阅读说明：本技术 提高水稻稻瘟病抗性的方法及其相关生物材料 (Method for improving rice blast resistance of rice and related biological material thereof ) 是由 李莉 李懿星 张大兵 邱牡丹 王天抗 宋书锋 于 2019-02-02 设计创作，主要内容包括：本发明公开了一种提高水稻稻瘟病抗性的方法及其相关生物材料。本发明提供了一种培育目的水稻的方法,包括如下步骤：抑制出发水稻中RAY1蛋白的活性,得到目的水稻；所述目的水稻与所述出发水稻相比,表现出对稻瘟病的抗性增强；所述RAY1蛋白为序列表中SEQ ID No.1所示的氨基酸序列组成的蛋白质。本发明利用CRISPR/Cas9技术,定点编辑水稻RAY1基因,通过移码突变,敲除了水稻了RAY1基因,使蛋白RAY1失活,获得了抗病性明显提高的新一代水稻新种质。(The invention discloses a method for improving rice blast resistance of rice and a related biological material thereof. The invention provides a method for cultivating target rice, which comprises the following steps: inhibiting the activity of RAY1 protein in the original rice to obtain the target rice; the target rice shows an enhanced resistance to rice blast as compared with the starting rice; the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table. The invention utilizes CRISPR/Cas9 technology to edit rice RAY1 gene at fixed point, and eliminates rice RAY1 gene through frame shift mutation, so that protein RAY1 is inactivated, and a new generation of new rice germplasm with obviously improved disease resistance is obtained.)

1. A method for cultivating rice blast resistance rice comprises the following steps: inhibiting the activity of RAY1 protein in the original rice to obtain the target rice;

the target rice shows an enhanced resistance to rice blast as compared with the starting rice;

the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

2. The method of claim 1, wherein: the inhibition of the activity of RAY1 protein in the starting rice is achieved by loss of function of the gene encoding the RAY1 protein.

3. The method of claim 2, wherein: the encoding gene of the RAY1 protein is 1) or 2) as follows:

1) a DNA molecule shown as SEQ ID No.2 in the sequence table;

2) a DNA molecule shown as SEQ ID No.3 in the sequence table.

4. A method according to claim 2 or 3, characterized in that: the method for losing the function of the encoding gene of the RAY1 protein is a CRISPR/Cas9 method.

5. The method of claim 4, wherein: the CRISPR/Cas9 method comprises the steps of introducing sgRNA expression vectors into the starting rice; the sgRNA target sequence is TCGTCGAGAGCTACGAGAT.

6. The application of the substance for inhibiting the activity of RAY1 protein in improving the rice blast resistance of rice:

the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

7. Use according to claim 6, characterized in that: the substance inhibiting RAY1 protein is any one of the following (1) to (3):

(1) a specific sgRNA, wherein the target sequence of the specific sgRNA is TCGTCGAGAGCTACGAGAT;

(2) a DNA molecule encoding the specific sgRNA of (1);

(3) and (2) a vector for expressing the specific sgRNA in (1).

An sgRNA having the target sequence in the rice genome of: TCGTCGAGAGCTACGAGAT are provided.

9. A recombinant plasmid containing a gene encoding Cas9 protein, a gene encoding a specific sgRNA, and a U3 promoter; the target sequence of the sgRNA is as follows: TCGTCGAGAGCTACGAGAT are provided.

Technical Field

The invention relates to the field of biotechnology breeding, in particular to a method for improving rice blast resistance of rice and a related biological material thereof.

Background

The rice blast is a major disease of rice caused by the infection of Magnaporthe oryzae (asexual generation: Pyricularia oryzae), and is one of the most important diseases in rice production in China and even the world. The rice blast commonly occurs in the world, the loss of the rice yield can reach 11% -30%, in the fields with serious diseases, the yield loss is as high as 80%, even the grains are not harvested, and the grain yield and quality are seriously affected. Plant diseases cause crop production to suffer from destructive yield loss, threatening global food safety. Repeated over-application of pesticides to control plant diseases has contaminated many environments worldwide. At present, genetic improvement of crop resistance is the most economical and environmentally friendly way to prevent disease outbreaks. Although many resistance (R) genes conferring race-specific resistance have been deployed in plant breeding, their resistance generally remains effective for only a few years, possibly due to strong selection pressure on the evolution of virulent races. In contrast, broad spectrum resistance controlled by multiple genes or Quantitative Trait Loci (QTLs) is generally persistent and more effective for disease management. Therefore, the cultivation of rice varieties with durable resistance and high efficiency becomes an urgent requirement on the current rice production.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the resistance of rice to rice blast.

