Application of pm61 protein and its coding gene in regulating wheat resistance to powdery mildew

Abstract

The application discloses application of Pm61 protein and a coding gene thereof in regulating wheat resistance to powdery mildew, and belongs to the field of genetic engineering, and particularly relates to application of Pm61 protein and a coding gene thereof in regulating wheat resistance to powdery mildew. The protein of the application is any one of the following: A1) a protein with an amino acid sequence as shown in SEQ ID No: 2; A2) a protein obtained by substitution, deletion and / or addition of amino acid residues on the protein of A1), the protein having more than 80% identity with the protein shown in A1) and having the same function; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). The Pm61 protein can positively regulate the resistance of wheat to powdery mildew, and can be used for wheat breeding with resistance to powdery mildew.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of Pm61 protein and its encoding gene in regulating wheat resistance to powdery mildew. Background Technology

[0002] Wheat (Triticum aestivum) Powdery mildew (L.) is one of the world's three major staple foods, providing basic nutrition for more than 35% of the population. Powdery mildew is caused by the specialized fungal pathogen *Erysiphe graminearum* (L.). Blumeria graminis f. sp. tritici Powdery mildew (Bgt) is caused by the fungus and is one of the major diseases threatening global wheat production. It causes significant yield losses by inhibiting the photosynthetic efficiency of the host wheat, reducing tillering and ear formation rates, and decreasing thousand-grain weight. Current strategies for controlling powdery mildew rely heavily on chemical agents, but these pose risks of environmental pollution and pathogen resistance. Therefore, developing wheat varieties with durable, broad-spectrum resistance has become a core solution that combines economic viability with ecological sustainability.

[0003] Through thousands of years of domestication and natural selection, local wheat varieties in my country have accumulated rich genetic diversity, serving as an important resource for the discovery of disease-resistant genes. Of the wheat resources currently existing in the National Crop Germplasm Bank, one-third (approximately 13,000 accessions) are local varieties. Historically, local wheat varieties have played a significant role in both production and breeding. Before the advent of modern improved varieties, local varieties dominated wheat production in my country, accounting for over 80% of the country's wheat planting area before the 1940s. Local wheat varieties contain abundant powdery mildew resistance genes; currently discovered powdery mildew resistance genes in local varieties include Pm2c, Pm3b, Pm5d, Pm5e, Pm24a, Pm24b, Pm45, Pm47, Pm59, Pm61, and Pm63. Furthermore, local varieties exhibit high hybridization compatibility with conventional wheat varieties, outstanding stress resistance (such as drought and salt tolerance), and great potential for agronomic trait improvement, making them suitable for wheat disease resistance breeding. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the powdery mildew resistance of plants.

[0005] To address the problems existing in the prior art, the present invention provides a protein.

[0006] The protein provided by this invention may be any of the following:

[0007] A1) A protein with the amino acid sequence shown in SEQ ID No:2;

[0008] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 75% identity with the protein shown in A1) and has the ability to regulate plant powdery mildew resistance.

[0009] For example, those skilled in the art can use conventional techniques in the field, such as the amino acid sequence shown in SEQ ID No:2 and the conservative substitution of amino acids, to obtain a protein mutant with the same function as the amino acid sequence shown in SEQ ID No:2 by substituting, deleting and / or adding one or more amino acids without affecting its activity.

[0010] A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0011] The protein described in A1 above is named Pm61.

[0012] To facilitate the purification or detection of the protein in A1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:2 in the sequence listing.

[0013] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0015] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein Pm61 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the protein Pm61 isolated in this invention, as long as they encode and function as protein Pm61, are derived from and equivalent to the nucleotide sequence of this invention.

[0016] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0017] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences or nucleotide sequences, then the identity value (%) can be obtained.

[0018] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0019] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0020] The protein mentioned above is derived from wheat (Triticum aestivum). L.).

[0021] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following:

[0022] B1) Nucleic acid molecules that encode the proteins described above;

[0023] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0024] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0025] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0026] 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);

[0027] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0028] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0029] C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above;

[0030] C2) expresses the gene encoding the nucleic acid molecule described in C1);

[0031] C3) contains an expression cassette containing the gene encoding described in C2);

[0032] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0033] C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0034] C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4);

[0035] C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4);

[0036] C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0037] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be a gene as shown in E1) or E2) below:

[0038] E1) The coding sequence is a cDNA molecule or DNA molecule of SEQ ID No:1;

[0039] E2) The nucleotide is a cDNA molecule or DNA molecule of SEQ ID No:3.

