WO2020182221A1 - 一种水稻抗白叶枯病蛋白及其编码基因与应用 - Google Patents

一种水稻抗白叶枯病蛋白及其编码基因与应用 Download PDF

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WO2020182221A1
WO2020182221A1 PCT/CN2020/082732 CN2020082732W WO2020182221A1 WO 2020182221 A1 WO2020182221 A1 WO 2020182221A1 CN 2020082732 W CN2020082732 W CN 2020082732W WO 2020182221 A1 WO2020182221 A1 WO 2020182221A1
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rice
resistant
gene
disease
bacterial blight
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French (fr)
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汪聪颖
朱小源
陈深
苏菁
曾列先
汪文娟
冯爱卿
杨健源
封金奇
陈炳
伍圣远
张梅英
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广东省农业科学院植物保护研究所
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Priority to AU2020235775A priority Critical patent/AU2020235775B2/en
Publication of WO2020182221A1 publication Critical patent/WO2020182221A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/125—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for bacterial resistance
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

Definitions

  • the invention belongs to the field of biotechnology, in particular a rice bacterial blight resistance protein and its coding gene and application.
  • Bacterial blight (Xanthomonas oryzae pv.oryzae (Xoo)) is one of the main diseases of rice in my country and the main rice-producing regions of the world in Southeast Asia, and it seriously threatens the safety of rice production.
  • the use of host resistance is an effective measure to control the disease.
  • variety resistance is often lost.
  • the persistence of variety resistance and its mechanism have become a key point in current disease resistance research.
  • Xa7 is considered to be a durable disease resistance gene that can effectively resist different pathogenic pathogens and has shown excellent and stable resistance in many countries around the world [Ona et al. ,1998, Epidemic development of bacterial blight on rice carrying resistance genes Xa-4, Xa-7, and Xa-10.
  • the Xa7 gene was originally identified by the International Rice Research Institute (IRRI) on the rice variety DV85 [Sidhu et al., 1978 Genetic analysis of bacterial blight resistance in lasty-four cultivars of rice, Oryza sativa L. Theor Appl Genet, 53:105 -111.].
  • IRRI International Rice Research Institute
  • Ogawa et al. introduced the gene Xa7 into the near-isogenic line IRBB7 [Ogawa et al.,1991, Breaking of near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen Xanthomonas v. campestris .Jpn J Breed,41:523-529.].
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a rice bacterial blight resistant protein.
  • Another object of the present invention is to provide a gene encoding the above-mentioned rice bacterial blight resistance protein.
  • Another object of the present invention is to provide pathogen-inducing regulatory elements in the promoter region of the aforementioned genes.
  • Another object of the present invention is to provide the application of the above-mentioned protein, gene and pathogen-inducing regulatory element in the promoter region.
  • a rice bacterial blight resistant protein named Xa7 protein, and its amino acid sequence is as follows:
  • the gene encoding the above-mentioned rice bacterial blight resistance protein is named Xa7 gene, and its nucleotide sequence is as follows:
  • the nucleotide sequence of the pathogen-inducing regulatory element in the promoter region of the gene encoding the above-mentioned rice bacterial blight resistance protein is as follows: TATAACCCCCCCCCCCAGATAACCA.
  • the rice bacterial blight resistant protein can be synthesized by chemical synthesis; or by cloning the gene encoding the rice bacterial blight resistant protein into an expression vector, the obtained recombinant expression vector is transformed into host cells, Purified after expression.
  • the preparation of the gene encoding rice bacterial blight resistance protein can be achieved by the following methods: obtaining by chemical synthesis; or designing primers, using DV85, IRBB7 or other rice varieties carrying Xa7 gene genomic DNA as templates, and PCR Obtained by amplification; or obtained by restriction digestion and screening from the plasmid carrying the Xa7 gene.
  • the application of the gene encoding rice bacterial blight resistance protein can be used to study the mechanism of rice resistance to bacterial blight, and can also be used to cultivate rice varieties resistant to rice bacterial blight or other disease resistance Crop, or use it as a molecular marker to select rice varieties that are resistant to rice bacterial blight.
  • the steps for cultivating rice varieties or other disease-resistant crops resistant to bacterial blight of rice are preferably as follows: the above-mentioned gene encoding rice bacterial blight resistant protein and the above-mentioned promoter region are induced and regulated The elements are introduced into susceptible rice or other crops to obtain disease-resistant rice or disease-resistant crops; or constitutive expression promoters or other pathogen-inducible promoters are connected in tandem with the coding sequences of the above genes to introduce susceptible Among diseased rice or other crops, disease-resistant rice or disease-resistant crops are obtained.
  • the steps for selecting rice varieties resistant to bacterial blight of rice are preferably as follows: the rice varieties carrying the above-mentioned genes are used as donor parents, and pollen crosses are carried out with rice varieties susceptible to bacterial blight A series of progeny obtained were screened using Xa7 as a molecular marker, and rice varieties resistant to bacterial blight were identified.
  • the donor parent is preferably DV85 or IRBB7.
  • the application of the pathogen-inducing regulatory element in the promoter region of the gene encoding the rice bacterial blight resistance protein can be used to study the mechanism of rice resistance to bacterial blight, and can also be used to cultivate rice resistant to bacterial blight Varieties or other disease-resistant crops.
  • the steps for cultivating rice varieties or other disease-resistant crops resistant to bacterial blight of rice are preferably as follows: the above-mentioned gene encoding rice bacterial blight resistant protein and the above-mentioned promoter region are induced and regulated The elements are introduced into susceptible rice or other crops to obtain disease-resistant rice or disease-resistant crops; or the above-mentioned pathogen-inducing regulatory elements in the promoter region are connected in tandem with other disease-resistant gene coding sequences to be introduced into susceptible rice Or other crops, get disease-resistant rice or disease-resistant crops.
