空载体水稻的纯合植株，以下简称转空载体水稻。
三、STV11-pa1过表达株系的鉴定
采用Trizol法分别提取生长至4周且叶片全展开的500ox L1和500ox L2的地上组织的总RNA，将该总RNA用逆转录酶反转录出第一链cDNA，简称为500ox L1的cDNA和500ox L2的cDNA。
按照上述方法，将500ox L1替换为转空载体水稻，得到空载对照cDNA。
以上述cDNA为模板，实时定量检测STV11-pa1基因的相对表达量(以Actin1基因为内参)。
鉴定STV11-pa1基因的引物为5’-TCTGCCACTGGATTGACTA-3’和5’-GGGTCCAATCTTGTCTATGA-3’。鉴定Actin1基因的引物为5’-CAGGCCGTCCTCTCTCTGTA-3’和5’-AAGGATAGCATGGGGGAGAG-3’。
实验结果表明(图6中B)：将转空载体水稻中STV11L基因的表达量作为1，500ox L1和500ox L2中STV11L基因的表达量分别为1629和1756，武育粳3号和转空载体水稻中STV11-pa1基因的表达量无显著差异。
四、STV11-pa1过表达株系的条纹叶枯抗性鉴定
以武陵粳1号、转空载体水稻、武育粳3号、500ox L1和500ox L2为实验 材料，鉴定水稻的条纹叶枯病抗性，鉴定方法同实施例2步骤四。
结果表明(图6中C和D)，在武育粳3号背景下过表达STV11-pa1基因能显著提高水稻的条纹叶枯病抗病性。由此证明蛋白质STV11-pa1同样可用于水稻条纹叶枯病的抗性改良。
实施例7、STV11L互作蛋白SIP1的筛选
为进一步明确STV11L介导条纹叶枯病抗性的分子机制，我们采用酵母双杂交的方法来进行STV11L互作蛋白的筛选。并最终筛选到STV11L的互作蛋白SIP1，具体步骤如下：
一、酵母双杂交载体的构建
1.诱饵载体的构建
a、PCR扩增特青基因组DNA获得DNA分子3。DNA分子3的核苷酸序列如序列表中序列2自5′末端起第128到727位所示(长度为600bp)。
b、通过InFusion克隆系统(Clontech)将DNA分子3连接到克隆载体pGBKT7上的限制性酶切位点NdeI和XhoI之间，得到重组质粒即为诱饵载体，命名为BD-STV11N。测序结果表明，该重组质粒中含有DNA分子3。
2.SIP1验证载体的构建
a.PCR扩增基因组DNA获得DNA分子4、5、6、和7。DNA分子4、5、6、和7的核苷酸序列分别如序列表中序列10自5′末端起第1到690位所示(长度690bp)、第688到1341位所示(长度为654bp)、第688位到960位所示(长度273bp)、第961到1341位所示(长度381bp)。
b.通过InFusion克隆系统(Clontech)将DNA分子4、5、6、和7分别连接到克隆载体pGADT7上的限制性酶切位点NdeI和PstI之间，得到重组质粒即为SIP1验证载体，对应命名为AD-STV11D1、AD-STV11D23、AD-STV11D2、和AD-STV11D3。测序结果表明，该重组质粒中分别含有DNA分子4、5、6、和7。二、诱饵载体的自激活检测
将BD-STV11N诱饵载体和pGBKT7空载体分别转化到酵母菌株Gold Yeast(Clontech)中，摇菌培养后，将转化菌落点在含有SD/-Trp/X-a-Gal平板培养基上，30℃培养3到4天。观察表明，同转化空载体的阴性对照一致，转化诱饵载体BD-STV11N的菌落在SD/-Trp/X-a-Gal培养基上能正常生长，且不显示蓝色，表明诱饵蛋白在酵母细胞中无毒性，且无自激活活性，可以用于酵母双杂交的文库筛选实验。
三、酵母文库的构建
取萌发两周左右的水稻幼苗，根据试剂盒说明(Invitrogen Life Technologies)提取高纯度和高完整性的RNA，参照Make Your Own“Mate&Plate TM”Library System User Mannual(Clontech)合成总cDNA并进行cDNA文库的构建。
四、STV11L互作蛋白SIP1的鉴定
通过酵母双杂交筛选，我们鉴定到STV11L的互作蛋白SIP1。通过酵母双杂交验证试验，我们发现SIP1羧基端的两个结构域(对应载体编号为AD-STV11D23)在STV11L与SIP1的互作中发挥不可替代的作用(图7)。
实施例8、STV11L和SIP1互作的体内、体外实验验证
为了验证STV11L和SIP1互作的真实性，我们分别采取了萤火虫荧光素酶互补实验(LCI，Firefly luciferase complementation imaging assays)、GST(Glutathione-S-transferase)pull-down实验、以及免疫共沉淀实验(Co-IP，coimmunoprecipitation assays)来验证上述互作。
一、蛋白互作载体的构建
以表达载体pCAMBIA1300-nLuc(下文简称为nLuc)为出发载体，在酶切位点SacI/SalI插入STV11L全长cDNA即序列表中序列2自5′末端起第128到5257位所示序列(长度为5130bp)。获得重组质粒在下文中简称为STV11L-nLuc。