[0040] The DNA molecule shown in SEQ ID No:1 (which regulates plant powdery mildew resistance) Pm61 The gene encodes protein Pm61, whose amino acid sequence is SEQ ID No:2.

[0041] The nucleotide sequence shown in SEQ ID No:1 is the nucleotide sequence of the protein Pm61 encoding gene (CDS).

[0042] The present invention Pm61 Genes can be any nucleotide sequence that encodes the protein Pm61. Considering codon degeneracy and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0043] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No:1.

[0044] B1) The nucleic acid molecule may also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No:1 and originate from the same species.

[0045] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0046] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0047] In this invention, the vector can be an expression vector. The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, can also be used. Specifically, vectors pCAMBIA1300 and pTPCK303 can be used.

[0048] Existing plant expression vectors can be used to construct structures containing... Pm61Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0049] use Pm61 When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0050] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0051] Using any vector capable of guiding the expression of exogenous genes in plants, the present invention can be used to... Pm61 Introducing genes or gene fragments into plant cells or recipient plants can yield transgenic cell lines and transgenic plants with altered resistance to powdery mildew. Pm61 Gene expression vectors can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0052] As a specific embodiment, the recombinant vector described above is the recombinant vector pCAMBIA1300-Pm61. 。

[0053] The structure of the pCAMBIA1300-Pm61 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:3 into the starting vector pCAMBIA1300, while keeping the other sequences of the vector pCAMBIA1300 unchanged. The pCAMBIA1300-Pm61 vector can express the Pm61 protein, whose amino acid sequence is SEQ ID No:2.

[0054] The recombinant vector may specifically be the recombinant vector pTPCK303-Pm61. The structure of the pTPCK303-Pm61 vector is described as follows: a DNA fragment with the sequence SEQ ID No:1 is inserted into the plasmid pTPCK303, while keeping other sequences of the pTPCK303 vector unchanged. The pTPCK303-Pm61 vector can express the Pm61 protein, whose amino acid sequence is SEQ ID No:2.

[0055] The microorganisms mentioned in this article may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from the genus *Escherichia* (…). Escherichia Erwinia ( Erwinia Agrobacterium tumefaciens ( ), Agrobacterium tumefaciens Agrobacterium Flavobacterium ( Flavobacterium Alcaligenes ( ) Alkaligenes ), Pseudomonas ( Pseudomonas ), Bacillus spp. ( Bacillus (e.g., Agrobacterium tumefaciens EHA105).

[0056] In one specific embodiment, the recombinant microorganism may be recombinant Agrobacterium EHA105 / pCAMBIA1300- Pm61 .

[0057] The recombinant Agrobacterium EHA105 / pCAMBIA1300-Pm61 is a recombinant bacterium obtained by introducing the recombinant vector pCAMBIA1300-Pm61 into Agrobacterium tumefaciens EHA105.

[0058] In one specific embodiment, the recombinant microorganism may be recombinant Agrobacterium EHA105 / pTPCK303-Pm61.

[0059] The recombinant Agrobacterium EHA105 / pTPCK303-Pm61 is a recombinant bacterium obtained by introducing the recombinant vector pTPCK303-Pm61 into Agrobacterium tumefaciens EHA105.

[0060] The present invention also provides the use of the protein Pm61 described above, or the expression substance of the gene regulating it, or the substance regulating the activity or content of the protein, in any of the following:

[0061] U1) Application in regulating plant powdery mildew resistance;

[0062] U2) Application in the preparation of products that regulate plant powdery mildew resistance;

[0063] U3) Application in cultivating plants resistant to powdery mildew;

[0064] U4) Application in the preparation of products for cultivating powdery mildew resistant plants;

[0065] U5) Applications in plant breeding.

[0066] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein Pm61.

[0067] In the above applications, the substance that regulates gene expression or the substance that regulates the activity or content of the protein can be a biological material related to the protein, and the biological material can be the biological material described above.

[0068] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation:

[0069] 1) Regulation occurring at the transcriptional level of the aforementioned gene;

[0070] 2) Regulation that occurs after the gene is transcribed (i.e., regulation of the splicing or processing of the primary transcript of the gene).

