阅读说明：本技术 抗病耐热相关蛋白TaRHP1及其相关生物材料与培育抗病耐热植物的方法 (Disease-resistant heat-resistant related protein TaRHP1, related biological material thereof and method for cultivating disease-resistant heat-resistant plants ) 是由 张增艳 王开 于 2019-04-15 设计创作，主要内容包括：本发明公开了抗病耐热相关蛋白TaRHP1及其相关生物材料与培育抗病耐热植物的方法。TaRHP1是如下A1)、A2)或A3)的蛋白质：A1)氨基酸序列是序列表中序列2的蛋白质；A2)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有90％以上的同一性且与植物耐热性和抗病性相关的蛋白质；A3)在A1)或A2)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。TaRHP1及其编码基因可用于提高植物对纹枯病和灰霉病的抗性,并可用于提高植物的耐热性。(The invention discloses a disease-resistant and heat-resistant related protein TaRHP1, a related biological material thereof and a method for cultivating disease-resistant and heat-resistant plants. TaRHP1 is a protein of A1), A2) or A3) as follows: A1) the amino acid sequence is protein of a sequence 2 in a sequence table; A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90% of identity with the protein shown in A1), and is related to the heat resistance and the disease resistance of plants; A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2). The TaRHP1 and the coding gene thereof can be used for improving the resistance of plants to sheath blight and gray mold and can be used for improving the heat resistance of the plants.)

1. The protein is the following protein A1), A2) or A3):

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90% of identity with the protein shown in A1), and is related to the heat resistance and the disease resistance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

2. The biomaterial related to the protein of claim 1, which is any one of the following B1) to B9):

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 containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

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

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces the expression of the protein of claim 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

3. The related biological material according to claim 2, wherein: B1) the nucleic acid molecule is a coding gene of the protein shown in the following b1) or b 2):

b1) the coding sequence is cDNA molecule or DNA molecule of 62-1219 site nucleotide of sequence 1 in the sequence table;

b2) the nucleotide is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.

4. A plant disease-resistant agent and/or a plant heat-resistant agent, characterized in that: the plant disease-resistant agent and/or plant heat-resistant agent contains the protein according to claim 1, or/and the biomaterial according to claim 2 or 3.

5. The protein of claim 1, or the biomaterial of claim 2 or 3 for use in any one of the following P1-P9:

use of P1, the protein of claim 1, or the biomaterial of claim 2 or 3 for modulating disease resistance in plants;

use of P2, the protein of claim 1, or the biomaterial of claim 2 or 3 for the preparation of a product for enhancing disease resistance in plants;

use of P3, the protein of claim 1, or the biomaterial of claim 2 or 3 for growing disease-resistant plants;

use of P4, the protein of claim 1, or the biomaterial of claim 2 or 3 for the preparation of a plant disease resistant product;

use of P5, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for modulating thermotolerance in a plant;

use of P6, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for the manufacture of a product for increasing the thermotolerance of a plant;

use of P7, the protein of claim 1, or the biomaterial of claim 2 or 3 for growing heat-resistant plants;

use of P8, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for the preparation of a heat resistant product of a plant;

use of P9, the protein of claim 1, or the biological material of claim 2 or 3 in plant breeding.

6. A method for producing a heat-resistant and/or disease-resistant plant, comprising increasing the expression level of the protein of claim 1 or a gene encoding the protein in a target plant to obtain a heat-resistant and/or disease-resistant plant; the heat resistance and/or disease resistance of the heat-resistant and/or disease-resistant plant is higher than that of the target seed plant.

7. A method for producing a transgenic plant having reduced disease resistance, which comprises reducing the expression of a gene encoding the protein of claim 1 in a target plant to obtain a transgenic plant having reduced disease resistance as compared to the target plant.

8. The anti-disease agent according to claim 4, or the use according to claim 5, or the method according to claim 6 or 7, wherein: the plant according to claim 4 or 5, the plant of interest according to claim 6 or claim 7 is a monocotyledonous plant or a dicotyledonous plant.