以表达载体pCAMBIA1300-cLuc(下文简称为cLuc)为出发载体，在酶切位点KpnI/PstI插入SIP1全长cDNA即序列表中序列10所示序列(长度为1344bp)。获得重组质粒在下文中简称为cLuc-SIP1。
以表达载体pCAMBIA2300为出发载体，在酶切位点插入如下表达盒：自上游至下游依次由CaMV 35S启动子、序列表中序列10所示的cDNA分子和polyA终止序列组成。获得重组质粒在下文中简称SIP1ox。
以表达载体pGEX-6P-1(GE Healthcare)为出发载体，在酶切位点插入STV11L全长cDNA即序列表中序列2自5′末端起第128到5260位所示序列(长度为5133bp)。获得重组质粒在下文中简称为GST-STV11L。
以表达载体pCAMBIA1300为出发载体，在酶切位点插入如下表达盒：自上游至下游依次由CaMV 35S启动子、序列表中序列2自5′末端起第128到5260位所示cDNA分子、3×Flag标签序列分子和polyA终止序列组成。获得重组质粒在下文中简称STV11L-Flag。
二、LCI蛋白互作验证
将含有重组质粒组合STV11L-nLUC/cLuc-SIP1、STV11L-nLUC/cLuc、nLUC/cLuc-SIP1、nLuc/cLuc的农杆菌分别注射到烟草叶片的对应区域。3天后，取注射过农杆菌的烟草叶片并在叶片表面喷洒1mM的Luciferin(Promega),暗下静置5分钟后置于low-light cooled CCD imaging apparatus(CHEMIPROHT1300B/LND,16bits,Roper Scientific)中成像，通过配套的IndiGo软件分析表明，注射含有重组质粒组合STV11L-nLUC/cLuc-SIP1的烟草叶片区域能检测了明显的互补荧光，而作为阴性对照组分别注射质粒组合STV11L-nLUC/cLuc、nLUC/cLuc-SIP1或nLuc/cLuc的叶片区域均没有检测到荧光信号(图8)。说明STV11L和SIP1在烟草体内存在互作。
三、pull-down互作验证
通过农杆菌侵染法，在烟草叶片中表达SIP1ox重组质粒。使用液氮将表达 SIP1蛋白的烟草叶片研磨均匀，随后加入适量的蛋白提取液(0.01M PBS[Ph7.3],0.1％Triton X-100、EDTA-free protease inhibitor cocktail[Roche]、1mM PMSF)。15000rpm离心30分钟后，取上清获得SIP1蛋白粗提液。将pGEX-6P-1空载体及GST-STV11L重组质粒分别转化到大肠杆菌BL21(DE3)pLysS(Promega)感受态细胞中。获得相应的大肠杆菌株系后，使用0.6mM的isopropylβ-D-1-thiogalactopyranoside(Pierce)在16℃过夜诱导蛋白表达。随后通过Glutathione Sepharose 4FF beads(GE Healthcare)纯化GST及GST-STV11L蛋白。将结合GST或GST-STV11L的Glutathione Sepharose 4FF beads分别与SIP1蛋白粗提液在4℃条件下共培养1个小时后，使用蛋白提取液洗涤上述beads并取适量洗涤后的beads溶于1×SDS(sodium dodecyl sulfate)上样液中进行SDS-PAGE(SDS-polyacrylamide gel electrophoresis)。使用购自Abmart的SIP1特异抗体进行的免疫检测结果表明，STV11L能够成功pull-down下SIP1蛋白(图9)，证实STV11L在体外直接结合SIP1。
四、Co-IP互作验证
将含有重组质粒组合STV11L-Flag/SIP1ox和Flag(STV11L-Flag重组质粒中去除STV11L全长cDNA后的空载体)/SIP1ox的农杆菌分别注射到烟草叶片中。3天后，取注射过农杆菌的烟草叶片提取总蛋白。并在4℃将两种蛋白提取液分别与适量anti-Flag magnetic beads(Sigma)共孵育两小时。接着使用4℃预冷的IP缓冲液洗涤上述beads至少五次。再取适量beads分别使用anti-Flag(Sigma)和anti-SIP1抗体进行免疫印迹分析。结果表明STV11L能够共沉淀SIP1(图10)，再次证明了STV11L与SIP1之间的互作。
实施例9、SIP1的功能验证
一、SIP1敲除载体SIP1Ri的构建
按照上述实施例3中480Ri的构建方法，将DNA分子1替换为DNA分子8，其它步骤均不变，得到重组质粒，命名为SIP1Ri。DNA分子8的核苷酸序列如序列表中序列9自5′末端起第228到545位所示(长度为318bp)。
二、SIP1沉默抑制了RSV病毒复制
SIP1是一种eEF1A(eukaryotic translation elongation factor 1alpha)蛋白。由基因组上四个高度同源的基因编码。基因编码区序列分别为序列表中序列10、序列11、序列12、和序列13。蛋白序列对应序列表中序列9。为了明确SIP1在RSV病毒侵染过程中的作用，我们先用携带RSV的灰飞虱侵染感病品种武育粳3号，随后用侵染后的武育粳3号幼苗制备水稻原生质体。在原生质体中分别转入pCAMBIA2301A空载体及SIP1Ri敲除载体。在转入后的0小时、24小时、及48小时取样提取RNA并制备相应的cDNA。以上述cDNA为模板，实时定量检测SIP1基因以及RSV基因RSVCP(NCBI登录号DQ333944)的相对表达量(以Actin1基因为内参)。