[0071] 3) Regulation of RNA transport of the gene (that is, regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm).

[0072] 4) Regulation of the translation of the aforementioned genes;

[0073] 5) Regulation of mRNA degradation of the aforementioned gene;

[0074] 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0075] The present invention also provides a method for regulating plant powdery mildew resistance, comprising regulating the activity and / or content of the proteins described above in the target plant, and / or the expression level of the gene encoding the proteins, to regulate plant powdery mildew resistance.

[0076] In the above method, regulating the activity and / or content of the protein Pm61 in the target plant, or / and the expression level of the gene encoding the protein, includes introducing the gene encoding the protein Pm61 into the recipient plant to alter the plant's powdery mildew resistance; the gene encoding the Pm61 encodes the protein Pm61.

[0077] The importation refers to the importation through recombination methods, including but not limited to Agrobacterium-mediated transformation, bio-projectile methods, electroporation, in-planta technology, and so on.

[0078] In the above applications and methods, the regulation can be to increase, enhance, or upregulate.

[0079] In the above applications and methods, the regulation can be suppression, reduction, or silencing.

[0080] The plants obtained by the above methods can be transgenic plants or plants obtained through conventional breeding techniques such as hybridization. In the above methods, the transgenic plants are understood to include not only first- and second-generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plants include seeds, callus tissue, complete plants, and cells.

[0081] This invention also provides a method for cultivating plants with altered resistance to powdery mildew, comprising:

[0082] 1) Increase, enhance and / or upregulate the expression level of the coding genes of the proteins mentioned above in the target plant, or / and increase, enhance and / or upregulate the activity and / or content of the coding genes of the proteins mentioned above, to obtain plants with improved resistance to powdery mildew.

[0083] 2) Inhibit, reduce, or silence the expression level of the coding genes of the proteins mentioned above in the target plant, or / and inhibit, reduce, or silence the activity and / or content of the coding genes of the proteins mentioned above, to obtain plants with reduced resistance to powdery mildew.

[0084] As one embodiment of the present invention, the method for cultivating plants with enhanced resistance to powdery mildew includes the following steps:

[0085] (1) Construct an expression vector containing the Pm61 gene coding sequence as shown in SEQ ID No:1;

[0086] (2) Introduce the expression vector constructed in step (1) into plants;

[0087] (3) Plants with improved resistance to powdery mildew were obtained through screening and identification.

[0088] In this invention, the purpose of plant breeding includes cultivating plants with increased / decreased resistance to powdery mildew.

[0089] In the above applications or methods, the plant is any one of the following:

[0090] N1) Monocotyledons;

[0091] N2) Plants of the order Poales;

[0092] N3) Gramineae plants;

[0093] N4) Plants of the Triticum genus;

[0094] N5) Wheat.

[0095] This invention utilizes recombinant vectors EHA105 / pCAMBIA1300-Pm61 and EHA105 / pTPCK303-Pm61 to transform the highly powdery mildew-susceptible wheat variety Fielder, respectively. After two generations of self-pollination and selection, three transgenic T2 families with the Pm61 self-promoter and three transgenic T2 families overexpressing Pm61 were obtained. Following the technical procedures for identifying powdery mildew resistance in wheat seedlings, the resistance of the above transgenic families and wild-type Fielder was evaluated. The results showed that all transgenic families exhibited powdery mildew immunity (IT=0) or high resistance (IT=0-1), while wild-type Fielder showed high susceptibility (IT=4). These results confirm that the Pm61 protein and its encoding gene can significantly improve wheat resistance to powdery mildew, and this invention provides important genetic resources and application prospects for wheat disease resistance breeding. Attached Figure Description

[0096] Figure 1 The Pm61 gene structure in Example 1 is shown, where CC is the CC domain of the R gene, NB-ARC is the NBS domain of the R gene, and LRR is the LRR domain of the R gene.

[0097] Figure 2 Phenotypic photographs of the Pm61 overexpression transgenic and self-promoter transgenic plants used in Example 2 to identify disease resistance. Detailed Implementation

[0098] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0099] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0100] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0101] In the following examples, the method for identifying wheat seedling resistance to powdery mildew is specifically referenced in the following literature: Liu ZY, Sun QX, Ni ZF, Yang TM. Development of SCAR markers linked to the Pm21 geneconferring resistance to powdery mildew in common wheat. Plant Breeding, 1999, 118: 215-219.