  • the present invention has the following advantages and effects:
  • the present invention is based on previous research, through the construction of a genomic BAC library of rice variety IRBB7, library screening, candidate insert sequencing, target insert sequence prediction of AvrXa7 recognition site, and a series of transgene function complementation tests and gene knockout The experiment finally completed the cloning of the Xa7 functional gene.
  • the present invention provides the sequence of Xa7 functional gene for the first time.
  • Figure 1 is a schematic diagram of the location of the subcloned fragments and the disease resistance phenotype diagram of the transgenic lines;
  • Figure A is a schematic diagram of the location and sequence of the overlapping regions of the subcloned fragments used in the transgene functional complementation experiment, and
  • Figure B is the subcloned transgenic rice line Photograph of the phenotype of resistance to bacterial blight pathogen PXO86;
  • Figure C shows the statistical results of the lesion length of subcloned transgenic rice lines against bacterial blight pathogen PXO86.
  • Figure 2 is a diagram showing the structural characteristics and resistance expression pattern of the Xa7 gene; among which, Figure A is a schematic diagram of the structural characteristics of the Xa7 gene sequence, and Figure B is a diagram showing the resistance expression pattern of the Xa7 gene to bacterial leaf blight PXO86.
  • Figure 3 is a diagram showing the results of the knock-out function verification of the pathogen-inducing element in the Xa7 gene promoter region;
  • Figure A is the sequence of the mutant homozygous line after gene editing of the pathogen-inducing element in the Xa7 gene promoter region, and
  • Figure B is the mutant line A diagram showing the expression pattern of resistance to bacterial blight pathogen PXO86, and
  • Figure C is a photograph of the disease resistance phenotype of each mutant strain to bacterial blight pathogen PXO86.
  • Figure 4 is a diagram showing the results of the knockout function verification of the coding region of the Xa7 gene;
  • Figure A shows the sequence of the mutant homozygous strain after gene editing of the coding region of the Xa7 gene, and
  • Figure 4 shows the resistance of each mutant strain to bacterial blight PXO86 Expression pattern,
  • Figure C shows the disease-resistant phenotype of each mutant strain against bacterial blight PXO86.
  • the present invention constructs the IRBB7 genomic BAC library of Xa7 resistant species, screens the library with close-linked molecular markers on both sides of Xa7, catches positive clones, and sequence the inserts of positive clones to obtain complete and accurate genomic sequences of the region.
  • the rice variety IRBB7 has been published in the literature "Ogawa et al., 1991, Breeding of near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen Xanthomonas campestris pv.oryzae.Japan) J Breed, 41: 523-529 public.
  • the plant material for constructing the genomic BAC library is the near-isogenic line IRBB7 containing the Xa7 gene, the vector is Epicentre’s CopyControl TM pCC1BAC TM , and the subcloning and transgene expression vector is pYLTAC747H/sacB (in the literature "Xu et al., 2008, Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis. Science in China (Series C: Life Sciences), (07): 604-613. "Open in).
  • TAC transformation-competent artificial chromosome
  • BAC library construction follows the experimental steps reported in the literature "Liu et al., 2002, Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning. Gene, 282(1):247-255.”
  • IRBB7 genomic DNA was extracted at the seedling stage, partially digested with HindIII restriction endonuclease, and 120-140kb DNA fragments were separated by pulse field electrophoresis, and then purified and ligated with the BAC vector pCC1BAC TM .
  • 75ng BAC vector was mixed with five sets of genomic DNA digestion products (A: 30ng, B: 60ng, C: 120ng, D: 160ng, E: 350ng), press T4 DNA ligase 50 ⁇ L system was used to prepare the reaction system, and the ligation reaction was carried out in the PCR machine using variable temperature ligation program: 10°C3min, 3min to 16°C, 16°C5min, 30s to 18°C, 18°C30s, 30s to 20 °C, 20°C for 30s, 8s to 4°C, 4°C for 3min, 5min from 4°C to 22°C, 22°C1min to 10°C, and so on for 20 times, finally 65°C for 5min.
  • variable temperature ligation program 10°C3min, 3min to 16°C, 16°C5min, 30s to 18°C, 18°C30s, 30s to 20 °C, 20°C for 30s, 8s to 4°C, 4°C for 3min, 5min from 4°
  • the ligation product was dialyzed with MILLIPORE TM VSWP membrane (0.025 ⁇ M) on a 1/4 ⁇ TE solution at 4°C for about 2 to 3 hours.
  • the dialysis product was electroporated to transform DH10B E. coli competent cells (Invitrogen TM ElectroMAX TM DH10B TM Cells). Take 1 ⁇ L of dialysis product and 20 ⁇ L of electrocompetent cells and mix them, transfer them into a pre-cooled 0.1cm electroporation cup, and place them in BioRad Electric shock conversion (parameters: voltage 2.0kV, resistance 200 ⁇ , capacitance 25 ⁇ F).
  • the total number of clones is about 45,000, and the average insert fragment of each clone is 100kb.
  • the library coverage is more than 10 times.
  • BAC plasmid sequencing and sequence analysis of positive clones Use Omega’s BAC/PAC DNA Maxi Kit to extract plasmids cloned from P1-10G, P3-12F and P2-9D, construct a 350bp small fragment library, and use HiSeq PE150 sequencing platform for second-generation sequencing , The amount of sequencing data per clone is 1Gb.
  • the sequencing data was assembled by Denovo without parameters, and the backbone sequence of the vector pCC1BAC TM was removed to obtain the insert sequence of the three positive plasmids.
  • the three insert sequences were assembled and assembled to obtain 307.5 kb fragments with overlapping and staggered end to end.
  • Galaxy’s TALgetter tool https://galaxy2.informatik.uni-halle.de:8976 was used to predict the recognition binding site (AvrXa7EBE) of AvrXa7 in the splicing sequence.
  • the P-Value of the four recognizing binding sites was less than 1.0E -6 , and only one AvrXa7EBE was located in the promoter region upstream of the ORF.
  • the ORF number is 52
  • the candidate gene is named CG52
  • its sequence is shown in SEQ ID NO.4.