鉴定SIP1基因的引物为5’-GGAGAAGACGCACATCAACA-3’和 5’-CTCTTGGGCTCGTTGATCTG-3’。鉴定RSVCP基因的引物为5’-TTTGCAGAAGGCAATCAATG-3’和5’-CTGGCATGGTCTTTTTGGTT-3’。鉴定Actin1基因的引物为5’-CAGGCCGTCCTCTCTCTGTA-3’和5’-AAGGATAGCATGGGGGAGAG-3’。
实验结果表明(图11中A)：转空载体原生质体中SIP1基因的表达量在0小时、24小时、及48小时较为一致，但转SIP1Ri的原生质体中，SIP1基因的表达量在12小时时就显著下调，48小时时进一步下调。相应地，转空载体原生质体中，RSV基因RSVCP的表达量在0小时、24小时、及48小时持续走高，而在转SIP1Ri的原生质体中，RSV基因RSVCP的表达量上升幅度显著减小(图11中B)。表明RSV病毒在水稻植株体内的复制依赖于SIP1蛋白的正常表达。
由此可见，在感病品种中，由于缺少(日本晴中缺失STV11L,图2)或低水平表达(Lemont老叶鞘及叶片中STV11L表达水平显著低于NIL39，图5中A)STV11L蛋白，SIP1的正常表达有利于RSV病毒的快速复制；但在抗病品种中，STV11L大量表达并与SIP1互作干扰了SIP1的正常功能，从而抑制了RSV病毒的有效复制(图11中B)。借助这一发现，本专利可提供这样一种方法，即在水稻中通过调节SIP1的表达来实现水稻对条纹叶枯病抗性的改良。
值得强调的是，大量的独立研究报道证实eEF1A在其它植物以及动物的RNA病毒侵染过程中，均发挥保守的关键作用(Sasikumar,AN,Perez WB,Kinzy TG.2012.The many roles of the eukaryotic elongation factor 1complex.Wiley Interdiscip.Rev.RNA.3:543-555；Li D,Wei T,Abbott CM,Harrich D.2013.The unexpected roles of eukaryotic translation elongation factors in RNA virus replication and pathogenesis.Microbiol.Mol.Biol.Rev.77:253-266)。由此，本专利同时提供了这样一种方法，即在不同的物种中，通过操纵STV11L蛋白的表达来调控eEF1A蛋白的功能，进而提高相应物种对RNA病毒的抗病性。
工业应用
利用本发明提供的蛋白质及其编码基因能调控植物RNA病毒病的抗病性。因此，本发明提供的蛋白质对在培育抗RNA病毒病的作物品种中具有广阔前景。同时本发明提供的蛋白质及其编码基因在人类以及禽畜RNA病毒病防治领域具有很大的应用潜力。

Claims (15)

  1. 蛋白质，为如下a1)或a2)或a3)或a4)或a5)或a6)：
    a1)氨基酸序列是序列表中序列4所示的蛋白质；
    a2)在序列表中序列4所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)氨基酸序列是序列表中序列7所示的蛋白质；
    a4)在序列表中序列7所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a5)将a1)或a2)或a3)或a4)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与植物抗病性相关的蛋白质；
    a6)与a1)或a2)或a3)或a4)所示的蛋白质具有70％或70％以上同一性的蛋白质。
  2. 编码权利要求1所述蛋白质的核酸分子。
  3. 根据权利要求2所述的核酸分子，其特征在于：所述核酸分子为如下(b1)或(b2)或(b3)或(b4)或(b5)或(b6)所示的DNA分子：
    (b1)核苷酸序列是序列表中序列2所示的DNA分子；
    (b2)核苷酸序列是序列表中序列2自5′末端起第128至5260位所示的DNA分子；
    (b3)核苷酸序列是序列表中序列1所示的DNA分子；
    (b4)核苷酸序列是序列表中序列5所示的DNA分子；
    (b5)与(b1)或(b2)或(b3)或(b4)限定的核苷酸序列具有50％或50％以上同一性，且编码权利要求1所述蛋白质的DNA分子；
    (b6)在严格条件下与(b1)或(b2)或(b3)或(b4)限定的核苷酸序列杂交，且编码权利要求1所述蛋白质的DNA分子。
  4. 含有或部分含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系。
  5. 如权利要求4所述的重组载体，其特征在于：所述重组载体为向表达载体或克隆载体插入权利要求2或3所述核酸分子得到的重组质粒。
