CN114805507B - Rice OsREIN1T219I protein and its encoding genes and applications - Google Patents

Abstract

The invention discloses rice OsREIN1 ^T219I Protein and its coding gene and application. OsREIN1 to be protected by the present invention ^T219I The protein is the protein shown in the sequence 1 in the sequence table or the protein which is derived from the sequence 1 or has more than 80 percent of identity with the protein shown in the sequence 1 and has the same function. The experiment of the invention proves that the invention contains OsREIN1 ^T219I Compared with rice material Nip containing wild OsREIN1 protein coding gene, rice material MC41 with protein homozygous coding gene has higher resistance to Pyricularia oryzae, osREIN1 ^T219I Can be applied to molecular design breeding for cultivating high disease-resistant rice.

Description

Rice OsREIN1T219I protein and its encoding genes and applications

Technical field

The invention relates to the field of biotechnology, specifically to rice OsREIN1 ^T219I protein and its encoding gene and application.

Background technique

Rice is one of the most important food crops. There are more than 300 kinds of invasive diseases of rice, and there are 29 kinds of rice diseases that occur all year round, such as rice blast, bacterial blight, rice sheath blight and the diseases that have gradually spread in recent years. Rice crank, rice smut, etc. The occurrence and prevalence of these diseases are the main factors affecting high and stable rice yields. Therefore, the discovery of rice disease resistance-related genes has received great attention.

Alleles refer to a pair of genes occupying the same locus on a pair of homologous chromosomes, which control a pair of relative traits. Different alleles cause changes in some genetic characteristics. Allelic variation makes it possible to use excellent alleles to breed superior varieties. Many effective molecular biology techniques have been developed to discover excellent alleles, such as SSR markers, association analysis of natural populations, etc. Many excellent alleles and their carriers related to traits such as stress tolerance, disease resistance, high quality and high yield have been identified using the above technology, and are widely used in cross breeding, the most commonly used and effective method for cultivating varieties.

Programmed cell death (PCD) refers to the active and orderly death of cells controlled by genes, and is a life phenomenon. PCD is commonly found in the differentiation, growth and development of plant tissues and organs. Its typical biochemical characteristics include cell shrinkage, nuclear condensation, chromatin marginalization, DNA being sheared and wrapped into apoptotic bodies by membranes, etc. In the interaction between plants and pathogenic bacteria, necrotic spots of various shapes often appear on the host site infected by pathogenic bacteria, which can be single cell death, or there can also be a larger necrotic area, in groups or strips, etc. This necrosis phenomenon was named hypersensitive response (HR) by Stakman in 1915, and it is a common reaction in plant disease resistance. Currently, the HR response is defined as a rapid death of plant cells that limits the growth of pathogenic bacteria.

Contents of the invention

The technical problem to be solved by the present invention is how to improve the disease resistance of rice or how to cultivate rice with high disease resistance.

In order to solve the above technical problems, the present invention first provides a protein. The protein is OsREIN1 ^T219I . The OsREIN1 ^T219I can be the protein of the following A1), A2) or A3):

A1) The amino acid sequence is the protein of sequence 1 in the sequence listing;

A2) The amino acid sequence shown in Sequence 1 in the sequence listing is obtained by substituting and/or deleting and/or adding one or several amino acid residues and having the same function derived from A1) or represented by A1) Proteins with more than 80% identity and the same function;

A3) A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2).

The protein described in A2) above is the amino acid sequence shown in Sequence 1 in the sequence listing. Except that the 219th amino acid is isoleucine, the other amino acid sequences can be substituted by one or several amino acid residues. and/or deleted and/or added proteins derived from A1) and having the same function or having more than 80% identity with the protein represented by A1) and having the same function.

Among the above-mentioned proteins, sequence 1 in the sequence listing consists of 981 amino acid residues.

The above-mentioned protein can be synthesized artificially, or its encoding gene can be first synthesized and then biologically expressed.

Among the above-mentioned proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio respectively to 11, 1 and 0.85 (default value) and search the identity of a pair of amino acid sequences to calculate, and then the identity value (%) can be obtained.

In the above-mentioned protein, the identity of more than 80% can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In order to solve the above technical problems, the present invention also provides biological materials related to the above-mentioned proteins. The biological material may be any one of the following B1) to B9):

B1) Nucleic acid molecules encoding the above-mentioned proteins;

B2) An expression cassette containing the nucleic acid molecule described in B1);

B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

B7) Transgenic plant organs containing the nucleic acid molecule described in B1), or transgenic plant organs containing the expression cassette described in B2);

B8) Nucleic acid molecules that increase or promote the expression of the protein described in claim 1;

B9) Expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

In the biological material described above, the nucleic acid molecule described in B1) may be the coding gene for the protein shown in b1), b2) or b3) below:

b1) A cDNA molecule or DNA molecule whose coding sequence is the nucleotide of sequence 2 in the sequence listing;

b2) The nucleotide is the DNA molecule of sequence 2 in the sequence listing;

b3) A cDNA molecule or DNA molecule that hybridizes to the cDNA or DNA molecule defined in b2) and encodes a protein with the same function.

Among the above-mentioned biological materials, the expression cassette containing nucleic acid molecules described in B2) refers to DNA that can express the protein described in the above-mentioned application in a host cell. This DNA can not only include a promoter for initiating the transcription of protein-encoding genes, but also can Includes terminators that terminate the transcription of protein-coding genes. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.

Existing plant expression vectors can be used to construct a recombinant expression vector containing the protein-encoding gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3' untranslated region of the foreign gene, that is, containing the poly(A) signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide poly(A) to be added to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed at the 3' end of storage protein genes all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be the same as the coding The reading frames of the sequences are identical to ensure correct translation of the entire sequence.

