阅读说明：本技术 一种水稻抗白叶枯病蛋白及其编码基因与应用 (Rice bacterial leaf blight-resistant protein and coding gene and application thereof ) 是由 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 于 2019-03-08 设计创作，主要内容包括：本发明公开了一种水稻抗白叶枯病蛋白及其编码基因与应用。该水稻抗白叶枯病蛋白的名称为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。本发明通过构建水稻品种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,其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,或是用于选育对水稻白叶枯病具有抗病性的水稻品种。(The invention discloses a rice bacterial leaf blight resistant protein, and a coding gene and application thereof. The rice bacterial leaf blight resistant protein is Xa7 protein, and the amino acid sequence of the rice bacterial leaf blight resistant protein is shown in SEQ ID NO. 1. The nucleotide sequence of the gene for coding the rice bacterial leaf blight resistant protein is shown as SEQ ID NO. 2. The cloning of the Xa7 functional gene is finally completed by constructing a genome BAC library of a rice variety IRBB7, screening the library, sequencing a candidate insert, predicting an AvrXa7 recognition site by a target insert sequence, and performing a series of transgenic function complementation tests and gene knockout tests. The invention provides the Xa7 functional gene sequence for the first time, which can be used for researching the mechanism of resisting bacterial leaf blight of rice, cultivating rice varieties with disease resistance to bacterial leaf blight of rice or other disease-resistant crops, or breeding rice varieties with disease resistance to bacterial leaf blight of rice.)

1. A rice bacterial leaf blight resistant protein is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.

2. A gene encoding the rice protein of claim 1, which is resistant to bacterial blight and characterized in that: the nucleotide sequence is shown as SEQID NO. 2.

3. The gene according to claim 2, characterized in that: also comprises a promoter region pathogen induction regulatory element with a nucleotide sequence shown as SEQ ID NO. 3.

4. Use of the gene of claim 2, wherein: the gene is used for researching a mechanism of resisting bacterial blight of rice, and is used for cultivating rice varieties with disease resistance to bacterial blight of rice or other disease-resistant crops, or breeding rice varieties with disease resistance to bacterial blight of rice.

5. Use of a gene according to claim 4, characterized in that: the steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are as follows: introducing the gene of claim 2 and the promoter region pathogen-induced regulatory element of claim 3 into susceptible rice or other crops to obtain disease-resistant rice or disease-resistant crops; or connecting a constitutive expression promoter or other pathogen inducible promoters with the gene of claim 2 in series, and introducing the gene into susceptible rice or other crops to obtain rice or disease-resistant crops.

6. Use of a gene according to claim 4, characterized in that: the steps for breeding the rice variety with disease resistance to the bacterial blight of the rice are as follows: using a rice variety carrying the gene of claim 2 as a donor parent, performing pollen hybridization with a rice variety susceptible to bacterial blight, screening a series of obtained offspring by using the gene of claim 2 as a molecular marker, and identifying to obtain the rice variety resistant to bacterial blight.

7. The promoter region pathogen-inducible regulatory element of the gene of claim 2, wherein: the nucleotide sequence is shown as SEQ ID NO. 3.

8. Use of the promoter region pathogen-inducible regulatory element of the gene of claim 7, wherein: the promoter region pathogen induction regulation and control element of the gene is used for researching a mechanism of resisting bacterial blight of rice, or is used for cultivating rice varieties with disease resistance to bacterial blight of rice or other disease-resistant crops.

9. Use of the promoter region pathogen-inducible regulatory element of the gene according to claim 8, wherein: the steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are as follows: introducing the gene of claim 2 and the promoter region pathogen-induced regulatory element of claim 7 into susceptible rice or other crops to obtain disease-resistant rice or disease-resistant crops; or the promoter region pathogen induction regulation element of claim 7 is connected in series with other disease-resistant gene coding sequences and is introduced into susceptible rice or other crops to obtain rice or disease-resistant crops.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a rice bacterial leaf blight resistant protein, and a coding gene and application thereof.