[0102] Wheat powdery mildew resistance is graded on a scale of 0-4. Resistance is determined based on the plant's response to powdery mildew fungus inoculation. Classifying the resistance, grades 0-2 indicate resistance, while grades 3-4 indicate susceptibility. The grading standards for various wheat powdery mildew conditions are as follows:

[0103] Grade 0: Immune, no disease spots on the entire plant, no mycelium attached.

[0104] Grade 0: Necrosis reaction, with necrotic spots on the leaves.

[0105] Grade 1: Highly resistant, with very small lesions, a thin mycelial layer, and visible green leaf surface. Occasionally, there are larger lesions, but they are still translucent green. The amount of spores produced is extremely low.

[0106] Level 2: Moderately resistant, with small leaf lesions, a relatively thick mycelial layer but weak three-dimensionality, and capable of producing a certain amount of spores.

[0107] Level 3: Moderately susceptible, with many leaf spots, thick mycelial layer with strong three-dimensionality, leaf loss of green color, large sporulation, but the spots do not merge.

[0108] Level 4: Highly susceptible, with numerous leaf spots, thick mycelial layer, high sporulation, and confluent lesions.

[0109] The powdery mildew-resistant parent *Xuxusanyuehuang*, the powdery mildew-susceptible parent *Mingxian 169*, and the powdery mildew physiological race E09 in the following examples are all described in: Sun HG, Hu JH, Song W, et al. Pm61: a recessive gene for resistance to powdery mildew in wheat landrace *Xuxusanyuehuang* identified by comparative genomics analysis. Theoretical and Applied Genetics, 2018, 131:2085-2097. *Xuxusanyuehuang* in this literature is the same material as the powdery mildew-resistant parent *Xuxusanyuehuang* described herein. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention; it may not be used for any other purpose.

[0110] The wheat variety Fielder used in the following examples is described in: Li, MM, Zhang, HZ, Xiao, HX, et al. A membrane associated tandem kinase from wild emmer wheat confers broad-spectrum resistance to powdery mildew. Nature Communications, 2024, 15:3124., which is a wheat variety highly susceptible to powdery mildew and is a transgenic recipient material. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0111] The pCAMBIA1300 plasmid used in the following examples is the base vector for constructing the Pm61 self-promoter transgenic vector. It was purchased from a biochemical reagent company and was double-digested with restriction endonucleases BamHI and HindIII. Then, the Pm61 self-promoter transgenic vector was constructed using homologous recombination.

[0112] The pTPCK303 plasmid used in the following examples is the basic vector for constructing the overexpression transgenic vector. It was purchased from a biochemical reagent company and was double-digested with restriction endonucleases KpnI and SpeI. Then, the overexpression transgenic vector was constructed using homologous recombination.

[0113] Example 1: Cloning of the Pm61 gene

[0114] 1. Take two-leaf stage leaves of *Gynostemma pentaphyllum* and extract genomic DNA.

[0115] 2. Using the genomic DNA obtained in step 1 as a template, the Pm61 gene was amplified using primers Pm61G-F: 5'-TACAGTGGTCAGCGCGTATC-3' (SEQ ID No:4) and Pm61G-R: 5'-CAGACATAGAGGCCAGAGCG-3' (SEQ ID No:5). Sequencing yielded a 9491 bp nucleotide sequence (nucleotide sequence shown in SEQ ID No:3). Gene structure prediction revealed the presence of a complete CC-NBS-LRR type gene. Figure 1 In this sequence, positions 1-2913 form the 5' UTR, positions 2914-6852 are a unique exon, and positions 6853-9491 form the 3' UTR. Its CDS sequence is shown in SEQ ID No:1 of the sequence listing, encoding a protein named Pm61. The amino acid sequence of this protein is shown in SEQ ID No:2 of the sequence listing.

[0116] 3. Take leaves of *Erysiphe simonii* var. *mongolica* 24 h after inoculation with physiological race E09 of powdery mildew and extract total RNA.

[0117] 4. After completing step 3, using the cDNA obtained from the reverse transcription of total RNA from *Eclipta prostrata* as a template, PCR amplification was performed using Pm61-1F: 5'-ATGGAAGTGGCATTAGCTACTG-3' (SEQ ID No: 6) and Pm61-1R: 5'-TCAACTAATACCCTTCCAGCGG-3' (SEQ ID No: 7). Simultaneously, the 5' and 3' ends of the Pm61 gene were amplified using the SMARTer® RACE 5' / 3' Kit. A final 3939 bp nucleotide sequence was obtained.