9. The method according to any one of claims 6-8, wherein: the improvement of the expression level of the protein of claim 1 or a gene encoding the protein in a plant of interest is achieved by introducing a gene encoding the protein of claim 1 into the plant of interest;

the reduction of the expression of the gene encoding the protein of claim 1 in the target plant is achieved by introducing a DNA molecule reverse-complementary to the DNA fragment represented by nucleotides 474 to 746 of sequence No. 1 in the sequence listing into the target plant.

10. The protein of claim 1, the anti-disease agent of claim 4 or 8, the use of claim 5 or 8, or the method of any one of claims 6-9, wherein: the disease resistance is banded sclerotial blight resistance and/or gray mold resistance.

Technical Field

The invention relates to a disease-resistant and heat-resistant related protein TaRHP1 and a related biological material thereof in the technical field of biology and a method for cultivating disease-resistant and heat-resistant plants.

Background

With global warming, natural disasters such as high temperature and the like are increasingly serious, the yield and the grain quality of important crops are seriously influenced (Trnka M,

RP,Ruiz-Ramos M,Kersebaum KC,Olesen JE,z, SennovMA. Adverse weather conditions for European meal production with less bed mole frequency Change. Nature Climate Change,2014,4: 637-643). Wheat flour and products thereof are used as staple food for about 50% of people in the world, so that high and stable yield of wheat plays a significant role in guaranteeing global food safety. England scientists predict that the temperature is higher than the optimum growth temperature by more than 2 ℃, which can cause the yield reduction of the wheat by 12-50% (Semenov)&Shewry.2011,Modelling predicts that heat stress,not drought,willincrease vulnerability of wheat in Europe.Scientific Reports,1:66)。

Sheath blight of wheat, also known as wheat sharp eyespot. The wheat sharp eyespot in China is mainly caused by can-1 which is a saprophytic nutritional pathogenic fungus, Rhizoctonia cerealis. The sheath blight disease can generally reduce the yield of the wheat by 10-30 percent, and the serious plot can reduce the yield of the wheat by more than 50 percent. Therefore, breeding and popularizing the new wheat variety resisting the sheath blight is the most economic, safe and effective way for preventing and treating the disease, and is very important for ensuring the stable and high yield of wheat in China. However, conventional breeding methods have been slow in breeding of sheath blight resistant wheat varieties due to the lack of readily available sheath blight resistant wheat germplasm resources. Important disease-resistant genes are discovered and cloned, and a new disease-resistant wheat strain (variety) is created through genetic engineering or gene editing, so that an important new way is opened for the breeding of the sheath blight resistance.

In addition, Botrytis cinerea (also known as Botrytis cinerea) is a broad-host fungus which is widely distributed in the air, not only can infect field crops, but also can cause great loss to the fruits at the post-harvest stage and the storage stage of plants. After the botrytis cinerea is infected, damping-off, fallen leaves, rotten flowers, rotten fruits and rotten cellars of various plant seedlings, fruits and storage organs can be caused, and the disease is called as the botrytis cinerea. By 2013, no plant has been found to be resistant to botrytis cinerea in the world. Gray mold was first only prevalent in europe and america, and spread in china began to occur since the 80's of the 20 th century. Due to the popularization of greenhouse and greenhouse planting technology, crop botrytis cinerea is serious and becomes a main limiting factor for the production of vegetable, flower and forestry seedling cultivation bases.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the disease resistance (such as the resistance of the plant to sheath blight and/or gray mold) and/or heat resistance of the plant.

In order to solve the technical problems, the invention provides a disease-resistant and heat-resistant protein derived from wheat, which is named as TaRHP1 and derived from a wheat line CI12633 resistant to banded sclerotial blight, and is A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90% of identity with the protein shown in A1), and is related to the heat resistance and the disease resistance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

In the protein, the sequence 2 in the sequence table consists of 385 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Among the above proteins, TaRHP1 can be derived from wheat.

Biomaterials associated with TaRHP1 are also within the scope of the present invention.