  • BAC subcloning library construction and functional complementation experiment The insert fragments of BAC plasmids P1-10G and P3-12F carrying the CG52 gene were incompletely digested with BamH I and Sau3A I, respectively, and ligated to the subcloning vector pYLTAC747H/sacB. The library was screened with CG52 promoter region and CDS region amplification primers, and the positive subclones were then extracted from the plasmids for end sequencing to determine the sequence of the insert.
  • BAC plasmids were selected and passed Agrobacterium EHA105 (purchased from Beijing Huayueyang Biotechnology Co., Ltd., and the steps for plasmid transformation Agrobacterium were in accordance with the literature "Hood et al., 1993, New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res, 2,208-218" operation) mediates, transforms the indica susceptible variety IR24 (the rice variety IR24 is in the document "Ogawa et al.1991.
  • the booting stage of transgenic rice adopts the "leaf cutting method” (according to the literature “Wu Shangzhong et al., 1985, Comparative Study on Pathogenicity of Bacterial Blight in Southern China and the Philippines, Acta Phytopathology, 15-2:65-72.”) Inoculate Xanthomonas oryzae PXO86 (the strain has been published in the document "Mew TW et al. 1982, Pathotypes of Xanthomonas compestris pv.oryzae in Asia. IRRI Research Paper Series, No 75.”), and the antibiotics were carried out 21 days after inoculation.
  • the insert position information of the four subclones used for functional complementary genetic transformation is shown in Figure 1A.
  • the L235 and L239 clones cover the full length of the CG52 gene.
  • the transgenic rice lines of these two clones are resistant to disease; the sequence of the L236 clone overlaps with the 5'end of the L235 fragment, and its 3'end contains only the partial promoter of the CG52 gene
  • the subregion, at the position of 213bp upstream of AvrXa7EBE, the transgenic rice line is susceptible; the 3'end of clone L240 overlaps with clone L235, and its 5'end only contains the CDS region of CG52 and the 13bp UTR sequence, and the AvrXa7EBE sequence is deleted.
  • RNA was extracted with the near-isogenic line IRBB7 carrying the Xa7 gene, and the 5'end and 3'end of Xa7 were amplified by Invitrogen TM GeneRacer TM Kit.
  • the specific primer for 5'RACE amplification is 5'-TGCCACCGATGAGGTAATCCTGC-3'
  • the specific primer for 3'RACE amplification is 5'-CCTCCTCGGAATCTGGCTCATGTC-3'.
  • RACE product passed PEASY TM- Blunt Zero Cloning Kit for cloning.
  • IRBB7 and IR24 were inoculated with Xanthomonas oryzae pylori (PXO86) at the booting stage by the "leaf clipping method" and samples were taken 0, 1, 3, and 5 days later to extract total RNA.
  • PXO86 Xanthomonas oryzae pylori
  • Reverse transcription into cDNA with Takara TM PrimeScript TM RT reagent Kit with gDNA Eraser, Premix Ex Taq TM II (Tli RNaseH Plus) reagent was used for gene quantitative analysis on Bio-Rad fluorescent quantitative PCR instrument CFX96 TM .
  • the 2- ⁇ CT method was used to calculate the relative expression of genes.
  • the amplification primer pairs of the target gene Xa7 are as follows:
  • Xa7 Fw 5’-GATCGTATGCCCGTTGCAGTTGC-3’;
  • the amplification primer pairs of the internal reference gene TF2 are as follows:
  • TF2 Fw 5’-GCCTGAAGTGTACTGTACCACCAC-3’;
  • Example 3 CRISPR/Cas9-mediated gene knockout to verify the key functional sites of the Xa7 gene
  • the present invention also uses the CRISPR/Cas9 system to construct gene knockout transgenic lines of these two functional regions.
  • the vector used for gene knockout is the binary expression vector pYLCRISPR/Cas9P ubi -H provided by the Liu Yaoguang laboratory of South China Agricultural University (Already in the document "Ma et al.2015, A robust CRISPR/Cas9system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”), the intermediate vector pYLsgRNA-OsU6aL (already in the document “Ma et al.2015, A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”), pYLsgRNA-Os
  • search PAM protospacer advanced motif sequences in the AvrXa7EBE and CDS regions of the Xa7 promoter region through CRISPR-P, select the edited target site, and design the sgRNA target linker according to the target site sequence.
  • the corresponding target adapter sequence is as follows:
  • Target1 (targeting AvrXa7EBE, the target sequence is located at positions -126 ⁇ -107 in Figure 3A):
  • OsU6aT1F 5’-gccgTATGTGGTTATCTGGGGGGG-3’;
  • OsU6aT1R 5’-aaacCCCCCCCAGATAACCACATA-3’;
  • Target2 (targeting AvrXa7EBE, the target sequence is located at positions -121 ⁇ -102 in Figure 3A):
  • OsU6aT2F 5’-gccgTTCGTATGTGGTTATCTGG-3’;
  • OsU6aT2R 5’-aaacCCAGATAACCACATACGAA-3’;
  • Target3 targeting the CDS region, the target sequence is located at positions +22 ⁇ +41 in Figure 4A:
  • OsU3T3F 5’-ggcaCTGCAACGGGCATACGATC-3’;
  • OsU3T3R 5’-aaacGATCGTATGCCCGTTGCAG-3’;
  • Target4 targeting the CDS region, the target sequence is located at positions +94 ⁇ +113 in Figure 4A:
  • OsU6cT4F 5’-tcagCGACTGCTGACCGTCAACTC-3’;
  • OsU6cT4R 5'-aaacGAGTTGACGGTCAGCAGTCG-3'.
  • primers 5'-GAACTGCTCTGCTCAAGTGCCTC-3'; QC Rv: 5'-TGCCACCGATGAGGTAATCCTGC-3'
  • the W6-4 and W7-4 homozygous strains obtained by editing the transgene by Target1 are produced in the ArvXa7EBE element in the Xa7 promoter region.