Among the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

Any of the following applications of the above-mentioned proteins and/or the above-mentioned biological materials also fall within the protection scope of the present invention:

Q1. Application of the protein and/or the biological material in regulating plant disease resistance,

Q2. Application of the protein and/or the biological material in preparing products that improve plant disease resistance,

Q3. Application of the protein and/or the biological material in cultivating disease-resistant plants,

Q4. Application of the protein and/or the biological material in preparing plant disease resistance products,

Q5. Application of the protein and/or the biological material in plant breeding.

In order to solve the above technical problems, the present invention also provides a method for cultivating disease-resistant plants. The method includes improving the activity of the above-mentioned protein or/and the expression level of the gene encoding the above-mentioned protein in the target plant to obtain a disease-resistant plant. The disease resistance of the disease-resistant plant is higher than the disease resistance of the target plant.

The improvement of the activity of the above-mentioned protein or/and the expression level of the above-mentioned protein coding gene in the target plant is achieved by introducing the above-mentioned protein coding gene into the target plant.

In the above method, the gene encoding the protein can be modified as follows first, and then introduced into the target plant to achieve better expression effects:

1) Connect to promoters expressed in various plants to facilitate their expression in plants; the promoters may include constitutive, inducible, temporal regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters ;The choice of promoter will vary with the temporal and spatial requirements for expression, and will also depend on the target species; e.g. tissue or organ specific expression promoters, depending on at what stage of development the receptor is required; although proven sources Many promoters for dicots will work in monocots and vice versa, but ideally, a dicot promoter is selected for expression in dicots and a monocot promoter for Expression in monocots;

2) Connecting with a suitable transcription terminator can also improve the expression efficiency of the gene of the invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to work in plants can be combined with The genes of the present invention are connected;

3) Introducing enhancer sequences, such as intron sequences (eg derived from Adhl and bronzel) and viral leader sequences (eg derived from TMV, MCMV and AMV).

In the above method, the plants sensitive to stress may be transgenic plants or plants obtained through conventional breeding techniques such as hybridization.

In the above method, the transgenic plants are understood to include not only the first to second generation transgenic plants, but also their progeny. In the case of transgenic plants, the gene can be propagated in the species or transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, intact plants and cells.

The plants and/or target plants mentioned above can be any of the following:

C1) Dicotyledonous plants;

D1) Monocotyledonous plants;

D3) Gramineae plants;

D4) Gramineae;

D5) Oryza plants;

D6) Rice.

The disease resistance mentioned above may be resistance to rice blast.

Any of the following applications of substances for detecting polymorphisms or genotypes of SNP1 in the rice genome also fall within the protection scope of the present invention:

F1. Application in identifying or assisting in identifying rice disease resistance;

F2. Application in preparing or assisting in the identification of rice disease resistance products;

F3. Application in detecting or assisting in detecting rice disease resistance;

F4. Application in preparing products for testing or assisting in testing rice disease resistance;

F6. Application in rice disease resistance breeding;

The SNP1 is a SNP site in the rice genome, corresponding to the 656th nucleotide of Sequence 2 in the sequence listing, which can be T or C. There are three genotypes at the SNP 1 locus, namely CC, TT or CT. Genotype TT is the homozygous type with T at the SNP1 locus, genotype CC is the homozygous type with the C locus, and genotype CT is the heterozygous type with SNP1 loci at C and T.

The disease resistance of rice whose genotype is genotype TT at the SNP molecular marker site is higher or can be higher than that of rice whose genotype is genotype CC at the SNP molecular marker site.

The rice disease resistance may be rice blast resistance.

This application screened and obtained ethylene mutants from more than 1,500 rice resources. Among them, Tang10 from Vietnam, numbered MC41, has obvious resistance to rice blast. The target gene was obtained using map-based cloning, which is REIN1, a member of the NBS-LRR family. The mutant gene OsREIN1 ^T219I of this gene can stimulate a hypersensitive response and increase the disease resistance of rice. Experiments of the present invention prove that OsREIN1 ^T219I protein has the function of improving rice disease resistance. Tobacco introduced with the OsREIN1 ^T219I gene can cause hypersensitivity reactions, but under the same conditions, tobacco introduced with the OsREIN1 gene and GFP gene cannot cause hypersensitivity reactions. OsREIN1 ^T219I protein and its encoding gene are of great significance for breeding disease-resistant rice varieties.

Description of drawings

Figure 1 shows that the ethylene-insensitive phenotype of MC41 is caused by a recessive mutation. Air means no ethylene treatment, and Ethylene means ethylene treatment.

Figure 2 shows the map-based cloning of rice REIN1 ^T219I .

Figure 3 shows that OsREIN1 ^T219I can stimulate hypersensitivity reactions.

Figure 4 shows that homozygous OsREIN1 ^T219I increases rice resistance to rice blast.

Figure 5 is a schematic diagram of the pCAMBIA2300-35S-OCS vector obtained after modification of the pCAMBIA2300 vector.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

The rice variety Nipponbare (O.Sativa L.spp.japonica, var nipponbare, AA genome, Nip) belongs to the japonica subspecies and is recorded in the following documents: Fu Xiulin et al., Rice variety Nipponbare, Agricultural Science and Technology Communications, 1973, 2, 11-15; Provided by Zhu Lihuang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Rice varieties Tang 10 (Oryza sativa L. Indica, Tang 10), numbered MC41 (from Vietnam) and Minghui 63 (MH63) (Oryza sativa L.MH63) were provided by researcher Chu Chengcai from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

Plant binary expression vector pCAMBIA2300: purchased from Enzyme Biotechnology, product number MY1383. Julie Leclercq etal.,Development of a new pCAMBIA binary vector using technology,Plasmid.2015Sep;81:50-4.

Homologous recombination kit: American Clone Smarter brand Seamless Assembly Cloning Kit, product number is C5891-50, domestically distributed by Sino-American Taihe Biotechnology Co., Ltd.