[0118] 5. The nucleotide sequence (3939 bp) obtained in step 3 was compared with the nucleotide sequence (3939 bp) obtained in step 1. It was found that the Pm61 gene has a full-length open reading frame (ORF) of exon with a length of 3939 bp (SEQ ID No:1).

[0119] Example 2: Verification of protein function via transgenic technology

[0120] I. Construction of Recombinant Plasmids

[0121] 1. Recombinant plasmid pCAMBIA1300- Pm61 Construction

[0122] (1) Take pCAMBIA1300 plasmid and digest it with restriction endonucleases BamHI and HindIII to recover the vector backbone of about 8900 bp.

[0123] (2) Using the genomic DNA obtained in step 1 of Example 1 as a template, PCR amplification was performed using three pairs of primers: ComPm61-1 (composed of ComPm61-1F and ComPm61-1R), ComPm61-2 (composed of ComPm61-2F and ComPm61-2R), and ComPm61-3 (composed of ComPm61-3F and ComPm61-3R). The PCR amplification products were recovered. The amplification product sequence of primer pair ComPm61-1 is shown in positions 1-2935 of SEQ ID No:3 in the sequence listing; the amplification product sequence of primer pair ComPm61-2 is shown in positions 2914-6852 of SEQ ID No:3 in the sequence listing; and the amplification product sequence of primer pair ComPm61-3 is shown in positions 6831-9491 of SEQ ID No:3 in the sequence listing.

[0124] ComPm61-1-F:5'-AATTCGAGCTCGGTACCCGGGTACAGTGGTCAGCGCGTATC-3' (SEQ ID No:8, positions 22-41 are identical to positions 1-20 of SEQ ID No:3);

[0125] ComPm61-1-R:5'-CAGTAGCTAATGCCACTTCCAT-3' (SEQ ID No:9, positions 1-22 are inversely complementary to positions 2914-2935 of SEQ ID No:3).

[0126] ComPm61-2-F:5'-ATGGAAGTGGCATTAGCTACTG-3' (SEQ ID No:6, positions 1-22 are identical to positions 2914-2935 of SEQ ID No:3);

[0127] ComPm61-2-R:5'-TCAACTAATACCCTTCCAGCGG-3' (SEQ ID No:7, positions 1-22 are inversely complementary to positions 6831-6852 of SEQ ID No:3)

[0128] ComPm61-3-F:5'-CCGCTGGAAGGGTATTAGTTGA-3' (SEQ ID No:10, positions 1-22 are identical to positions 6831-6852 of SEQ ID No:3);

[0129] ComPm61-3-R:5'-TGTAAAACGACGGCCAGTGCCACAGACATAGAGGCCAGAGCG-3' (SEQ ID No:11, positions 23-42 are inversely complementary to the sequence of positions 9472-9491 of SEQ ID No:3).

[0130] (3) Using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit, the PCR amplification product obtained in step (2) and the vector backbone recovered in step (1) were homologously recombinated to obtain the recombinant plasmid pCAMBIA1300-Pm61. In the recombinant plasmid pCAMBIA1300-Pm61, the Pm61 gene is driven by its own promoter (as shown in positions 1-2913 from the 5' end of SEQ ID No:3 in the sequence listing).

[0131] pCAMBIA1300- Pm61 The structure of the vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:3 between the 5'-AATTCGAGCTCGGTACCCGGG-3' (SEQ ID No:12) and 5'-TGTAAAACGACGGCCAGTGCCA-3' (SEQ ID No:13) fragments of the starting vector pCAMBIA1300, while keeping the other sequences of the vector pCAMBIA1300 unchanged. The pCAMBIA1300-Pm61 vector can express the Pm61 protein, whose amino acid sequence is SEQ ID No:2.

[0132] 2. Construction of recombinant plasmid pTPCK303-Pm61

[0133] (1) Take the pTPCK303 plasmid and digest it with restriction endonucleases KpnI and SpeI to recover the vector backbone of about 14000 bp.

[0134] (2) Using the cDNA shown in SEQ ID No:1 in step 4 of Example 1 as a template, PCR amplification was performed using primer set OE-Pm61 (composed of OE-Pm61-F and OE-Pm61-R), and the amplification product was recovered.