  • the gene expression analysis of these transgenic lines showed (Figure 3B) that the absence of the ArvXa7EBE element will cause the loss of the function of Xa7 induced expression by the pathogen, and thus become susceptible to the pathogen ( Figure 3C).
  • the W12-1, W12-6, W13-4 and W15-3 homozygous strains obtained by editing the transgenes of Target3 and Target4 produced different types of base deletion, insertion and substitution mutations in the CDS region of the Xa7 gene, respectively ( Figure 4A) , Resulting in early termination, frameshift, substitution and other mutations in the encoded protein after Xa7 mutation. Although the CDS region of the gene is mutated, its transcription is still activated and expressed by pathogens (Figure 4B), but the resistance of these mutant homozygous strains to pathogens is still lost (Figure 4C).

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Abstract

提供一种水稻抗白叶枯病蛋白Xa7蛋白，其氨基酸序列如SEQ ID NO.1所示，编码该水稻抗白叶枯病蛋白的基因的核苷酸序列如SEQ ID NO.2所示，可用于研究水稻抗白叶枯病的机制，用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物。

Description

一种水稻抗白叶枯病蛋白及其编码基因与应用 技术领域
本发明属于生物技术领域，特别一种水稻抗白叶枯病蛋白及其编码基因与应用。
背景技术
白叶枯病(Xanthomonas oryzae pv.oryzae(Xoo))是我国以及世界稻米主产区东南亚水稻的主要病害之一，严重威胁稻米安全生产。利用寄主抗性是控制该病害的有效措施。然而，由于病菌致病性变异，常造成品种抗性丧失，品种抗性的持效性及其机制成为当今抗病性研究的一个关键点，深入了解持久抗病性分子机制，对水稻品种获得持久抗性，持续有效地控制病害有着重要意义[伍尚忠,1982,《水稻白叶枯病及其防治》,上海科技出版社；Mew,1987,Current status and future prospects of research on bacterial blight of rice.Ann.Rev.Phytopathol.,25:359-382.]。在已鉴定的白叶枯病抗性基因中，Xa7被认为是一个持久抗病基因，能有效抵抗不同的致病型病原菌，在世界众多国家表现出优异而稳定的抗性[Ona et al.,1998,Epidemic development of bacterial blight on rice carrying resistance genes Xa-4,Xa-7,and Xa-10.Plant Dis.,82:1337-1340.；Adhikari et al.,1999,Virulence of Xanthomonas oryzae pv.oryzae on rice lines containing single resistance genes and gene combinations.Plant Dis.,83:46-50；Vera et al.,2000,Predicting durability of a disease resistance gene based on an assessment of the fitness loss and epidemiological consequences of avirulence gene mutation.Proc.Natl.Acad.Sci.,97:13500-13505.；曾列先等,2006,水稻抗白叶枯病近等基因系对华南菌系的抗性研究.植物病理学报,36:177-180]。
Xa7基因最初是由国际水稻所(IRRI)在水稻品种DV85上鉴定出来的[Sidhu et al.,1978Genetic analysis of bacterial blight resistance in seventy-four cultivars of rice,Oryza sativa L.Theor Appl Genet,53:105-111.]。Ogawa等通过DV85与IR24重复回交，把基因Xa7导入近等基因系IRBB7中[Ogawa et al.,1991,Breeding of near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen Xanthomonas campestris pv.oryzae.Jpn J Breed,41:523-529.]。Hopkins等研究表明Xa7是与一个无毒基因家族直接相对应的显性抗性基因[Hopkins et al.,1992,Identification of a family of avirulence genes from Xanthomonas oryzae pv.oryzae.Mol Plant Microbe Interact,5:451-459.]，Kaji and Ogawa将该基因标记定位于第6染色体RGP图谱107.5cM位置，与G1091标记的重组率为8％[Kaji R and Ogawa T.Identification of the located chromosome of the resistance gene,Xa-7,to bacterial leaf blight in rice.Breed.Sci.,1995,45(Suppl.1):79.]，Porter等对Xa7进行了精细定位，但由于当时水稻基因组测序尚为完善，无法对目标基因区域的候选基因进行分析及预测[Porter et al.,2003,Development and mapping of markers linked to the rice bacterial blight resistance gene Xa7.Crop Science,43:1484-1493.]。本课题组通过大群体分析，将Xa7基因更精细地界定于分子标记GDSSR02和RM20593之间，同样由于水稻基因组已测序品种代表性的局限，该基因定位的区域存在较大间隙(Gap)，仍然未能克隆目的基因[Chen et al.，High-resolution mapping and gene prediction of Xanthomonas oryzae pv.Oryzae resistance gene Xa7.Molecular Breeding,2008,3:433-441.]。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种水稻抗白叶枯病蛋白。