The biological material related to the TaRHP1 provided by the invention is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding TaRHP 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

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

B5) a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule of B1);

B6) a nucleic acid molecule that reduces expression of TaRHP 1;

B7) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B6).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, the nucleic acid molecule according to B1) may specifically be a gene represented by 1) or 2) below:

1) the coding sequence (ORF) is DNA molecule of 62 th-1219 th nucleotides of sequence 1 in the sequence table;

2) the nucleotide sequence is a DNA molecule of a sequence 1 in a sequence table.

In the above biological material, the nucleic acid molecule of B6) may specifically be a DNA molecule reverse-complementary to any one of the DNA fragments represented by nucleotides 1 to 1219 of sequence 1 in the sequence table, such as a DNA molecule reverse-complementary to the DNA fragment represented by nucleotides 474 to 746 of sequence 1 in the sequence table.

Wherein, the sequence 1 in the sequence table is composed of 1219 nucleotides, and the coding sequence is the protein shown by the sequence 1 and the sequence 2 in the sequence table.

In the above biological material, the expression cassette containing a nucleic acid molecule encoding TaRHP1 (TaRHP1 gene expression cassette) described in B2) refers to a DNA capable of expressing TaRHP1 in a host cell, and the DNA may include not only a promoter that initiates transcription of the TaRHP1 gene, but also a terminator that terminates transcription of TaRHP 1. Further, 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. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, e.g. the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters(e.g., the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al GenesDev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant expression vector containing the TaRHP1 gene expression cassette can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In order to solve the technical problems, the invention also provides a plant disease-resistant agent and/or a plant heat-resistant agent.

The plant disease-resistant agent and/or the plant heat-resistant agent provided by the invention contain the protein or/and biological materials related to the protein.

The active ingredients of the plant disease-resistant agent and/or the plant heat-resistant agent can be the protein or biological materials related to the protein, and the active ingredients of the plant disease-resistant agent and/or the plant heat-resistant agent can also contain other biological ingredients or/and non-biological ingredients, and the other active ingredients of the agent can be determined by a person skilled in the art according to the disease-resistant and/or heat-resistant effect of the plant.

In the plant disease-resistant agent and/or the plant heat-resistant agent, the plant disease-resistant agent can be an agent for resisting plant sheath blight and/or plant gray mold.

The protein or the biological material can be applied to any one of the following P1-P9:

use of P1, the protein or the biomaterial for modulating disease resistance in plants;

the application of P2, the protein or the biological material in preparing products for improving plant disease resistance;

the use of P3, the protein or the biological material for growing disease-resistant plants;

the application of P4, the protein or the biological material in preparing plant disease-resistant products;

the use of P5, said protein or said biomaterial for modulating thermotolerance in plants;

the use of P6, the protein or the biomaterial for the manufacture of a product for increasing the thermotolerance of a plant;

use of P7, the protein or the biological material for growing heat-resistant plants;

the use of P8, the protein or the biomaterial for the preparation of a plant heat resistant product;

use of P9, the protein or the biological material in plant breeding.

In order to solve the technical problems, the invention also provides a method for cultivating heat-resistant and/or disease-resistant plants.

The method for cultivating the heat-resistant and/or disease-resistant plant comprises the steps of improving the expression level of the protein or the coding gene thereof in a target plant to obtain the heat-resistant and/or disease-resistant plant; the heat resistance and/or disease resistance of the heat-resistant and/or disease-resistant plant is higher than that of the target seed plant.

In the above method, the improvement of the expression level of the protein or the gene encoding the protein in the target plant can be achieved by introducing the gene encoding the protein into the target plant.

In the method, the coding gene of the protein can be modified as follows and then introduced into a target plant to achieve better expression effect:

1) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

4) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition).

In the above method, the heat-resistant and/or disease-resistant plant may be a transgenic plant, or may be a plant obtained by a conventional breeding technique such as crossing.

In order to solve the technical problems, the invention also provides a method for cultivating transgenic plants with reduced disease resistance

The method for cultivating the transgenic plant with reduced disease resistance provided by the invention comprises the step of reducing the expression of the coding gene of the protein in a target plant to obtain the transgenic plant with the disease resistance lower than that of the target plant.