[0135] OE-Pm61-F:5'-TGCAGCCCTAGGCCTACTAGGGATCCATGGAAGTGGCATTAGCTACTG-3' (SEQ ID No:14, positions 27-48 are inversely complementary to positions 1-22 of SEQ ID No:1).

[0136] OE-Pm61-R:5'-TGATACGAACGAAAGCTCTGAGCTCTCAACTAATACCCTTCCAGCGG-3' (SEQ ID No:15, positions 26-47 are inversely complementary to positions 3918-3939 of SEQ ID No:1)

[0137] (3) Using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit, the recovered Pm61 gene cDNA fragment product and the vector backbone recovered by enzyme digestion of pTPCK303 plasmid were homologously recombinated to obtain the recombinant plasmid pTPCK303-Pm61. In the recombinant plasmid pTPCK303-Pm61, the Pm61 gene is driven by the maize Ubiquitin promoter.

[0138] The structure of the pTPCK303-Pm61 vector is described as follows: It is a recombinant vector obtained by inserting a cDNA fragment with the sequence SEQ ID No:1 between the 5'-TGCAGCCCTAGGCCTACTAGGGATCC-3' (SEQ ID No:16) and 5'-TGATACGAACGAAAGCTCTGAGCTC-3' (SEQ ID No:17) fragments of the starting vector pTPCK303, while keeping the other sequences of the pTPCK303 vector unchanged. The pTPCK303-Pm61 vector can express the Pm61 protein, whose amino acid sequence is SEQ ID No:2.

[0139] II. Obtaining Recombinant Agrobacterium

[0140] 1. Strain EHA105 / pCAMBIA1300-Pm61: The recombinant plasmid pCAMBIA1300-Pm61 (containing the Pm61 gene promoter sequence) was introduced into Agrobacterium tumefaciens EHA105 using the heat shock transformation method. Positive transformants were screened in LB medium containing 50 mg / L kanamycin and 25 mg / L rifampin to obtain recombinant Agrobacterium, which was named EHA105 / pCAMBIA1300-Pm61.

[0141] 2. Strain EHA105 / pTPCK303-Pm61: The overexpression recombinant plasmid pTPCK303-Pm61 (driven by the Ubi ubiquitin promoter to generate the Pm61 gene) was introduced into Agrobacterium tumefaciens EHA105 using the heat shock transformation method. The recombinant Agrobacterium was obtained by screening and named EHA105 / pTPCK303-Pm61.

[0142] III. Transfer Pm61 The creation of genetically modified wheat

[0143] 1. Pm61 Self-Promoter Transgenic Line: Using Agrobacterium-mediated transformation, EHA105 / pCAMBIA1300-Pm61 was transformed into the highly powdery mildew-susceptible wheat variety Fielder. The resulting T0 generation plants were self-crossed once to obtain the T1 generation plants. Genomic DNA was extracted from the leaves of the T1 generation plants. Using the genomic DNA as a template, PCR amplification was performed using ComPm61S (composed of ComPm61S-F and ComPm61S-R) and ComPm61E (composed of ComPm61E-F and ComPm61E-R). The presence of bands amplified by both primer pairs indicated that the plant was a transgenic line of pCAMBIA1300-Pm61. Pm61 The plants were transplanted into flowerpots and propagated for one generation to form T2 generation plants. A total of three transgenic T2 families with the Pm61 self-promoter were screened and named T2-Pm61. - Com1 to T2-Pm61-Com3.

[0144] ComPm61S-F: 5'-CTAGCCAATACGCAAACCGC-3' (SEQ ID No: 18);

[0145] ComPm61S-R: 5'-GGGAAGCTTGGCTCCATCTT-3' (SEQ ID No: 19);

[0146] ComPm61E-F: 5'-GACAACGTTGGGACCGTTTG-3' (SEQ ID No: 20);

[0147] ComPm61E-R: 5'-TTGGGTAACGCCAGGGTTTT-3' (SEQ ID No: 21).