本发明的另一目的在于提供编码上述水稻抗白叶枯病蛋白的基因。
本发明的再一目的在于提供上述基因的启动子区病原诱导调控元件。
本发明的再一目的还在于提供上述蛋白、基因以及启动子区病原诱导调控元件的应用。
本发明的目的通过下述技术方案实现：一种水稻抗白叶枯病蛋白，名称为Xa7蛋白，其氨基酸序列如下所示：
Figure PCTCN2020082732-appb-000001
编码上述水稻抗白叶枯病蛋白的基因，名称为Xa7基因，其核苷酸序列如下所示：
Figure PCTCN2020082732-appb-000002
编码上述水稻抗白叶枯病蛋白的基因的启动子区病原诱导调控元件，其核苷酸序列如下所示：TATAACCCCCCCCCCCCCAGATAACCA。
所述的水稻抗白叶枯病蛋白可通过化学合成法合成得到；或是通过将编码所述的水稻抗白叶枯病蛋白的基因克隆入表达载体中，得到的重组表达载体转化宿主细胞，表达后纯化得到。
所述的编码水稻抗白叶枯病蛋白的基因的制备，可通过以下方式实现：通过化学合成方式获得；或者设计引物，以DV85、IRBB7或其他携带Xa7基因的水稻品种基因组DNA为模板，PCR扩增获得；或者从携带Xa7基因的质粒中酶切、筛选获得。
所述的编码水稻抗白叶枯病蛋白的基因的应用，可用于研究水稻抗白叶枯病的机制，也可用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物，或是将其作为分子标记，用于选育对水稻白叶枯病具有抗病性的水稻品种。
所述的用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物的步骤优选如下：将上述编码水稻抗白叶枯病蛋白的基因和上述启动子区病原诱导调控元件导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物；或者将组成型表达启动子或其它病原诱导型启动子与上述基因的编码序列串联，导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物。
所述的用于选育对水稻白叶枯病具有抗病性的水稻品种的步骤优选如下：将携带上述基因的水稻品种作为供体亲本，与易感白叶枯病的水稻品种进行花粉杂交，得到的一系列后代利用Xa7作为分子标记进行筛选，鉴定得到抗白叶枯病水稻品种。
所述的供体亲本优选为DV85或IRBB7。
编码上述水稻抗白叶枯病蛋白的基因的启动子区病原诱导调控元件的应用，可用于研究水稻抗白叶枯病的机制，也可用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物。
所述的用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物的步骤优选如下：将上述编码水稻抗白叶枯病蛋白的基因和上述启动子区病原诱导调控元件导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物；或者将上述启动子区病原诱导调控元件与其它抗病基因编码序列串联，导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物。
本发明相对于现有技术具有如下的优点及效果：
本发明是在前期研究基础上，通过构建水稻品种IRBB7的基因组BAC文库，文库筛选、候选插入片段测序、目标插入片段序列对AvrXa7识别位点预测、以及一系列的转基因功能互补试验、基因敲除试验，最终完成了Xa7功能基因的克隆。本发明首次提供了Xa7功能基因的序列。
附图说明
图1是亚克隆片段位置示意图及其转基因株系抗病表型图；其中，图A为用于转基因功 能互补实验的亚克隆片段重叠区位置与序列示意图，图B为亚克隆转基因水稻株系对白叶枯病菌PXO86的抗性表型照片图；图C为亚克隆转基因水稻株系对白叶枯病菌PXO86的病斑长度统计结果图。
图2是Xa7基因结构特征与抗性表达模式图；其中，图A为Xa7基因序列结构特征示意图，图B为Xa7基因对白叶枯病菌PXO86的抗性表达模式图。
图3是Xa7基因启动子区病原诱导元件敲除功能验证结果图；其中，图A为Xa7基因启动子区病原诱导元件进行基因编辑后的突变纯合株系序列，图B为各突变株系对白叶枯病菌PXO86的抗性表达模式图，图C为各突变株系对白叶枯病菌PXO86的抗病表型照片图。
图4是Xa7基因编码区敲除功能验证结果图；其中，图A为Xa7基因编码区进行基因编辑后的突变纯合株系序列，图4为各突变株系对白叶枯病菌PXO86的抗性表达模式，图C为各突变株系对白叶枯病菌PXO86的抗病表型。
具体实施方式
以下结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式并不限于此。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。在本发明的实施例部分，阐述了Xa7基因的分离克隆、功能特征及其功能验证。
实施例1 Xa7基因的分离克隆
在本发明前期研究基础上，发现Xa7基因位点附近的基因组序列与常见的水稻参考基因组日本晴、9311、明恢63和珍汕97等均存在很大差异。明确该区域基因组序列是实现该基因克隆的前提基础。本发明通过构建Xa7抗源品种IRBB7基因组BAC文库，用Xa7两侧紧密连锁分子标记筛选文库，钓取阳性克隆，并对阳性克隆插入片段进行测序，从而获得该区域完整、精确的基因组序列。
水稻品种IRBB7已在文献“Ogawa et al.,1991,Breeding of near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen Xanthomonas campestris pv.oryzae.Japan J Breed,41:523-529.”)中公开。
构建基因组BAC文库的植物材料为含有Xa7基因的近等基因系IRBB7，载体为Epicentre公司的CopyControl TM pCC1BAC TM，亚克隆与转基因表达载体为pYLTAC747H/sacB(已在文献“Xu et al.,2008,Construction and characterization of the transformation-competent artificial chromosome(TAC)libraries of Leymus multicaulis.Science in China(Series C:Life Sciences),(07):604-613.”中公开)。
BAC文库构建：按照文献“Liu et al.,2002,Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning.Gene,282(1):247-255.”报道的实验步骤操作。于苗期提取IRBB7基因组DNA，用HindⅢ限制性内切酶部分酶切，用脉冲场电泳分离120-140kb长度的DNA片段，纯化后与BAC载体pCC1BAC TM进行连接。75ng BAC载体分别与五组基因组DNA酶切产物混合(A:30ng、B:60ng、C:120ng、D:160ng、E:350ng)，按