In the above method, the reduction of the expression of the gene encoding the protein in the target plant can be achieved by introducing a DNA molecule reverse-complementary to the DNA fragment represented by nucleotides 474 to 746 of sequence No. 1 in the sequence listing into the target plant.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

As described above, the plant and the plant of interest are both monocotyledonous or dicotyledonous plants. The monocotyledon can be wheat, and the dicotyledon can be cruciferous plants such as Arabidopsis thaliana.

As above, the disease resistance may be resistance to sheath blight and/or resistance to gray mold.

As described above, the sheath blight disease can be caused by Rhizoctonia cerealis (Rhizoctonia cerealis). The gray mold can be caused by gray mold (Botrytis cinerea).

Transgenic experiments for introducing the TaRHP1 gene into arabidopsis thaliana prove that compared with acceptor arabidopsis thaliana, the transgenic arabidopsis thaliana expressing the TaRHP1 gene has obviously improved heat resistance and resistance to gray mold; transgenic experiments for introducing the TaRHP1 gene into wheat prove that the resistance of transgenic wheat expressing the TaRHP1 gene to banded sclerotial blight is obviously improved compared with receptor wheat, which indicates that the TaRHP1 gene is related to the resistance of plants to banded sclerotial blight, gray mold and heat resistance, and the TaRHP1 and the coding gene thereof can be used for improving the resistance of plants to banded sclerotial blight and gray mold and can be used for improving the heat resistance of plants.

Drawings

FIG. 1 shows the analysis of the expression level of TaRHP1 gene after wheat is inoculated with Rhizoctonia cerealis.

FIG. 2 is TaRHP1 gene expression analysis after wheat heat treatment.

FIG. 3 is T₃Analysis of the relative level of transcription of TaRHP1 in the transgenic TaRHP1 gene Arabidopsis positive strain.

FIG. 4 shows the phenotype of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana expressed by TaRHP1 against high temperature stress.

FIG. 5 shows the increased resistance of transgenic Arabidopsis thaliana expressed by TaRHP1 to gray mold.

FIG. 6 shows PCR detection of TaRHP1 transgenic wheat.

FIG. 7 shows RT-qPCR detection of silencing of TaRHP1 gene in wheat.

FIG. 8 shows that silencing of the TaRHP1 gene significantly reduced the ability of wheat CI12633 to defend Rhizoctonia solani.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

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

Wheat CI12633 is germplasm from the agricultural germplasm resource protection and utilization platform (germplasm resource bank of the agricultural academy of sciences of Jiangsu province), and wheat CI12633 exhibits resistance to sheath blight. Yangmai 16 is a wheat variety with Rhizoctonia solani, and comes from the institute of agricultural science in the Riyuwa region of Jiangsu; the wenmai 6 is germplasm from a national plant germplasm resource sharing platform (germplasm resource library of Chinese academy of agricultural sciences), and the wenmai 6 is high in banded sclerotial blight.

The wild type Arabidopsis thaliana in the following examples was Col-0 ecotype Arabidopsis thaliana (Arabidopsis thaliana, Columbia-0(Col-0)) (Zhao Xiao, Wang Jin, Yuan Jin, Wang Xi-li, Zhao Qi ng, Kong Pei-tao, Zhang Xiao, NITRIC OXIDE-ASSOCTEIAD PROTEIN1(AtNOA1) isoessianal for salicylic acid-induced root wasing in Arabidopsis thaliana. New Phytocologist 2015,207:211-224), which was awarded by the researchers in the institute of crop science, Chin academy of agriculture, China. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes. Col-0 ecotype Arabidopsis thaliana is sensitive to Botrytis cinerea and high temperature.

The wheat sharp eyespot pathogenic bacteria in the following examples, Rhizoctonia cerealis R0301 (agricultural academy of sciences in Jiangsu province) (Cold Sufeng, Zhang Aixiang, Liwei, Chenhuai Gu, New wheat variety (series) in Jiangsu province for resistance analysis of sharp eyespot. Jiangsu agricultural bulletin, 2010, 26 (6): 1176-1180); wheat sharp blight pathogen WK207(Ji L, Liu C, Zhang L, Liu A, Yu J. variation of rDNA internal transformed space sequences in Rhizoctonia cerealis. Current microbiology.2017,74, 877-884), introduced from Shandong agricultural university in professor Jinfeng. The biological material is only used for repeating the relevant experiments of the invention and can not be used for other purposes.