Background

Bacterial leaf blight (Xanthomonas oryzae pv. oryzae) is one of the main diseases of rice in southeast Asia in China and the main rice producing areas in the world, and seriously threatens the safe rice production. The utilization of host resistance is an effective measure for controlling the disease. However, due to pathogenic variation of pathogenic bacteria, the variety resistance is often lost, the persistence of the variety resistance and the mechanism thereof become a key point of the current disease resistance research, the persistent disease resistance molecular mechanism is deeply known, the persistent resistance is obtained for rice varieties, and the important significance is provided for continuously and effectively controlling diseases [ Wushangkai, 1982, rice bacterial blight and prevention thereof, Shanghai science and technology publishers; mew,1987, Current status and future protocols of research on bacterial light of rice, Ann.Rev.Phytopathol, 25:359, 382 ]. Among the identified bacterial blight resistance genes, Xa7 is considered to be a persistent disease resistance gene effective against different pathogenic bacteria and exhibiting excellent and stable resistance in numerous countries throughout the world [ Ona et al, 1998, Epidemic level of bacterial lighting restriction genes Xa-4, Xa-7, and Xa-10.Plant Dis.,82:1337-1340 ]; adhikari et al, 1999, Virus of Xanthomonas oryzae pv. oryzae on cellulose associations single resistance genes and gene combinations plant Dis.,83: 46-50; vera et al, 2000, differentiating duration of a discrete resistance generated on an assessment of the fine loss and elementary local control of the error gene mutation, Proc. Natl.Acad.Sci.,97: 13500-; the study on the resistance of a rice bacterial strain to a southern China bacterial strain by a bacterial strain near isogenic line for resisting bacterial blight, previously listed, 2006, 36:177-180 ℃.

The Xa7 gene was originally identified by the International Rice institute (IRRI) on rice variety DV85 [ Sidhu et al, 1978Genetic analysis of bacterial clearance resistance in the foundation-grassroots of rice, Oryza sativa L.the or apple Genet,53:105-111 ]. Ogawa et al introduced gene Xa7 into the near isogenic line IRBB7 by repeated backcrosses of DV85 with IR24 [ Ogawa et al, 1991, Breedenggof near-isogenic lines of rice with single genes for resistance to microbial light strain J Breed,41:523 pn 529 ]. Research by Hopkins et al showed that Xa7 is a dominant resistance gene directly corresponding to an avirulence gene family [ Hopkins et al, 1992, Identification of a family of genes from Xanthomonas oryzae pv. oryzae. mol Plant Microbe Interact,5: 451. sup. -, Kaji and Ogawa located the gene marker at position 107.5cM on chromosome 6 RGP map, the recombination rate with the G1091 marker was 8% [ Kaji R and ogawa T.identification of the located chromosome of the resistance gene, Xa-7, to bacterial leaf height in rice.Breed.Sci.,1995,45(suppl.1):79.], Porter et al fine-positioned Xa7, however, since the sequencing of rice genomes was now perfect, candidate genes for the target gene region could not be analyzed and predicted [ Porter et al, 2003, Development and mapping of markers linking and the line background gene Xa7.crop Science,43: 1484-. The subject group, by large population analysis, more finely bound the Xa7 gene between the molecular markers GDSSR02 and RM20593, also because of the limitation of representativeness of the sequenced varieties in the rice genome, the gene has a large Gap (Gap) in the located region, and the target gene still cannot be cloned [ Chen et al, High-resolution mapping and gene prediction of Xanthomonas oryzae pv. Oryzae resistance gene Xa7.molecular Breeding,2008,3: 433-.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a rice protein for resisting bacterial leaf blight.

Another object of the present invention is to provide a gene encoding the above rice protein against bacterial blight.

It is still another object of the present invention to provide a promoter region pathogen-inducible regulatory element of the above-mentioned gene.

The invention also aims to provide application of the protein, the gene and the promoter region pathogen induction regulatory element.

The purpose of the invention is realized by the following technical scheme: a rice bacterial leaf blight resistant protein is an Xa7 protein, and the amino acid sequence of the rice bacterial leaf blight resistant protein is as follows:

MAAADHPDRMPVAVAGLRHHYAFPANLRPAARLLTVNSGVFLISTAGAIVLVHTAGNPPAIDNDPAYALVAFVLFLLGIWLMSIALVADQFPRAAGVAVAIARALQDYLIGGN。

the gene for coding the rice bacterial blight resistant protein is named as Xa7 gene, and the nucleotide sequence of the gene is shown as follows:

ATGGCGGCCGCTGATCATCCTGATCGTATGCCCGTTGCAGTTGCAGGCTTGCGCCACCATTACGCCTTCCCTGCAAACCTTCGCCCCGCCGCTCGACTGCTGACCGTCAACTCCGGCGTCTTCCTCATCTCCACCGCCGGGGCCATCGTCCTCGTCCACACCGCCGGTAACCCACCCGCCATCGACAACGATCCAGCCTACGCCTTGGTCGCATTCGTGCTCTTCCTCCTCGGAATCTGGCTCATGTCTATTGCCCTCGTCGCCGACCAGTTCCCGCGCGCCGCTGGGGTCGCCGTGGCCATTGCCAGGGCGCTGCAGGATTACCTCATCGGTGGCAATTAA。

the promoter region pathogen induction regulatory element of the gene for coding the rice bacterial blight-resistant protein has the following nucleotide sequence: TATAACCCCCCCCCCCCCAGATAACCA are provided.