Figure PCTCN2020082732-appb-000003
T4 DNA连接酶50μL体系配制反应体系，在PCR仪里用变温连接程序进行连接反应：10℃3min，3min升至16℃，16℃5min，30s升至18℃，18℃30s，30s升至20℃，20℃30s，8s降至4℃，4℃3min，5min从4℃升温至22℃，22℃1min降到10℃，如此循环20次，最后65℃5min。连接产物用MILLIPORE TM的VSWP膜(0.025μM)在4℃，1/4×TE溶液上透析约2～3h。透析产物电击转化DH10B大肠杆菌感受态细胞(为Invitrogen TM的ElectroMAX TM DH10B TM Cells)。取1μL透析产物和20μL电转感受态细胞混匀，转入预冷的0.1cm电击杯，在BioRad
Figure PCTCN2020082732-appb-000004
电击仪上电击转化(参数为：电压2.0kV，电阻200Ω，电容25μF)。电击后，迅速加入1mL S.O.C.培养基，37℃、200rpm摇床恢复培养1小时。取10～100μL之间十个梯度的菌液量分别涂于含25μg/mL卡那霉素和5％蔗糖的LB半固体培养基，于37℃培养12～16h，计算克隆数。然后以每平板约500个克隆的菌液量按相同方式涂板培养，并整板刮取混合菌落，分装到96孔深孔板构建“BAC克隆混合池”。每孔 按照大概450个原始单克隆计，总克隆数约为45000个，平均每个克隆插入片段100kb，相对于水稻全基因组430Mb来推算，该文库覆盖率大于10倍。
BAC阳性克隆筛选：用碱裂解法提取各个“BAC克隆混合池”质粒DNA，以混合池质粒为模板，扩增Xa7两侧紧密连锁分子标记U05-indel和POZ-indel，其中，扩增分子标记U05-indel的引物对为U05-indel Fw和U05-indel Rv，扩增分子标记POZ-indel的引物对为POZ-indel Fw和POZ-indel Rv。检测到阳性的混合池后，将阳性池稀释40000倍，按每孔5×倍率分装到384孔板，培养过夜后取菌液为模板，再次用对应的分子标记引物进行PCR扩增筛选，直至筛选到阳性单克隆。经过多轮筛选，鉴定分离到三个阳性克隆：P1-10G、P3-12F和P2-9D。经酶切和电泳检测，这三个BAC克隆插入片段分别为150kb、125kb和107kb。
U05-indel Fw:5’-CAGACAAGTGTTGTTCATGTTCG-3’；
U05-indel Rv:5’-GAAGTCCGAGCTGGGGACGATGTAC-3’；
POZ-indel Fw:5’-CCAAGAAAGGTCCAACTCGCTTAG-3’；
POZ-indel Rv:5’-GAACAGTCCTCAGAATTCGACCAC-3’。
阳性克隆BAC质粒测序及序列分析：用Omega公司的BAC/PAC DNA Maxi Kit提取P1-10G、P3-12F和P2-9D克隆的质粒，构建350bp小片段文库，采用HiSeq PE150测序平台进行二代测序，每个克隆的测序数据量为1Gb。测序数据进行Denovo无参组装，并剔除载体pCC1BAC TM的骨架序列，分别获得这三个阳性质粒的插入片段序列。这三个插入片段序列经拼接组装后得到相互间首尾有重叠交错的307.5kb片段。与国际通用的水稻基因组序列比对，发现该片段对应于粳稻品种日本晴基因组增加了101.1kb，对应于籼稻品种明恢63基因组增加了80kb。利用Softberry的Fgenesh工具(https://www.softberry.com/berry.phtml？topic＝fgenesh&group＝programs&subgroup＝gfind)对307.5kb的拼接序列进行预测，得到82个开放阅读框(ORF)。为进一步缩小目标范围，通过Galaxy的TALgetter工具(https://galaxy2.informatik.uni-halle.de:8976)在拼接序列中预测AvrXa7的识别结合位点(AvrXa7EBE)。结果检索到四个识别结合位点的P-Value小于1.0E -6，其中仅有一个AvrXa7EBE位于ORF上游的启动子区。这个ORF编号为52，该候选基因命名为CG52，其序列如SEQ ID NO.4所示。
BAC亚克隆文库构建与功能互补实验：将携带有CG52基因的BAC质粒P1-10G和P3-12F的插入片段分别用BamH Ⅰ和Sau3A Ⅰ不完全酶切，连接到亚克隆载体pYLTAC747H/sacB上。分别用CG52启动子区和CDS区扩增引物筛选文库，阳性亚克隆再分别提取质粒进行末端测序，确定插入片段的序列。根据序列信息挑选四个亚克隆BAC质粒通过农杆菌EHA105(购于北京华越洋生物科技有限公司，质粒转化农杆菌步骤按文献“Hood et al.,1993,NewAgrobacterium helper plasmids for gene transfer to plants.Transgenic Res,2,208-218”操作)介导，转化籼稻感病品种IR24(水稻品种IR24在文献“Ogawa et al.1991.Breeding of near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen Xanthomonas campestris pv.oryzae..Japan J Breed.41:523-529.”中公开)，籼稻品种的遗传转化按照文献“Lin and Zhang,2005,Optimising the tissue culture conditions for high efficiency transformation of indica rice.Plant Cell Rep,23:540-547.”报道的方法操作。转基因水稻孕穗期以“剪叶法”(按文献“伍尚忠等,1985,华南及菲律宾稻白叶枯病病原菌株致病性比较研究,植物病理学报,15-2:65-72.”操作)接种水稻白叶枯菌PXO86(菌株已在文献“Mew TW et al.1982,Pathotypes of Xanthomonas compestris pv.oryzae in Asia.IRRI Research Paper Series,No75.”中公开)，于接菌后21天进行抗病表型调查，参照国际水稻所INGER的方法调查评价抗性(按文献“INGER Genetic Resources Center,1996,Standard Evaluation System for rice(4th edition)[M].Philippines:International Rice Research Institute Press,p20-21.”操作)。