  6. 如权利要求4所述的重组微生物，其特征在于：所述重组微生物为将权利要求4或5所述重组载体导入出发微生物得到的重组菌。
  7. 与权利要求1所述蛋白质结合的抗体。
  8. d1)或d2)或d3)的应用：
    d1)权利要求1所述蛋白质，或，权利要求2或3所述核酸分子，或，含有或部分含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在调控植物抗病性中的应用；
    d2)权利要求1所述蛋白质，或，权利要求2或3所述核酸分子，或，含有或部分含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物 或转基因细胞系，在培育抗病性提高或降低的转基因植物中的应用；
    d3)权利要求1所述蛋白质，或，权利要求2或3所述核酸分子，或，含有或部分含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在制备抗病性药物中的应用。
  9. 一种培育转基因植物的方法，包括使权利要求1所述蛋白质在受体植物中表达或过表达、或提高受体植物中权利要求1所述蛋白质的活性，得到转基因植物的步骤；与所述受体植物相比，所述转基因植物的抗病性增强。
  10. 一种培育转基因生物的方法，包括抑制或提高受体中eEF1A蛋白质的表达量或受体中eEF1A蛋白质的活性，得到转基因生物的步骤；与所述受体相比，所述转基因生物的抗病性增强。
  11. 一种培育转基因植物的方法，包括将通过权利要求9或10所述的方法获得的转基因植物与待改良植物杂交，得到后代转基因植物的步骤；所述后代转基因植物与所述转基因植物具有相同的抗病性。
  12. 如权利要求1所述的蛋白质，或，权利要求8所述的应用，或，权利要求9至11任一所述的方法，其特征在于：所述抗病性为抗水稻条纹病毒引起的病害。
  13. 如权利要求1或12所述的蛋白质，或，权利要求8或12所述的应用，或，权利要求9至12任一所述的方法，其特征在于：所述植物为c1)至c14)中的任一种：c1)双子叶植物；c2)单子叶植物；c3)禾本科植物；c4)水稻；c5)大麦；c6)小麦；c7)烟草；c8)番茄；c9)燕麦；c10)高粱；c11)玉米；c12)棉花；c13)大豆；c14)油菜。
  14. 权利要求1或12或13所述的蛋白质的应用，为如下H1)至H4)中的至少一种：
    H1)与eEF1A蛋白质互作；
    H2)能够检测eEF1A蛋白质；
    H3)用于富集eEF1A蛋白质；
    H4)用于调控生物体内eEF1A蛋白质的生物学功能。
  15. 一种调控生物体内源eEF1A蛋白质生物学功能的方法，包括通过导入生物体内权利要求1所述蛋白质或提高生物体内所述蛋白质的表达或活性，利用所述蛋白质与eEF1A蛋白质的互作来调控生物体内eEF1A蛋白质生物学功能。
PCT/CN2017/096359 2016-08-09 2017-08-08 水稻条纹叶枯病抗性基因Stv-bi及其应用 WO2018028553A1 (zh)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112899274A (zh) * 2019-12-04 2021-06-04 中国科学院遗传与发育生物学研究所 miR1432在调控水稻白叶枯病抗性中的应用
CN112979775A (zh) * 2021-03-18 2021-06-18 中国农业科学院作物科学研究所 抗穗发芽转基因小麦的培育方法及其相关生物材料
CN114107369A (zh) * 2021-12-02 2022-03-01 河南农业大学 一种myc标签融合表达载体的制备方法及其应用
CN114134170A (zh) * 2021-12-02 2022-03-04 河南农业大学 一种ha标签融合表达载体的制备方法及其应用
CN114150008A (zh) * 2021-12-02 2022-03-08 河南农业大学 一种3×flag标签融合表达载体的制备方法及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104561085A (zh) * 2013-10-18 2015-04-29 北京大学 OsAGO18基因在提高水稻对水稻条纹叶枯病抗性中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112012027504B1 (pt) * 2010-04-28 2022-05-24 Evogene Ltd Método de aumento de produção, biomassa, taxa de crescimento, e/ou tolerância ao estresse abiótico de uma planta, e, construção de ácidos nucleicos

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104561085A (zh) * 2013-10-18 2015-04-29 北京大学 OsAGO18基因在提高水稻对水稻条纹叶枯病抗性中的应用