In order to solve the technical problems, the invention firstly provides a method for cultivating rice blast resistant rice.

The method for cultivating rice blast resistance rice provided by the invention comprises the following steps: inhibiting the activity of RAY1 protein in the original rice to obtain the target rice; the target rice shows an enhanced resistance to rice blast as compared with the starting rice; the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

In the above method, the inhibition of the activity of the RAY1 protein in the original rice may be inhibition of the whole or part of the activity of the RAY1 protein in the original rice.

In the above method, the inhibition of the activity of RAY1 protein in the starting rice may be achieved by the loss of function of the gene encoding the RAY1 protein.

The encoding gene of the RAY1 protein can be 1) or 2) as follows: 1) a DNA molecule shown as SEQ ID No.2 in the sequence table; 2) a DNA molecule shown as SEQ ID No.3 in the sequence table.

In the above method, the loss of function of the gene encoding the RAY1 protein can be achieved by any method known in the art, such as deletion mutation, insertion mutation or base change mutation of the gene, and further the loss of function of the gene.

In the above method, the loss of function of the gene encoding the RAY1 protein may be the loss of function of all or part of the gene encoding the RAY1 protein.

In the above-mentioned methods, the gene encoding the RAY1 protein is disabled by chemical mutagenesis, physical mutagenesis, RNAi, gene site-directed editing, homologous recombination, or the like.

In any case, the entire gene encoding the RAY1 protein may be targeted, or each element regulating the expression of the gene encoding the RAY1 protein may be targeted, as long as the loss of gene function can be achieved. For example, exon 1, exon 2, exon 3 and/or exon 4 of the gene encoding RAY1 may be targeted.

In the above-mentioned genome site-directed editing, Zinc Finger Nuclease (ZFN) technology, Transcription activator effector-like nuclease (TALEN) technology, clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats/CRISPR associated, CRISPR/Cas9system) technology, and other technologies capable of realizing genome site-directed editing can be adopted.

In the specific embodiment of the invention, the CRISPR/Cas9 technology is adopted, wherein the target sequence involved is TCGTCGAGAGCTACGAGAT, and the coding gene of the sgRNA (guide RNA) used is shown as SEQ ID No.4 in the sequence table.

More specifically, the invention uses a recombinant vector pYLCRISPR/Cas9-MT-RAY1 capable of expressing a guide RNA and Cas 9. The recombinant vector pYLCRISPR/Cas9-MT-RAY1 is a recombinant vector obtained by replacing a fragment between two Bsa I enzyme cutting sites on the vector pYLCRISPR/Cas9-MTmono with a DNA fragment containing a specific sgRNA coding gene and a U3 promoter and keeping other nucleotides of pYLCRISPR/Cas9-MTmono unchanged; in particular to a DNA molecule shown in SEQ ID No.5 in a sequence table replacing a fragment between two Bsa I enzyme cutting sites of a vector pYLCRISPR/Cas 9-MTmono. The above method is applicable to any rice, such as: japonica rice (Oryza sativa subsp. japonica) or indica rice (Oryza sativa subsp. indica) as long as it contains the above target sequence. An example of the present invention is rice Nipponbare (Oryza Sativa L.spp.japonica).

In order to solve the technical problems, the invention also protects the application of the substance inhibiting the activity of the RAY1 protein in improving the rice blast resistance of rice; the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

In the above application, the inhibition of the activity of the RAY1 protein may be the inhibition of the whole activity or part activity of the RAY1 protein.

In the above application, the substance inhibiting the RAY1 protein may be any one of the following (1) to (3): (1) a specific sgRNA, wherein the target sequence of the specific sgRNA is TCGTCGAGAGCTACGAGAT; (2) a DNA molecule encoding the specific sgRNA of (1); (3) and (2) a vector for expressing the specific sgRNA in (1).

In the application, the coding gene of the specific sgRNA is shown as SEQ ID No.4 in a sequence table.