用作功能互补遗传转化的四个亚克隆的插入片段位置信息如图1A所示。L235和L239克隆覆盖率CG52基因全长，这两个克隆的转基因水稻株系均表现抗病；L236克隆的序列与L235片段的5’端重合，其3’端只包含了CG52基因的部分启动子区，在AvrXa7EBE上游213bp位置，其转基因水稻株系表现感病；而L240克隆3’端与L235克隆重合，其5’端仅包含CG52 的CDS区和13bp的UTR序列，缺失了AvrXa7EBE序列，其转基因水稻株系表现感病。由此可知，CG52基因就是抗病功能基因，而且启动子区AvrXa7EBE和完整的CDS序列都是该基因功能不可或缺的部分。(图1B和图1C)。
实施例2 Xa7基因的序列结构与表达特征
Xa7基因的序列结构分析：用携带Xa7基因的近等基因系IRBB7提取总RNA，通过Invitrogen TM的GeneRacer TM Kit分别扩增Xa7的5’端和3’端全长。其中，5’RACE扩增特异引物为5’-TGCCACCGATGAGGTAATCCTGC-3’，3’RACE扩增特异引物为5’-CCTCCTCGGAATCTGGCTCATGTC-3’。RACE产物通过
Figure PCTCN2020082732-appb-000005
的pEASY TM-Blunt Zero Cloning Kit进行克隆。测序后确定Xa7的转录起始位点(TSS)位于起始密码子(ATG)上游的104bp处；同时确定了Xa7的3’UTR长度为253bp(图2A)。
Xa7基因的表达模式分析：IRBB7和IR24在孕穗期以“剪叶法”接种白叶枯菌(PXO86)后分别于0、1、3、5天后取样，提取总RNA。用Takara TM的PrimeScript TM RT reagent Kit with gDNA Eraser反转录成cDNA，通过
Figure PCTCN2020082732-appb-000006
Premix Ex Taq  TMⅡ(Tli RNaseH Plus)试剂在Bio-Rad荧光定量PCR仪CFX96 TM上进行基因定量分析。采用2 －△△CT方法计算基因的相对表达量。
目的基因Xa7的扩增引物对如下：
Xa7 Fw:5’-GATCGTATGCCCGTTGCAGTTGC-3’；
Xa7 Rv:5’-GGAGTTGACGGTCAGCAGTCGAG-3’。
内参基因TF2的扩增引物对如下：
TF2 Fw:5’-GCCTGAAGTGTACTGTACCACCAC-3’；
TF2 Rv:5’-CAAAGGGTTCAGAAATGAGGAA GG-3’。
结果如图2B所示，Xa7基因在接菌前表达水平很低，接菌1天后表达量开始上调，3天后达到峰值，5天后表达量开始回落。因此，Xa7基因的表达模式是病原菌诱导激活型的。
实施例3 由CRISPR/Cas9介导的基因敲除验证Xa7基因的关键功能位点
为进一步验证Xa7启动子区AvrXa7EBE和CDS区序列的功能，本发明还利用CRISPR/Cas9系统构建这两个功能区域的基因敲除转基因株系。基因敲除所用的载体是华南农业大学刘耀光实验室提供的双元表达载体pYLCRISPR/Cas9P ubi-H(已在文献“Ma et al.2015,A robust CRISPR/Cas9system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”中公开)，中间载体pYLsgRNA-OsU6aL(已在文献“Ma et al.2015,A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”中公开)，pYLsgRNA-OsU3aL(已在文献“Ma et al.2015,A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”中公开)和pYLsgRNA-OsU6c(已在文献“Ma et al.2015,A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”中公开)。编辑位点sgRNA靶向序列的选择和设计是采用在线工具CRISPR-P(https://crispr.hzau.edu.cn/CRISPR/)辅助完成。
首先通过CRISPR-P在Xa7启动子区AvrXa7EBE和CDS区检索PAM(protospacer adjacent motif)序列，选择编辑的靶位点，根据靶位点序列设计sgRNA靶标接头。相应靶标接头序列如下：
Target1(靶向AvrXa7EBE，靶序列位于图3A的-126～-107位置)：
OsU6aT1F:5’-gccgTATGTGGTTATCTGGGGGGG-3’；
OsU6aT1R:5’-aaacCCCCCCCAGATAACCACATA-3’；
Target2(靶向AvrXa7EBE，靶序列位于图3A的-121～-102位置)：
OsU6aT2F:5’-gccgTTCGTATGTGGTTATCTGG-3’；
OsU6aT2R:5’-aaacCCAGATAACCACATACGAA-3’；
Target3(靶向CDS区，靶序列位于图4A的+22～+41位置)：
OsU3T3F:5’-ggcaCTGCAACGGGCATACGATC-3’；
OsU3T3R:5’-aaacGATCGTATGCCCGTTGCAG-3’；
Target4(靶向CDS区，靶序列位于图4A的+94～+113位置)：
OsU6cT4F:5’-tcagCGACTGCTGACCGTCAACTC-3’；
OsU6cT4R:5’-aaacGAGTTGACGGTCAGCAGTCG-3’。
按照文献“Ma et al.2015,A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.Mol.Plant.8,1274-1284.”报道的实验步骤，分别将靶标接头连接到相应的Bsa Ⅰ酶切sgRNA中间载体，经过两轮PCR反应及产物退火、巢式PCR扩增得到特异的sgRNA表达盒模板。表达盒再通过连接反应组装进双元表达载体。其中，Target1和Target2分别独立构建双元表达载体，Target3和Target4一起构建到同一个双元表达载体。这三个基因编辑表达载体分别通过EHA105农杆菌介导，转入IRBB7品种中。
各基因敲除转基因家系用引物(QC Fw:5’-GAACTGCTCTGCTCAAGTGCCTC-3’；QC Rv:5’-TGCCACCGATGAGGTAATCCTGC-3’)PCR扩增靶向位点后测序，在T 0代、T 1代筛选编辑成功的纯合株系。
如图3A所示，Target1编辑转基因获得的W6-4和W7-4纯合株系，Target2编辑转基因获得的W8-7和W9-6纯合株系，都分别在Xa7启动子区ArvXa7EBE元件产生了碱基缺失。这些转基因株系的基因表达分析表明(图3B)，ArvXa7EBE元件的缺失会造成Xa7受病原诱导表达的功能丧失，从而对病原菌表现感病(图3C)。
Target3和Target4编辑转基因获得的W12-1、W12-6、W13-4和W15-3纯合株系，分别在Xa7基因CDS区产生了不同类型的碱基缺失、插入和替换突变(图4A)，导致Xa7突变后的编码蛋白发生提前终止、移码、替换等突变。尽管基因CDS区突变后，其转录仍受病原菌激活表达(图4B)，但这些突变纯合株系对病原菌的抗性仍旧丧失了(图4C)。
本实施例一方面反向验证了Xa7的功能基因，另一方面阐明了Xa7基因的抗病功能由两个必要因素构成：一是其启动子区AvrXa7EBE元件在病原诱导下的转录激活功能，另一个是完整的Xa7基因编码蛋白执行抗病反应。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 一种水稻抗白叶枯病蛋白，其特征在于：氨基酸序列如SEQ ID NO.1所示。