Magnaporthe oryzae Guy11: provided by Liu Jun, Institute of Microbiology, Chinese Academy of Sciences. S Fukiya et al., Establishment of a new cross of the rice blast fungus derived from Japanese differential strain Ina168 and hermaphroditic rice pathogen Guy11, BiosciBiotechnol Biochem. 2001Jul;65(7):1464-73.

Example 1. Screening and genetic analysis of rice materials with defective ethylene response

In the process of screening 1,500 rice resources for ethylene-reactive deficient materials, it was found that Tang10, a material numbered MC41, had yellow seedlings when treated with ethylene compared with the control Nipponbare (Oryza sativa L.cv. Nipponbare, Nip). The ethylene response of the F1 generation was similar to that of Nip, that is, its root length was significantly shorter under ethylene treatment (Figure 1). Therefore, it is speculated that the abnormal ethylene response trait of MC41 may be determined by a recessive single gene.

Further analysis will be conducted on the apparent and recessive nature of the abnormal variation in ethylene response of MC41. When MC41 was used as the female parent and MH63 was used as the male parent, MH63 had the same normal ethylene response as Nip. The F1 generation etiolated seedlings had the same phenotype as MH63, indicating that the abnormal ethylene response of MC41 was a recessive mutation. Phenotypic analysis of the F2 generation etiolated seedlings found a 3:1 (MH63 phenotype: MC41 phenotype) separation, which was consistent with the χ ² test (Table 1). The above results indicate that the variation of MC41 is a recessive mutation controlled by a single gene.

Table 1 Explicit and recessive analysis of MC41 abnormal ethylene response phenotypes

Note: “+” means MC41 ethylene-insensitive phenotype; “-” means MH63 normal ethylene-sensitive phenotype; critical value (0.05, 1)=3.84.

Example 2. Identification of MC41 variant alleles

The MC41 mutation site was determined in a 73k interval on the long arm of chromosome 7 through map-based cloning, and a specific SNP (named SNP1) located in the rice gene LOC_Os07g40810 was identified on one of the genes, which can lead to amino acid variation. Non-synonymous substitutions list the gene as a candidate gene. The protein encoded by this gene is REIN1, a member of the NBS-LRR family, and the gene is named OsREIN1 gene. Design primers according to the rice genome reference gene sequence:

REIN1F: 5’-ATGGAACATGCTGTTGTTAGTGC-3’

REIN1R: 5’-TCAGTCTTCGTCGAAAGATGAAGATAGT-3’

Using MC41 total mRNA as a template, a DNA band of approximately 3Kb was obtained by amplification with the above primers. After sequencing, it was confirmed that it is the REIN1 gene belonging to the NBS-LRR family, but it is different from the sequence of LOC_Os07g40810 in the rice genome reference sequence. OsREIN1 in MC41 has a single base change, which is located in the NB-ARC domain P-loop ( The base at position 656 in ATP/ADP binding was mutated from C to T (C656T), causing a single amino acid change at position 219 in the encoded protein OsREIN1, from threonine to isoleucine (T219I) (Figure 2) . Therefore, the mutated protein in rice MC41 was named OsREIN1 ^T219I , and the amino acid sequence of the OsREIN1 ^T219I protein is as shown in Sequence 1 in the sequence listing. The CDS sequence of the coding chain of the gene encoding OsREIN1 ^T219I protein (OsREIN1 ^T219I ) is the DNA molecule shown in Sequence 2 in the sequence listing. In the OsREIN1 ^T219I gene of MC41, the nucleotide of the SNP1-specific mutation site (position 656 of sequence 2 in the sequence listing) is T, which corresponds to the amino acid at this site of the OsREIN1 ^T219I protein (position 219 of sequence 1 in the sequence listing). ) is isoleucine (I).

In rice MH63 and Nip, the nucleotide of the SNP1 site is C (corresponding to position 656 of sequence 2 in the sequence listing), and the corresponding amino acid is threonine (T) (corresponding to position 656 of sequence 1 in the sequence listing). No. 219). Therefore, in the rice genome, the nucleotide of the SNP1 site is C or T, and the corresponding amino acid is threonine (T) or isoleucine (I); there are three genotypes of this site, namely CC, TT or CT, the genotype TT is the homozygous type with T at the SNP1 locus, the genotype CC is the homozygous type with the C locus, and the genotype CT is the heterozygous type with the SNP1 locus at C and T.

Next, the alleles of OsREIN1 were detected in 5518 rice materials, and it was found that only the OsREIN1 gene in MC41 had a point mutation at the SNP1 site, so this mutation was a rare mutation.

Example 3. OsREIN1 ^T219I is involved in the regulation of disease resistance

HR (hypersensitivity response) cell death blocks pathogenic bacteria from acquiring nutrients and limits the growth and reproduction of pathogenic bacteria. It can also stimulate specific defense responses in adjacent tissues and systemic acquired resistance (SAR) of plants. Hypersensitivity therefore plays an important role in plant disease resistance.

Testing whether OsREIN1 ^T219I can initiate plant hypersensitivity responses in the tobacco system. The specific experimental steps are as follows:

1. Construction of hypersensitive reaction vector:

1) Use the following primers and use the cDNA of Nip and MC41 as templates to amplify the target fragment through PCR:

F:5’-TGGAGAGGACAGGGTACCCGGATGGAACATGCTGTTGTTAGTGC-3’

(The underlined sequence is the homologous recombination arm sequence, and the bolded sequence is the 3xMYC tag sequence)

2) Transform the plant expression vector pCAMBIA2300: insert the 35S promoter sequence (sequence 3 in the sequence listing) between the NcoI and KpnI restriction sites, and insert the OCS terminator sequence (sequence in the sequence listing) between PstI and HindIII. 4) Obtain the modified vector pCAMBIA2300-35S-OCS (Figure 5); then use BamHI to digest the pCAMBIA2300-35S-OCS vector and recover the linearized vector.

