A powdery mildew-resistant protein Pm57 from Aegilops searsii, its encoding gene, and applications thereof

Abstract

The present invention discloses a powdery mildew-resistant related protein Pm57 from Aegilops searsii, its encoding gene and applications. The protein is any one of the following proteins: (a1) a protein with an amino acid sequence of SEQ ID No. 3; (a2) a protein with the amino acid sequence shown in SEQ ID No. 3 having substitution and / or deletion and / or addition of one or several amino acid residues and having the same function; (a3) a protein having more than 80% identity with the amino acid sequence defined in any one of (a1)-(a2) and having the same function; (a4) a fusion protein obtained by connecting a tag to the end of the protein defined in any one of (a1)-(a3). Introducing the nucleic acid molecule encoding the protein into powdery mildew-susceptible wheat can obtain transgenic plants with significantly increased disease resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the powdery mildew resistance-related protein Pm57 of Aegilops searsii, its coding gene, and applications thereof. Background Art

[0002] Wheat is one of the most important food crops in China, and its production safety and supply stability are closely related to China's food security. However, wheat production is extremely vulnerable to various diseases. Among them, wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) has currently become one of the most serious diseases affecting wheat yield and quality. Wheat powdery mildew is a fungal airborne disease that mainly harms leaves, and can also harm leaf sheaths, stems, and ears in severe cases. After the disease occurs, Blumeria graminis f. sp. tritici will plunder the nutrients of the plant, causing serious losses in wheat yield. In general epidemic years, the yield can be reduced by 10%, and in severely diseased years, it can be as high as 40%. In addition, powdery mildew can also seriously affect the quality of wheat. In China, with the increase in the use of water and fertilizer, the improvement of planting density, and the large-scale planting of dwarf and semi-dwarf varieties, the harm of wheat powdery mildew is becoming increasingly serious, and it has become one of the major catastrophic problems that need to be solved urgently in wheat production.

[0003] Currently, the prevention and control of wheat powdery mildew mainly focuses on prevention. There are mainly two methods in production: chemical control and planting disease-resistant varieties. Chemical control is to spray pesticides. Although spraying pesticides can achieve certain control effects, it increases costs and pollutes the ecological environment. Cultivating and planting new wheat varieties resistant to powdery mildew has become the most economical, effective, and environmentally friendly method for controlling wheat powdery mildew recognized at present. However, Blumeria graminis f. sp. tritici has a high degree of variability, and the resistance of varieties is easily overcome by new powdery mildew mutant races and the resistance is lost. Therefore, continuously exploring new genes resistant to powdery mildew and applying them to the breeding of disease-resistant varieties is an urgent need for the current breeding and utilization of wheat disease-resistant varieties.

[0004] Aegilops searsii (2n = 2x = 14, S s S s ) is a diploid species of the genus Aegilops in the Triticeae tribe, which contains excellent genes lacking in cultivated varieties and is an important gene source for wheat breeding improvement. Summary of the Invention

[0005] One of the purposes of the present invention is to provide the powdery mildew resistance-related protein Pm57 of Aegilops searsii, its coding gene, and applications thereof.

[0006] The present invention provides a protein, and the protein is any one of the following proteins:

[0007] (a1) A protein with the amino acid sequence of SEQ ID No. 3;

[0008] (a2) A protein with the amino acid sequence shown in SEQ ID No. 3, which has been subjected to substitution and / or deletion and / or addition of one or several amino acid residues and has the same function;

[0009] (a3) A protein with more than 80% identity to the amino acid sequence defined in any of (a1)-(a2) and having the same function;

[0010] (a4) A fusion protein obtained by linking a tag to the end of the protein defined in any of (a1)-(a3).

[0011] The above-mentioned protein is named Pm57 protein. The Pm57 protein can be derived from Aegilops searsii.

[0012] The present invention also provides related biological materials of the above-mentioned protein, and the related biological materials are any of the following:

[0013] c1) A nucleic acid molecule encoding the above-mentioned protein;

[0014] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0015] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2), such as the G4 gene plant expression vector prepared in the following examples;

[0016] c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3);

[0017] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0018] c6) A transgenic plant tissue containing the nucleic acid molecule described in c1), or a transgenic plant tissue containing the expression cassette described in c2);

[0019] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2).

[0020] Optionally, according to the above-mentioned related biological materials, the nucleic acid molecule described in c1) is a DNA molecule shown as any of the following:

[0021] d1) A DNA molecule with the nucleotide sequence shown in SEQ ID NO. 1 in the sequence listing;

[0022] d2) The coding sequence is the DNA molecule shown as SEQ ID NO.2 in the sequence listing;

[0023] d3) A DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2), is derived from Aegilops searsii and encodes the above-mentioned protein;

[0024] d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the above-mentioned protein.

[0025] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained through biological expression.

[0026] In the above-mentioned protein, the tag refers to a polypeptide or protein that is expressed in fusion with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and / or SUMO tag, etc.

[0027] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined by using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website.

[0028] In this article, the identity of more than 80% 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.