[0148] 2. Overexpression Transgenic Lines: Using Agrobacterium-mediated transformation, EHA105 / pTPCK303-Pm61 was transformed into the highly powdery mildew-susceptible wheat material Fielder. The resulting T0 generation plants were self-crossed once to obtain the T1 generation plants. Genomic DNA was extracted from the leaves of the T1 generation plants. Using the genomic DNA as a template, PCR amplification was performed using OE-Pm61S (composed of OE-Pm61S-F and OE-Pm61S-R) and OE-Pm61E (composed of OE-Pm61E-F and OE-Pm61E-R). The presence of bands with both primer pairs indicated that the plant was a pTPCK303-Pm61 transgenic plant. These were then transplanted into flowerpots for propagation to form the T2 generation plants. A total of three transgenic lines were obtained through screening. Pm61 The T2 gene overexpression families were named T2-Pm61-OE1 to T2-Pm61-OE3, respectively.

[0149] OE-Pm61S-F: 5'-TGCTCACCCTGTTGTTTGGT-3' (SEQ ID No: 22);

[0150] OE-Pm61S-R: 5'-GAGCTCACGCACCATATCGA-3' (SEQ ID No: 23);

[0151] OE-Pm61E-F: 5'-GATCTGCCATCATCCCTCCG-3' (SEQ ID No: 24);

[0152] OE-Pm61E-R: 5'-CTGGTGTGTGCGCAATGAAA-3' (SEQ ID No: 25).

[0153] IV. Identification of powdery mildew resistance in genetically modified wheat

[0154] The wheat tested included: control varieties were the highly powdery mildew-resistant local variety Xuxu Sanyuehuang and the highly powdery mildew-susceptible variety Fielder; the transgenic line with its own promoter was T2-Pm61. - Com1, T2-Pm61 - Com2 and T2-Pm61 - Com3; overexpression transgenic lines T2-Pm61-OE1, T2-Pm61-OE2 and T2-Pm61-OE3.

[0155] The test plants were cultured to the two-leaf stage. Then, *Zhongzuo 9504* plants infected with powdery mildew strain E09 and fully diseased were inoculated onto the test plants. After the infected materials fully developed disease, the phenotype of the transgenic plants was observed and photographed. The test results are shown below. Figure 2 The results showed that the three transgenic T2 families with their own Pm61 promoters and the three Pm61 overexpression T2 families all exhibited high resistance to powdery mildew, with a reaction grade of 1. Wheat Fielder plants showed high susceptibility to powdery mildew, with a reaction grade of 4.

[0156] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A protein, wherein the protein is any of the following: A1) A protein with the amino acid sequence shown in SEQ ID No:2; A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1).

2. The protein according to claim 1, characterized in that: The protein is derived from wheat.

3. A biomaterial relating to the protein of claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 1 or 2; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule described is a gene as shown in E1) or E2) below: E1) The coding sequence is a DNA molecule of SEQ ID No:1; E2) The nucleotide is the DNA molecule of SEQ ID No:

3.

5. The use of the protein or gene expression regulator or substance regulating the activity or content of said protein as described in claim 1 or 2 in any of the following: U1) Application in improving plant resistance to powdery mildew; U2) Application in the preparation of products that enhance plant resistance to powdery mildew; Application of U3 in cultivating plants with improved resistance to powdery mildew; U4) Application in the preparation of products for cultivating plants with enhanced resistance to powdery mildew; The substance regulating gene expression or the substance regulating protein activity or content is a biological material related to the protein, and the biological material is any one of B1) to B7) below: B1) A nucleic acid molecule encoding the protein described in claim 1; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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 molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); The plant in question is wheat.

6. A method for improving plant resistance to powdery mildew, characterized in that: The method includes increasing, enhancing, or upregulating the activity and / or content of the protein described in claim 1 or 2 in the recipient plant, or / or the expression level of the gene encoding the protein described in claim 1 or 2, to improve the powdery mildew resistance of the plant; the plant is wheat.

7. The method according to claim 6, characterized in that: The improvement of the activity and / or content of the protein described in claim 1 or 2 in the recipient plant and / or the expression level of the gene encoding the protein described in claim 1 or 2 includes introducing the gene encoding the protein into the recipient plant to obtain a target plant with higher resistance to powdery mildew than the recipient plant; the gene encoding the protein described in claim 1 or 2.

8. Methods for cultivating plants with altered resistance to powdery mildew, including: The expression level of the gene encoding the protein described in claim 1 in the recipient plant is increased, enhanced, and / or upregulated, or the activity and / or content of the gene encoding the protein described in claim 1 is increased, to obtain a plant with improved powdery mildew resistance; the plant is wheat.

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