  2. 编码权利要求1所述的水稻抗白叶枯病蛋白的基因，其特征在于：核苷酸序列如SEQ ID NO.2所示。
  3. 根据权利要求2所述的基因，其特征在于：还包括核苷酸序列如SEQ ID NO.3所示的启动子区病原诱导调控元件。
  4. 权利要求2所述的基因的应用，其特征在于：将所述的基因用于研究水稻抗白叶枯病的机制，用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物，或是用于选育对水稻白叶枯病具有抗病性的水稻品种。
  5. 根据权利要求4所述的基因的应用，其特征在于：所述的用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物的步骤如下：将权利要求2所述的基因和权利要求3所述的启动子区病原诱导调控元件导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物；或者将组成型表达启动子或其它病原诱导型启动子与权利要求2所述的基因串联，导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物。
  6. 根据权利要求4所述的基因的应用，其特征在于：所述的用于选育对水稻白叶枯病具有抗病性的水稻品种的步骤如下：将携带权利要求2所述的基因的水稻品种作为供体亲本，与易感白叶枯病的水稻品种进行花粉杂交，得到的一系列后代通过权利要求2所述的基因作为分子标记进行筛选，鉴定得到抗白叶枯病水稻品种。
  7. 权利要求2所述的基因的启动子区病原诱导调控元件，其特征在于：核苷酸序列如SEQ ID NO.3所示。
  8. 权利要求7所述的基因的启动子区病原诱导调控元件的应用，其特征在于：将所述的基因的启动子区病原诱导调控元件用于研究水稻抗白叶枯病的机制，或是用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物。
  9. 根据权利要求8所述的基因的启动子区病原诱导调控元件的应用，其特征在于：所述的用于培育对水稻白叶枯病具有抗病性的水稻品种或者其它抗病性作物的步骤如下：将权利要求2所述的基因和权利要求7所述的启动子区病原诱导调控元件导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物；或者将权利要求7所述的启动子区病原诱导调控元件与其它抗病基因编码序列串联，导入易感病的水稻或其它作物中，得到具有抗病性的水稻或抗病性作物。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438100A (zh) * 2022-03-01 2022-05-06 云南省农业科学院生物技术与种质资源研究所 一种高效分离带有野生稻血缘的抗白叶枯病基因及其家族成员的方法
CN117070531A (zh) * 2023-08-31 2023-11-17 中国科学院华南植物园 水稻OsWAK123基因及其编码蛋白的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112301035B (zh) * 2020-09-10 2022-12-27 浙江师范大学 高抗水稻白叶枯病基因Xa7及其用途
CN112266921A (zh) * 2020-11-13 2021-01-26 上海交通大学 水稻白叶枯病抗病基因Xa7及其应用
CN114350687B (zh) * 2022-03-01 2023-08-22 云南省农业科学院生物技术与种质资源研究所 一种水稻抗白叶枯病基因、蛋白及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101451141A (zh) * 2008-12-18 2009-06-10 上海交通大学 激发水稻产生抗病反应的无毒基因avrxa5
CN101974079A (zh) * 2010-08-17 2011-02-16 中国农业科学院作物科学研究所 激发水稻产生抗病反应的avrXa23蛋白及其编码基因
CN103333231A (zh) * 2013-05-24 2013-10-02 中国农业科学院作物科学研究所 植物抗病蛋白Xa23及其编码基因与应用
CN107475210A (zh) * 2017-09-07 2017-12-15 四川农业大学 一种水稻白叶枯病抗性相关基因OsABA2及其应用
CN108220327A (zh) * 2016-12-12 2018-06-29 中国科学院遗传与发育生物学研究所 培育抗白叶枯病植物的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1175594A (ja) * 1997-09-08 1999-03-23 Norin Suisansyo Nogyo Seibutsu Shigen Kenkyusho チオニン遺伝子を用いた複数病害抵抗性植物の作出方法
CN100381465C (zh) * 2005-01-19 2008-04-16 中国科学院遗传与发育生物学研究所 一种检测水稻白叶枯病抗性的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101451141A (zh) * 2008-12-18 2009-06-10 上海交通大学 激发水稻产生抗病反应的无毒基因avrxa5
CN101974079A (zh) * 2010-08-17 2011-02-16 中国农业科学院作物科学研究所 激发水稻产生抗病反应的avrXa23蛋白及其编码基因
CN103333231A (zh) * 2013-05-24 2013-10-02 中国农业科学院作物科学研究所 植物抗病蛋白Xa23及其编码基因与应用
CN108220327A (zh) * 2016-12-12 2018-06-29 中国科学院遗传与发育生物学研究所 培育抗白叶枯病植物的方法
CN107475210A (zh) * 2017-09-07 2017-12-15 四川农业大学 一种水稻白叶枯病抗性相关基因OsABA2及其应用

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438100A (zh) * 2022-03-01 2022-05-06 云南省农业科学院生物技术与种质资源研究所 一种高效分离带有野生稻血缘的抗白叶枯病基因及其家族成员的方法
CN114438100B (zh) * 2022-03-01 2023-11-10 云南省农业科学院生物技术与种质资源研究所 一种高效分离带有野生稻血缘的抗白叶枯病基因及其家族成员的方法
CN117070531A (zh) * 2023-08-31 2023-11-17 中国科学院华南植物园 水稻OsWAK123基因及其编码蛋白的应用
CN117070531B (zh) * 2023-08-31 2024-01-26 中国科学院华南植物园 水稻OsWAK123基因及其编码蛋白的应用

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