3) Mix and ligate the PCR recovery product with Nip as the template and the PCR recovery product with MC41 as the template in step 1) with the linearized vector obtained in step 2) using a homologous recombination kit to construct respectively Recombinant vectors pCAMBIA2300-REIN1 and pCAMBIA2300-REIN1 ^T219I . Among them, pCAMBIA2300-REIN1 is a recombinant vector that connects the OsREIN1 gene derived from Nip to a linearized vector to express the OsREIN1 protein (the OsREIN1 protein replaces I with T at position 219 of sequence 1 in the sequence list and maintains the other amino acids of sequence 1 Protein obtained with unchanged residues); pCAMBIA2300-REIN1 ^T219I is a recombinant vector that connects the OsREIN1 ^T219I gene derived from MC41 to a linearized vector, and expresses the OsREIN1 ^T219I protein whose amino acid sequence is sequence 1 in the sequence list.

4) Transform the two recombinant vectors pCAMBIA2300-REIN1 and pCAMBIA2300-REIN1 ^T219I constructed in step 3) individually into E.coli.DH5α competent cells, and spread them into solid LB medium containing 50 μl/ml kanamycin. Medium, incubate overnight.

5) Pick the single clones grown on the solid medium for colony PCR identification. Extract the plasmids from the positive recombinant colonies and sequence them to verify that the pCAMBIA2300-REIN1 recombinant plasmid and pCAMBIA2300-REIN1 ^219I recombinant plasmid are obtained before proceeding to the next experiment.

The recombinant vector pCAMBIA2300-GFP of the GFP gene was constructed according to the above vector construction method and used as a control for subsequent experiments. The only difference between pCAMBIA2300-GFP and pCAMBIA2300-REIN1 ^T219I is that the OsREIN1 ^T219I gene in pCAMBIA2300-REIN1 ^T219I is replaced by the GFP gene, and other nucleotides are the same.

2.HR response detection:

1) Nicotiana benthamiana was cultured for about 4 weeks under the conditions of 14 hours of light/10 hours of darkness, a temperature of 25°C, and a relative humidity of 70%.

2) Transfer the pCAMBIA2300-REIN1, pCAMBIA2300-REIN1 ^T219I and GFP control recombinant vector pCAMBIA2300-GFP obtained in the above 1 into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1, EHA105/pCAMBIA2300-REIN1 ^219I and EHA105/pCAMBIA2300-GFP.

3) Select three types of recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1, EHA105/pCAMBIA2300-REIN1 ^219I and EHA105/pCAMBIA2300-GFP for monoclonal culture and amplification. Collect the bacterial cells, and suspend the recombinant Agrobacterium cells in an infection solution (containing 10mM MgCl ₂ , 10mM MES, 150 μM acetosyringone, and the rest is water, pH=5.6) to obtain the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 infection solution, recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution and recombinant Agrobacterium EHA105/pCAMBIA2300-GFP infection solution. The OD _600nm of each recombinant Agrobacterium infection solution is 1.0 (based on infection liquid is a blank control). Rest at room temperature for 2 to 3 hours (at least 0.5 h, no more than 3 hours.)

4) Mix various recombinant Agrobacterium infection solutions individually or in different proportions, inject them on the back of the tobacco with a 1mL syringe, and mark the water-soaked area of the tobacco leaves with a marker. The experiment was repeated three times, with the following 9 treatments set for each repetition, and 10 tobacco leaves were injected into each treatment.

Treatment of tobacco infected by recombinant Agrobacterium REIN1-3Xmyc alone: Use a 1mL syringe to inject the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 infection solution in 3) on the back of the tobacco, and mark the water-soaked area of the tobacco leaves with a marker.

Recombinant Agrobacterium REIN1 ^219I -3Xmyc alone infects tobacco treatment: Use a 1mL syringe to inject the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution in 3) on the back of the tobacco, and mark the water-soaked area of the tobacco leaves with a marker pen.

Recombinant Agrobacterium tumefaciens GFP-3Xmyc alone infects tobacco treatment: Use a 1mL syringe to inject the recombinant Agrobacterium tumefaciens EHA105/pCAMBIA2300-GFP infection solution in 3) on the back of the tobacco, and mark the water-soaked area of the tobacco leaves with a marker.

1:1 treatment of recombinant Agrobacterium REIN1 ^219I -3Xmyc and recombinant Agrobacterium REIN1-3Xmyc (REIN1 ^219I -3Xmyc/REIN1-3Xmyc 1:1): Combine the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution from 3) and The recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 infection solution was mixed at a volume ratio of 1:1 to obtain a recombinant Agrobacterium mixed infection solution. Use a 1 mL syringe to inject the recombinant Agrobacterium mixed infection solution on the back of tobacco, and use a marker pen to mark the water-soaked area of the tobacco leaves.

1:0.5 treatment of recombinant Agrobacterium REIN1 ^219I -3Xmyc and recombinant Agrobacterium REIN1-3Xmyc (REIN1 ^219I -3Xmyc/REIN1-3Xmyc 1:0.5): Combine the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution from 3) and The recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 infection solution was mixed at a volume ratio of 1:0.5 to obtain a recombinant Agrobacterium mixed infection solution. Use a 1 mL syringe to inject the recombinant Agrobacterium mixed infection solution on the back of tobacco, and use a marker pen to mark the water-soaked area of the tobacco leaves.

1:0.1 treatment of recombinant Agrobacterium REIN1 ^219I -3Xmyc and recombinant Agrobacterium REIN1-3Xmyc (REIN1 ^219I -3Xmyc/REIN1-3Xmyc 1:0.1): Combine the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution from 3) and The recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 infection solution was mixed at a volume ratio of 1:0.1 to obtain a recombinant Agrobacterium mixed infection solution. Use a 1 mL syringe to inject the recombinant Agrobacterium mixed infection solution on the back of tobacco, and use a marker pen to mark the water-soaked area of the tobacco leaves.