The rice bacterial leaf blight resistant protein can be obtained by chemical synthesis; or cloning the gene for encoding the rice bacterial leaf blight resistant protein into an expression vector to obtain a recombinant expression vector, transforming host cells by the obtained recombinant expression vector, and purifying after expression.

The preparation of the gene for coding the rice bacterial leaf blight resistant protein can be realized by the following modes: obtained by a chemical synthesis mode; or designing a primer, and carrying out PCR amplification by using DV85, IRBB7 or other rice variety genome DNA carrying Xa7 gene as a template; or obtained by enzyme digestion and screening from a plasmid carrying the Xa7 gene.

The application of the gene for coding the protein resisting bacterial blight of rice can be used for researching a mechanism of resisting bacterial blight of rice, can also be used for cultivating rice varieties with disease resistance to bacterial blight of rice or other disease-resistant crops, or can be used as a molecular marker for breeding the rice varieties with disease resistance to bacterial blight of rice.

The steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are preferably as follows: introducing the gene for coding the rice bacterial leaf blight resistant protein and the promoter region pathogen induction regulation element into susceptible rice or other crops to obtain rice or disease resistant crops; or connecting the constitutive expression promoter or other pathogenic inducible promoters with the coding sequence of the gene in series, and introducing the promoter into susceptible rice or other crops to obtain rice or disease-resistant crops.

The steps for breeding the rice variety with disease resistance to the bacterial blight of the rice are preferably as follows: the rice variety carrying the gene is used as a donor parent and is subjected to pollen hybridization with a rice variety susceptible to bacterial blight, and a series of obtained offspring are screened by using Xa7 as a molecular marker, so that the bacterial blight resistant rice variety is identified.

The donor parent is preferably DV85 or IRBB 7.

The application of the promoter region pathogen induction regulation element of the gene for coding the rice bacterial leaf blight resistant protein can be used for researching a rice bacterial leaf blight resistant mechanism and can also be used for cultivating rice varieties with disease resistance to rice bacterial leaf blight or other disease resistant crops.

The steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are preferably as follows: introducing the gene for coding the rice bacterial leaf blight resistant protein and the promoter region pathogen induction regulation element into susceptible rice or other crops to obtain rice or disease resistant crops; or the promoter region pathogen inducing and regulating element is connected serially with other disease resisting gene coding sequence and introduced into rice or other crop susceptible to disease to obtain rice or other disease resisting crop with disease resistance.

Compared with the prior art, the invention has the following advantages and effects:

on the basis of earlier stage research, the cloning of the Xa7 functional gene is finally completed by constructing a genome BAC library of a rice variety IRBB7, library screening, candidate insert sequencing, prediction of an AvrXa7 recognition site by a target insert sequence, and a series of transgenic function complementation tests and gene knockout tests. The invention provides the sequence of the Xa7 functional gene for the first time.

Drawings

FIG. 1 is a schematic diagram of the position of subcloned fragments and the phenotype of disease resistance of transgenic lines; wherein, the picture A is a schematic diagram of the position and the sequence of an overlapping region of a subcloned fragment used for a transgenic function complementation experiment, and the picture B is a photo picture of resistance phenotype of a subcloned transgenic rice line to the bacterial blight strain PXO 86; FIG. C is a statistical result chart of lesion length of the subcloned transgenic rice line against P.albuginea PXO 86.

FIG. 2 is a diagram showing structural features and resistance expression patterns of Xa7 gene; wherein, the picture A is a schematic diagram of the structural characteristics of the Xa7 gene sequence, and the picture B is a pattern diagram of the resistance expression of the Xa7 gene to P.albuginea PXO 86.

FIG. 3 is a diagram showing the result of the knockout function verification of the pathogenic inducing element in the promoter region of the Xa7 gene; wherein, the picture A is a mutation homozygous line sequence after gene editing is carried out on the Xa7 gene promoter region pathogen inducing element, the picture B is a resistance expression pattern picture of each mutation line to the bacterial blight strain PXO86, and the picture C is a picture of a disease-resistant phenotype picture of each mutation line to the bacterial blight strain PXO 86.

FIG. 4 is a diagram showing the result of verifying the knock-out function of the coding region of Xa7 gene; wherein, the picture A is the mutation homozygous line sequence after the Xa7 gene coding region carries on the gene editing, the picture 4 is the resistance expression pattern to the bacterial blight PXO86 of each mutant line, the picture C is the disease-resistant phenotype to bacterial blight PXO86 of each mutant line.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited to these examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. In the examples section of the present invention, isolated cloning of the Xa7 gene, functional characterization and functional verification thereof are illustrated.