In the application, the vector for expressing the specific sgRNA is a recombinant vector pYLCRISPR/Cas9-MT-RAY 1. The recombinant vector pYLCRISPR/Cas9-MT-RAY1 is a recombinant vector obtained by replacing a fragment between two Bsa I enzyme cutting sites of the vector pYLCRISPR/Cas9-MTmono with a DNA fragment containing a specific sgRNA coding gene and a U3 promoter and keeping other nucleotides of pYLCRISPR/Cas9-MTmono unchanged; in particular to a DNA molecule shown in SEQ ID No.5 in a sequence table replacing a fragment between two Bsa I enzyme cutting sites on a vector pYLCRISPR/Cas 9-Mtmono.

In the above application, the rice is a japonica rice variety (Oryza sativa subsp. japonica) or an indica rice variety (Oryza sativa subsp. indica). The japonica rice variety may be Nipponbare (Oryza sativa L.spp.japonica).

In order to solve the above technical problems, the present invention also provides a specific sgRNA having a target sequence in a rice genome of: TCGTCGAGAGCTACGAGAT are provided.

In order to solve the technical problems, the invention also provides a recombinant plasmid, which contains a Cas9 protein coding gene, a sgRNA coding gene and a U3 promoter; the target sequence of the sgRNA is as follows: TCGTCGAGAGCTACGAGAT are provided.

The invention utilizes CRISPR/Cas9 technology to edit rice RAY1 gene at fixed point, and eliminates rice RAY1 gene through frame shift mutation, so that protein RAY1 is inactivated, and a new generation of new rice germplasm with obviously improved disease resistance is obtained. The obtained RAY1 site-directed editing lines showed an increased resistance to rice blast compared to wild-type controls. Therefore, the invention has important significance for improving the disease resistance of the rice and provides a new material for developing new disease-resistant varieties.

Drawings

FIG. 1 is a gel electrophoresis diagram of the full-length sequence of RAY1cDNA amplified by PCR.

FIG. 2 is a map of the intermediate vector pYLgRNA-U3.

FIG. 3 is a diagram showing the alignment of the sequencing sequence of pYLgRNA-U3-RAY1 and the sequence of the intermediate vector pYLgRNA-U3.

FIG. 4 is an amplification electrophoresis detection map of the expression cassette of the intermediate vector pYLgRNA-U3-RAY 1.

FIG. 5 is a map of the genome editing vector pYLCRISPR/Cas9-MTmono vector.

FIG. 6 is an electrophoresis diagram showing the result of PCR detection of a single colony of E.coli transformed with the recombinant vector pYLCRISPR/Cas9-MT-RAY 1.

FIG. 7 shows the mutation pattern of RAY1 and the amino acid pattern encoded by the mutation.

FIG. 8 shows the results of the rice blast inoculation identification of the line L-46 rice plant and Nip at the seedling stage; wherein ZA18, ZB10, ZB13, ZB20, ZC2, ZC10 and ZG1 are physiological races of rice blast.

FIG. 9 shows the relative expression amounts of the rice blast resistance-related genes OsPR1a, OsPR10 and PBZ1 in the rice plants of lines L-46, L-47 and L-48 and NiP.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The expression vector pYLgRNA-U3 is used for editing a RICE ear development Osal gene at a fixed point in a document of Shijiang Wei, Li yi star, Song Shufeng, Qiu peony, Deng Yao, Li. CRISPR/Cas 9. HYBRID RICE (HYBRID RICE), 2017 and 32 (3): 74-78, the biological material is only used for repeating the experiments related to the present invention and is not used for other purposes.

Expression vector pYLCRISPR/Cas9-MTmono in the literature "Shijiang Wei, Li-yi star, Song front, Qiu peony, Du Yao, Li. CRISPR/Cas9 fixed-point editing RICE tasal gene HYBRID RICE (HYBRID RICE), 2017, 32 (3): 74-78, publicly available from the research center for hybrid rice in Hunan, the biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.

Nipponbare (NIP) rice variety "MP, A Robust CRISPR/Cas9System for convention, High-Efficiency Multiplex Genome Editing in Mono cot and dicotplants. mol plant.2015Aug 3; 1274-84.doi 10.1016/j. molp.2015.04.007.Epub2015Apr 24. the biomaterial, which is publicly available from the research center for hybrid rice in Hunan, is used only for repeating the relevant experiments of the present invention and is not used for other purposes.

Physiological races of rice blast fungus (Magnaporthe oryzae) ZA18, ZB10, ZB13, ZB20, ZC2, ZC10 and ZG1 are described in the literature: "Characterisation of molecular identity and probability of probability blast in Hunan pr overlap of China. plant Disease,2017,101 (4): 557 561 ", publicly available from the research center for hybrid rice in Hunan, the biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.