1:1 treatment of recombinant Agrobacterium REIN1 ^219I -3Xmyc and recombinant Agrobacterium GFP-3Xmyc (REIN1 ^219I -3Xmyc/GFP-3Xmyc 1:1): Combine the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution from 3) and The recombinant Agrobacterium EHA105/pCAMBIA2300-GFP infection solution was mixed at a volume ratio of 1:1 to obtain a recombinant Agrobacterium mixed infection solution. Use a 1 mL syringe to inject the recombinant Agrobacterium mixed infection solution on the back of tobacco, and use a marker pen to mark the water-soaked area of the tobacco leaves.

1:0.5 treatment of recombinant Agrobacterium REIN1 ^219I -3Xmyc and recombinant Agrobacterium GFP-3Xmyc (REIN1 ^219I -3Xmyc/GFP-3Xmyc 1:0.5): Combine the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution from 3) and The recombinant Agrobacterium EHA105/pCAMBIA2300-GFP infection solution was mixed at a volume ratio of 1:0.5 to obtain a recombinant Agrobacterium mixed infection solution. Use a 1 mL syringe to inject the recombinant Agrobacterium mixed infection solution on the back of tobacco, and use a marker pen to mark the water-soaked area of the tobacco leaves.

1:0.1 treatment of recombinant Agrobacterium REIN1 ^219I -3Xmyc and recombinant Agrobacterium GFP-3Xmyc (REIN1 ^219I -3Xmyc/GFP-3Xmyc 1:0.1): Combine the recombinant Agrobacterium EHA105/pCAMBIA2300-REIN1 ^219I infection solution from 3) and The recombinant Agrobacterium EHA105/pCAMBIA2300-GFP infection solution was mixed at a volume ratio of 1:0.1 to obtain a recombinant Agrobacterium mixed infection solution. Use a 1 mL syringe to inject the recombinant Agrobacterium mixed infection solution on the back of tobacco, and use a marker pen to mark the water-soaked area of the tobacco leaves.

5) HR response can be observed about 40 hours after injection.

Figure 3 shows the HR detection results. Transient expression experiments in tobacco showed that when the recombinant Agrobacterium containing control GFP-3XMyc and REIN1-3xMyc were injected, there was no obvious change in the leaves, indicating that neither could initiate HR. However, when the Agrobacterium containing REIN1 ^T219I -3XMyc was injected, the leaves changed color. Can stimulate strong HR. Agrobacterium containing REIN1 ^T219I -3XMyc and GFP-3Xmyc can stimulate HR regardless of whether they are mixed at 1:1, 1:0.5 or 1:0.1 (shown in the left picture in Figure 3). The right picture in Figure 3 shows that when Agrobacterium containing REIN1 ^T219I -3XMyc and Agrobacterium containing REIN1-3xMyc are mixed and injected at 1:0.1, HR is not affected, HR decreases at 1:0.5, but does not occur at 1:1. HR, the above results indicate that the REIN1 ^T219I -3Xmyc recombinant Agrobacterium can cause plant HR, but the REIN1-3Xmyc recombinant Agrobacterium cannot cause plant HR. The increase of REIN1-3xMyc inhibits the HR response, and OsREIN1 can inhibit the HR caused by OsREIN1 ^T219I1 . It further illustrates that sREIN1 ^T219I is a recessive mutation, and only when it is homozygous for recessive mutation, it triggers the HR response and confers disease resistance to the plants. OsREIN1 ^T219I can cause plant HR and can be used to regulate plant disease resistance. Homozygous OsREIN1 ^T219I can improve rice disease resistance.

3. Detection of rice blast resistance in rice MC41 and Nip

3.1 Preparation of blast fungus:

Inoculate the tested Magnaporthe oryzae Guy11 onto the yeast starch slant medium (10g of water-soluble starch, 2g of yeast extract, 20g of agar plus water to 1000ml). After the mycelium is fully grown, use 1.5ml of sterile water to gently inoculate it. Scrape the mycelium and move it to the oatmeal culture medium (30g oatmeal, 0.6g CaCl ₂ , 15g agar, add water to 1000ml), spread it evenly and place it at 28°C. After about 10 days, the culture dish will be filled with bacteria. Silk, culture it under light at 25-28°C for about 2 days. When it is fully sporulated, wash the blast fungus spores with sterile distilled water. After filtering with three layers of lens paper, calculate the spore concentration under a 100x microscope and adjust the spore concentration. to 5×10 ⁵ /ml (containing 0.02% Tween-20) to obtain a spore suspension for later use.

3.2 Disease resistance detection of test plants:

Place the four-week-old Nipponbare and MC41 rice seedlings in an inoculation box, use a sprayer to inoculate them (each inoculation box is inoculated with 30 mL of the spore suspension obtained in 3.1), and then incubate them in a dark and humidified environment at 28°C for 24 hours. The inoculation box was moved to a normal light (16h light/8h dark) incubator for growth. After 5 days, photos were taken and the disease of the rice was recorded.

Figure 4 shows that the lesions of Nip were obvious 6 days after inoculation. Both new and old leaves began to wilt, and the leaf tips were obviously withered. The affected area was about 40-70%. However, although there were needle-like spots on the old leaves of MC41, the leaf tips were slightly There is withered yellow, but it is still green. The results showed that the disease resistance of MC41 was significantly higher than that of the control Nip.