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
CHEN, TAO ET AL.: "Improving Resistance to Rice Stripe Disease and Eating Quality of Wuyujing 3 by Pyramiding Stv-bi and Wx-mq", CHINESE JOURNAL OF RICE SCIENCE, vol. 29, no. 5, 31 December 2015 (2015-12-31), pages 467 - 474, XP055463925 *
DATABASE GENBANK [O] 1 March 2016 (2016-03-01), XP055463914, Database accession no. XP-015615307.1 *
DATABASE GENBANK [O] 23 March 2015 (2015-03-23), XP055463913, Database accession no. EEE52167.1. *
DATABASE GENBANK [O] 5 May 2011 (2011-05-05), XP055463910, Database accession no. ABA93881.1 *
DATABASE GENBANK [O] Oryza sativa Indica Group; 23 March 2015 (2015-03-23), XP055463908, Database accession no. EAY81073.1. *
DATABASE GENBANK [O] Oryza sativa Japonica Group; 5 May 2011 (2011-05-05), XP055463906, Database accession no. ABA93879.1 *
WANG, Q. ET AL.: "STV11 encodes a sulphotransferase and confers durable resistance to rice stripe virus", NATURE COMMUNICATIONS, vol. 5, 9 September 2014 (2014-09-09), pages 2, XP055463919 *
WANG, QI., MAP-BASED CLONING AND FUNCTIONAL ANALYSIS OF RICE STRIPE VIRUE RESISTANCE GENE IN RICE. CDFD, 15 May 2016 (2016-05-15) *
WU, X. J. ET AL.: "Fine mapping of qSTVllTQ, a major gene conferring resistance to rice stripe disease", THEOR. APPL. GENET., vol. 122, 8 December 2010 (2010-12-08), pages 915 - 923, XP019884671 *
ZHANG, Y. X. ET AL.: "Fine mapping of qSTVllKAS, a major QTL for rice stripe disease resistance", THEOR. APPL. GENET., vol. 122, 8 March 2011 (2011-03-08), pages 1591 - 1604, XP055463921 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112899274A (zh) * 2019-12-04 2021-06-04 中国科学院遗传与发育生物学研究所 miR1432在调控水稻白叶枯病抗性中的应用
CN112899274B (zh) * 2019-12-04 2022-06-07 中国科学院遗传与发育生物学研究所 miR1432在调控水稻白叶枯病抗性中的应用
CN112979775A (zh) * 2021-03-18 2021-06-18 中国农业科学院作物科学研究所 抗穗发芽转基因小麦的培育方法及其相关生物材料
CN112979775B (zh) * 2021-03-18 2022-05-10 中国农业科学院作物科学研究所 抗穗发芽转基因小麦的培育方法及其相关生物材料
CN114107369A (zh) * 2021-12-02 2022-03-01 河南农业大学 一种myc标签融合表达载体的制备方法及其应用
CN114134170A (zh) * 2021-12-02 2022-03-04 河南农业大学 一种ha标签融合表达载体的制备方法及其应用
CN114150008A (zh) * 2021-12-02 2022-03-08 河南农业大学 一种3×flag标签融合表达载体的制备方法及其应用

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