The Botrytis cinerea in the following examples is Botrytis cinerea (Abuqamar S, Chai MF, LuoH, Song F, Mengolite T.tomato protein kinase 1b media signalling of plants to biological fuels and of plants in plant cell 200820: 1964-1983), which is publicly available from the institute of crop science of Chinese academy of agricultural sciences to repeat the experiments of the present application and is not useful for other purposes.

The monocot plant expression vector pWMB123 (ref, Wang Ke, Liu Huiyun, Du Lipu, Ye Xingguo. Generation of marker-free transgenic wheat straw microorganism Agrobacterium-mediated co-transformation strategy in commercial maize straw industries. plant Biotechnology journal.2017,15, 614-623) in the examples described below was publicly available from the institute of crop science of Chinese Agrochemical institute to repeat the experiments of the present application and was not used for other applications.

The dicot expression vector pCAMBIA1300(Zhao Xiao, Wang Jin, Yuanjin, Wang Xi-li, Zhao Qi ng-ping, Kong Pei-tao, Zhang Xiao. NITRIC OXIDE-ASSOCIATED PROTEIN1(AtNOA1) is an expression for a salicylic acid-induced rootwalking in Arabidopsis thaliana. New Phytology. 2015,207:211-224) in the examples below. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes.

The 3 components of the BSMV viral vector in the following examples BSMV-alpha, BSMV-beta and BSMV-gamma plasmids (Lu X-D (Liu Xiao Dong), Zhang Z-Y (Zhang Zengyan), Yao W-L (Yao Wulan), XinZ-Y (Shixinzhiyong). augmentation of barleys stereoscopic vision-base induced gene diagnosis in wyeat. ACTA Agron Sin (crop academic newspaper), 2005,31(11): 1518-1520; Zhao Dan, Zhao Cheng, Huang Rubi, Lining, Liu Yan, Huang Ju Jing, Zhang Zengyan. the BSMV-VIGS technique was used to rapidly analyze the anti-Huang dwarf function of wheat TNBL1 gene. crop academic newspaper ACTA AGNOMICA, 2011 ICA, 37(11):2106-2110), which was obtained from the institute of Chinese sciences, and was applied for other agricultural non-repeatable uses.

The sheath blight disease level criteria of wheat (Lensdeep, Li Anfei, Li Xianxin et al 1997, early declaration of resistance of wheat germplasm to sheath blight. crop variety resources (4):31-33) are shown in Table 1.

TABLE 1 grade Standard of sheath blight disease of wheat

Sheath blight disease grade of wheat (IT)	Sheath blight disease of wheat
		Level 0	The leaf sheath and stem of the plant have no disease spot
Level 1	The leaf sheath of the plant has lesion but does not invade the stem
		Stage
2	The leaf sheath and stem of the plant have scabs, and the annular stem of the scab is more than 0 and less than or equal to 1/2
		Grade 3	The leaf sheath and stem of the plant have scabs, and the ring and stem of the scab are more than 1/2 and less than or equal to 3/4
4 stage	The leaf sheath and stem of the plant have disease spots, and the ring and stem of the disease spots are more than 3/4 and less than or equal to 1
		Grade 5	The plant has lesion on leaf sheath and stem, and withered booting ear or withered white ear

Wherein, 0 grade represents immunity, 1 grade represents resistance, 2 grade represents resistance, 3 grade-4 grade represents feeling, and 5 grade represents high feeling.

The Disease Index (DI) is [ (Σ number of diseased plants per stage × representative value per stage)/(total number of plants × highest representative value) ] × 100.

The preparation method of the fungi wheat grain of the rhizoctonia cerealis comprises the following steps: cooking wheat grains for 5-6 hours for 20 minutes, filling a triangular flask with 250-500 ml, preparing an MS liquid culture medium, sterilizing, inoculating newly cultured hypha blocks of rhizoctonia cerealis R0301 and WK207 into the triangular flask, and culturing at a constant temperature of 25 ℃ until the hypha is densely distributed on the wheat grains.