The above experiments show that the OsREIN1 mutant gene OsREIN1 ^T219I from MC41 (Tang 10) is related to the regulation of rice disease resistance. Its homozygous rice variety MC41 (Tang 10) has increased disease resistance compared with the control Nip containing the unmutated OsREIN1 gene. . Therefore, OsREIN1 ^T219I can be used in molecular design breeding to breed highly disease-resistant rice.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

sequence list

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

<120> Rice OsREIN1T219I protein and its encoding genes and applications

<130>GNCSQ210207

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 981

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Glu His Ala Val Val Ser Ala Ala Glu Gly Ala Ile His Thr Leu

1 5 10 15

Leu Gly Lys Leu Gly Thr Ile Val Leu Gln Glu Ala Gln Leu Leu Gly

20 25 30

Gly Ile Arg Gly Glu Leu Gln His Leu Lys Asp Glu Leu Glu Ser Met

35 40 45

Thr Ala Phe Leu Gln Asp Leu Ser Gly Arg Asp Glu Cys Gly Lys Gln

50 55 60

Val Lys Ile Trp Lys Lys His Val Arg Glu Ile Ala Tyr Asp Ile Glu

65 70 75 80

Asp Cys Ile Asp Glu Phe Lys His Gln Leu Gly Asp Ser Ser Ser Ala

85 90 95

Gly Gly Ser Gly Pro Val Val Phe Phe Arg Lys Ala Thr His Ile Leu

100 105 110

Gln Thr Thr Arg Val Arg His Gln Ile Ala Lys Gln Ile Gln Glu Leu

115 120 125

Lys Arg Arg Thr Met Asn Ile Ser Ala Arg Asn Ser Arg Tyr Ser Ala

130 135 140

Asn His Leu Ile Ser Gly Thr Ala Gly Asn Ser Met Ala Ala Tyr Asp

145 150 155 160

Ser Gln Ala Asn Leu Leu Asn Val Asp Thr Arg Ile Thr Ala Leu Phe

165 170 175

Pro Glu Arg Arg Gln Leu Val Gly Ile Glu Pro Arg Gln Gly Asn Leu

180 185 190

Val His Trp Leu Leu Glu Ala His Val Gln Gln Leu Arg Val Val Ser

195 200 205

Ile Phe Gly Phe Gly Gly Leu Gly Lys Thr Ile Leu Ala Met Thr Thr

210 215 220

Tyr Gln Ser Leu Ser Gly Arg Asn Gly Pro Phe Gln Cys Gln Ala Phe

225 230 235 240

Val Thr Val Ser Gln Ser Phe Asp Val Lys Val Leu Met Arg Asp Ile

245 250 255

Leu Leu Gln Ile Thr Gln Pro Val Asn Gln Pro Ser Ser Pro Ser Thr

260 265 270

Gly Ala Gly Lys Gly Pro Met Glu Gly Leu Leu Lys Gly Met Glu Ala

275 280 285

Trp Asn Val Val Gln Leu Ala Ser Ile Leu Arg Gln Gln Leu Glu Asn

290 295 300

Lys Arg Tyr Leu Ile Val Leu Asp Asp Ile Trp Ser Met Thr Ala Trp

305 310 315 320

Glu Gly Ile Arg Phe Ser Leu Pro Asp Ser Asn Asn Gly Ser Arg Ile

325 330 335

Val Val Thr Thr Arg Ile Arg Ala Val Ala His Thr Cys Cys Phe His

340 345 350

Glu Tyr Asp Arg Ala Tyr Glu Ile Lys Pro Leu Thr Asp Cys Glu Ser

355 360 365

Arg Asp Leu Phe Phe Lys Arg Ile Phe Gly Ser Ser Ile Cys Pro Glu

370 375 380

His Leu Glu Asp Ile Ser Ala Lys Ile Leu Gly Lys Cys Gly Gly Thr

385 390 395 400

Pro Leu Ser Ile Val Ser Ile Ala Gly Leu Leu Ala Ser Lys Pro Val

405 410 415

His Ser Lys Asp Leu Trp Glu Lys Ile Tyr Ser Ser Leu Gly Ser Glu

420 425 430

Ile Glu Thr Asn Pro Ser Leu Asp Arg Leu Lys Lys Ile Leu Glu Leu

435 440 445

Ser Tyr Asn Asp Leu Pro Tyr His Leu Lys Thr Cys Phe Leu Tyr Leu

450 455 460

Ser Ile Tyr Pro Glu Asp His Asn Ile Arg Arg Lys Thr Ile Leu Arg

465 470 475 480

Arg Trp Val Ala Glu Arg Phe Val Thr Gly Lys Arg Gly Leu Ser Val

485 490 495

Phe Glu Val Ala Glu Ser Tyr Phe Asp Glu Phe Ile Asn Arg Ser Ile

500 505 510

Ile Gln Pro Val Thr Thr Ser Phe Thr Gly Lys Val Lys Thr Phe Arg

515 520 525

Val His Asp Val Met Leu Glu Ile Ile Val Ser Lys Ser Ile Glu Asp

530 535 540

Asn Phe Ile Thr Leu Val Gly Glu Gln Asn Thr Leu Phe Pro Gln Glu

545 550 555 560

Lys Ile Arg Arg Leu Thr Val His Ser Arg Gly Val Lys Tyr Ile Ala

565 570 575

Thr Arg Glu Ile Leu Cys His Val Arg Ser Leu Ser Ile Phe Ala Asp

580 585 590

Gly Glu Thr Leu Gln Phe Gly Trp Met Lys Leu Met Arg Ile Leu Asp

595 600 605

Leu Glu Gly Tyr Glu Phe Leu Arg Asn Arg Asp Leu Lys Asp Leu Cys

610 615 620

Arg Leu Phe Gln Leu Glu Tyr Leu Asn Leu Arg Arg Thr His Ile Thr

625 630 635 640

Glu Leu Pro Ala Gln Ile Gly Asn Leu Lys Lys Leu Glu Thr Leu Asp

645 650 655

Ile Arg Asp Thr Ala Ile Lys His Leu Pro Pro Gly Ile Thr Asn Leu

660 665 670

Pro His Leu Ala Asn Leu Leu Gly Gly Arg Arg Ser Tyr Asn His Thr

675 680 685

Gly Arg Trp Pro Ile Ser Glu Phe Trp Gly Leu His Ile Pro Asn Glu

690 695 700

Leu Arg Lys Met Asp Ser Leu Thr Thr Leu Ala Gln Val Glu Ile Thr

705 710 715 720

Thr Ser Thr Ser His Tyr Ile Ser Glu Leu Ser Lys Leu Ser Arg Leu

725 730 735

Arg Lys Leu Gly Val Leu Met Phe Val Asp Asp Asp Ser Thr Trp Ala

740 745 750

Ser Leu Ile Ser Ala Leu Glu Lys Leu Ser Gly Ser Leu Arg Ser Leu

755 760 765

Leu Leu Trp Arg Pro Asp Gly Ala Met Asn Phe Asn Ile Val Asn Ser

770 775 780

Leu Ser Ser Pro Pro Ile Phe Thr Lys Ser Met Asn Leu Arg Gly Gln

785 790 795 800

Leu Thr Gln Leu Pro Cys Trp Phe Pro Leu Leu Ser Asn Ile Thr Glu

805 810 815

Leu Thr Leu Arg Ala Thr Glu Leu Ser Ala Glu Glu Asp Leu Lys Val

820 825 830

Leu Gly Ser Leu Pro Ser Leu Leu Tyr Leu Arg Leu His His Asn Ala

835 840 845

Tyr Ile Gly Thr Glu Phe Ser Ala Ser Ala Gly Glu Phe Pro Ser Leu

850 855 860

Arg Leu Leu Val Ile His Leu Asp Met Ser Glu Asp Trp Glu Ala Arg

865 870 875 880

Phe Glu Glu Gly Ala Leu Pro Lys Leu Ala Arg Leu Glu Leu Ser Leu

885 890 895

Phe Glu Glu Ala Ser Ile Gln Glu Ile Thr Gly Ile Glu Phe Leu Pro

900 905 910

Ser Leu Lys Glu Val Ser Ile Arg Ala Cys His Ser Asn Ile Val Asn

915 920 925

Val Glu Glu Ile Ala Thr Ser Leu Arg Ala Asp Ala Glu Lys Asn Ile

930 935 940

Asn Lys Pro Ile Val Thr Phe Glu Glu Lys Gln Trp Val Pro Met Arg

945 950 955 960

Ser Arg Thr Asp Pro Pro Leu Asp His Met Gly Asn Leu Leu Ser Ser

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Ser Phe Asp Glu Asp

980

<210> 2

<211> 2946

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggaacatg ctgttgttag tgccgcggaa ggtgcgatcc acactctctt gggaaagctc 60

ggcacaatcg tcctccaaga agcccagctt ctgggaggta ttcggggtga actgcaacac 120

ttgaaggatg aactggagag catgacagcc tttcttcagg acctttctgg gagagatgaa 180

tgcggcaagc aagttaagat ttggaagaag catgtgcgtg agattgcata tgatatcgag 240

gattgtattg atgagttcaa acatcaactt ggtgacagca gtagtgccgg tggcagcggc 300

cctgtagtgt ttttccgcaa ggccacccac atattgcaga ccaccagagt gaggcatcag 360

attgccaaac aaatccaaga actaaaaagg cgtactatga atatcagtgc ccgaaattca 420

aggtatagtg ccaatcatct catctctgga actgctggga atagcatggc tgcatatgat 480

agccaagcta atcttttaaa tgttgatact cgcattactg cactctttcc agagagaagg 540

cagcttgttg gcattgaacc acgtcaggga aatcttgtgc actggttatt ggaggcacat 600

gtgcaacaat tacgggtggt gtctatattt ggttttggtg gtttgggcaa gacaatactt 660

gctatgacaa catatcaaag tctatctgga agaaatggac cttttcaatg tcaagctttt 720

gtaactgtgt cccagagttt tgatgtcaag gttctgatga gagatattct tctccaaatc 780

actcaaccag ttaatcaacc gagttctcca tcaactggag ctggcaaagg tcccatggaa 840

ggcctactca agggcatgga agcatggaat gtggtacaac ttgcgagcat cctcaggcag 900

caattggaaa ataagagata tctgattgtt cttgatgata tctggagcat gactgcatgg 960

gaaggtattc ggttttcttt gccggactca aataatggta gcagaatagt ggttactaca 1020

cgaatcagag ctgtagcaca cacctgttgt ttccatgagt acgaccgagc ttacgaaatc 1080

aaacctctca ctgattgtga atccagagac ttatcttca aaagaatatt tggcagctca 1140

atttgtcctg agcacttaga agatatttca gctaagattc tgggaaaatg tggtggcaca 1200

cctttatcca tagtcagtat agcaggcctc ttggctagca agcctgtgca cagtaaagat 1260

ctttgggaga agatttatag ctctcttggt tcagagattg aaaccaatcc atcacttgat 1320

agattgaaga aaatacttga gcttagctat aatgatcttc cttaccactt gaagacttgc 1380

ttctttatatt taagcattta ccctgaggac cataatatca gaaggaaaac tatactcagg 1440

cgatgggtag cagaacgctt tgtgactggc aaacgtggac tgagtgtttt tgaggtggca 1500

gaaagctact ttgatgaatt cattaaccgg agcattattc agccagtgac cactagcttt 1560

acagggaagg ttaaaacatt ccgagttcat gatgtgatgt tggagatcat tgtgtcaaag 1620

tcaattgaag ataattttat cactctagta ggagaacaga acactttgtt tccgcaagag 1680

aagataaggc ggctaactgt tcatagcaga ggtgtgaaat atattgccac aagagaaata 1740

ttatgccatg tccggtcctt gagcatattt gccgatggag aaacattgca gtttggttgg 1800

atgaaattaa tgagaatatt ggacttagaa ggctacgaat tcctgagaaa tagagatctc 1860

aaagacttgt gcaggctgtt tcaattggaa taccttaatc tgagaagaac acatattacg 1920

gaactccctg cacaaatagg aaacctgaag aagttggaga ctctggatat aagggacaca 1980

gccataaagc atttgcctcc tggcataacc aatcttccac atttagcaaa cttacttgga 2040

ggaagaagat cctataacca cactgggcgg tggcctattt ctgagttctg gggtttacat 2100

atccctaacg aacttcgaaa aatggattca cttacaacac ttgcacaggt agaaatcacg 2160

acctctacat cacattatat cagcgagttg tcgaagctat cccggttgag gaaacttgga 2220

gtactgatgt ttgttgatga cgacagcacc tgggcatcct tgatctctgc tcttgagaag 2280

ctcagcggca gccttcgctc gttattactt tggcgacctg atggcgcgat gaacttcaac 2340

attgtcaatt cattatccag tccaccaata tttacgaaaa gtatgaacct tcgaggtcag 2400

ttaacacaac taccatgttg gtttcctttg ctttccaaca tcacagaatt aacccttcgc 2460

gcaaccgaac taagcgccga ggaagacctg aaggtactag gaagtttacc tagcctgcta 2520

tacctcaggt tgcatcacaa tgcatacata ggaacagaat tttctgcttc agcaggggaa 2580

ttcccatctc ttagactgct tgtcattcat ctcgacatgt ctgaagattg ggaggcaaga 2640

tttgaagaag gggcactacc aaagctagca aggctagagc tgtctctgtt cgaagaagcc 2700

tccatccaag agatcactgg catagagttc cttccgagtc tgaaggaagt ctcgattcgt 2760

gcttgtcaca gcaatatcgt gaatgtggaa gagatagcta cctctctgag ggctgatgcc 2820

gagaagaaca tcaacaaacc catcgtcact ttcgaggaga aacagtgggt gccgatgagg 2880

tcaaggacgg atcctccttt agatcacatg ggaaacctac tatcttcatc tttcgacgaa 2940

gactga 2946

<210> 3

<211> 531

<212> DNA

<213> Artificial Sequence

<400> 3

ccatggagtc aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac 60

agttcataca gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg 120

agcacgacac gcttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg 180

caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag 240

ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc 300

attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg 360

gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 420

aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 480

cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggaca g 531

<210> 4

<211> 233

<212> DNA

<213> Artificial Sequence

<400> 4

gcatgccctg ctttaatgag atatgcgaga cgcctatgat cgcatgatat ttgctttcaa 60

ttctgttgtg cacgttgtaa aaaacctgag catgtgtagc tcagatcctt accgccggtt 120

tcggttcatt ctaatgaata tatcacccgt tactatcgta tttttatgaa taatattctc 180

cgttcaattt actgattgta ccctactact tatatgtaca atattaaaat gaa 233

Claims (10)

1. A protein, characterized in that: the protein is OsREIN1 ^T219I The method comprises the steps of carrying out a first treatment on the surface of the The OsREIN1 ^T219I The amino acid sequence is protein with a sequence 1 in a sequence table.

2. A biological material associated with the protein of claim 1, which is any one of the following B1) to B4):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

3. The biomaterial according to claim 2, characterized in that: b1 The coding sequence of the nucleic acid molecule is a sequence 2 in a sequence table.

4. Use of a protein as claimed in claim 1 and/or a biomaterial as claimed in claim 2 or 3 for any of the following:

use of Q1, the protein of claim 1 and/or the biological material of claim 2 or 3 for modulating disease resistance in plants;

use of Q2, a protein according to claim 1 and/or a biomaterial according to claim 2 or 3 for the preparation of a product for improving disease resistance in plants;

use of Q3, the protein of claim 1 and/or the biological material of claim 2 or 3 for growing disease resistant plants;

use of Q4, the protein of claim 1 and/or the biomaterial of claim 2 or 3 for the preparation of a plant disease resistant product;

use of Q5, a protein according to claim 1 and/or a biological material according to claim 2 or 3 in plant disease-resistant breeding.

5. A method for growing a disease-resistant plant, comprising increasing the activity of the protein of claim 1 or/and the expression level of the gene encoding the protein of claim 1 in a plant of interest to obtain a disease-resistant plant; the disease resistance of the disease resistant plant is higher than the disease resistance of the target plant.

6. The method according to claim 5, wherein: the improvement of the activity of the protein of claim 1 and/or the expression level of the gene encoding the protein of claim 1 in the target plant is achieved by introducing the gene encoding the protein of claim 1 into the target plant.

7. The use according to claim 4 and/or the method according to claim 5 or 6, characterized in that: the plant or/and the target plant is a monocotyledonous plant.

8. The use according to claim 4 and/or the method according to claim 5 or 6, characterized in that: the plant or/and the target plant is rice; the disease resistance is rice blast resistance.

9. Use of any one of the following substances for detecting polymorphism or genotype of SNP1 in rice genome:

f1, application in identification or auxiliary identification of rice disease resistance;

f2, application in preparing identification or auxiliary identification rice disease resistance products;

f3, application in detecting or assisting in detecting rice disease resistance;

f4, application in preparing products for detecting or assisting in detecting rice disease resistance;

f5, application in rice disease-resistant breeding;

the SNP1 is a SNP locus of a rice genome, corresponds to nucleotide 656 of a sequence 2 in a sequence table, is T or C, and is higher than or candidate to be higher than rice with genotype CC, wherein the genotype of the SNP molecular marker locus is genotype TT.

10. The use according to claim 9, characterized in that: the disease resistance of the rice is rice blast resistance.