[0029] In this article, the identity of more than 90% can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0030] The stringent conditions are hybridization and membrane washing twice in a solution of 2×SSC and 0.1% SDS at 68°C for 5 minutes each time, and then hybridization and membrane washing twice in a solution of 0.5×SSC and 0.1% SDS at 68°C for 15 minutes each time.

[0031] A recombinant vector containing the nucleic acid molecule can be constructed using existing expression vectors. When constructing a recombinant vector using the nucleic acid molecule, any one of enhancer-type, constitutive, tissue-specific or inducible promoters can be added before the transcription start nucleotide, and they can be used alone or in combination with other plant promoters; in addition, when constructing a recombinant vector using the nucleic acid molecule, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. For the purpose of facilitating the identification and screening of transgenic plants or transgenic microorganisms, the used expression vector can be processed, such as adding a gene that can produce a color change enzyme or a luminescent compound in plants or microorganisms, an antibiotic marker with resistance, or an anti-chemical reagent marker gene, etc. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants or microorganisms can be directly screened by phenotype.

[0032] The recombinant vector can specifically be a recombinant expression vector. The recombinant expression vector can specifically be a recombinant plasmid obtained by inserting the nucleic acid molecule into an existing plant expression vector. The existing plant expression vector can specifically be the pWMB110 vector.

[0033] The applications of the above-mentioned protein also fall within the protection scope of the present invention, and the applications are any one of the following

[0034] (1) Regulating the powdery mildew resistance of plants;

[0035] (2) Cultivating transgenic plants with changed powdery mildew resistance.

[0036] The applications of the above-mentioned related biological materials also fall within the protection scope of the present invention, and the applications are any one of the following

[0037] (1) Regulating the powdery mildew resistance of plants;

[0038] (2) Cultivating transgenic plants with changed powdery mildew resistance.

[0039] The regulation of the powdery mildew resistance of plants can be to improve the powdery mildew resistance of plants. The cultivation of transgenic plants with changed powdery mildew resistance can be to cultivate transgenic plants with enhanced powdery mildew resistance.

[0040] The present invention also protects a method for plant breeding, including increasing and / or enhancing the expression and / or the content and / or the activity of the coding gene of the above-mentioned protein in a starting plant, to obtain a plant with the following characteristics:

[0041] Compared with the starting plant, the powdery mildew resistance of the plant is improved.

[0042] The present invention also protects a method for preparing a transgenic plant, which includes the step of introducing a substance that enhances and / or increases the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein into the starting plant to obtain a transgenic plant;

[0043] Compared with the starting plant, the transgenic plant has the following characteristics:

[0044] Compared with the starting plant, the powdery mildew resistance of the transgenic plant is improved.

[0045] The above-mentioned introduction can be to transform a plant with a vector carrying the nucleic acid molecule of the present invention by any known transformation method such as chemical transformation method or electroporation method. The introduced nucleic acid molecule can be single-copy or multi-copy. The introduction can be to integrate the foreign gene into the plant chromosome, or it can be the expression of the plasmid outside the chromosome.

[0046] Optionally, according to the above preparation method, the substance that enhances and / or increases the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein is any one of the biological materials in the above c1)-c7).

[0047] In this article, the plant can be any one of the following:

[0048] (1) Monocotyledonous plants;

[0049] (2) Gramineous plants;

[0050] (3) Triticum plants;

[0051] (4) Wheat Fielder.

[0052] Any of the above-mentioned powdery mildew can be the powdery mildew caused by powdery mildew pathogens. The powdery mildew pathogen can be Blumeria graminis f. sp. tritici. Specifically, the powdery mildew pathogen can be physiological race E09.

[0053] The present invention has obtained the powdery mildew resistance gene Pm57 contained in the wheat relative species Aegilops searsii, providing a richer powdery mildew resistance gene resource for wheat breeding work. Introducing the nucleic acid molecule encoding this protein into powdery mildew-susceptible wheat can obtain transgenic plants with significantly increased disease resistance. The present invention has great application and popularization value for plant powdery mildew breeding. Description of the Drawings

[0054] Figure 1Analysis results of the Pm57 gene of Aegilops searsii for wheat powdery mildew resistance; observe and photograph the leaves 7 days after inoculation (7 dpi); at the same time, perform DAB staining on the leaves 2 days after inoculation, and brown indicates the accumulation of H 2 O 2 accumulation; perform TPN staining on the leaves 7 days after inoculation to observe the fungal structure and cell death of the leaves.

[0055] Figure 2 Map-based cloning of the wheat powdery mildew resistance gene Pm57; among them, L1-16 are molecular markers; black indicates the Aegilops searsii chromosome 2S s , gray indicates the Chinese Spring wheat chromosome; CS on the left is Chinese Spring, 89-88 is the Chinese Spring-Aegilops searsii translocation line carrying Pm57, and types I-VI are 6 recombinant genotypes screened; the powdery mildew identification results are on the right, R represents resistant, S represents susceptible; the dotted middle section is the Pm57 mapping interval; the pentagon represents the annotated gene.

[0056] Figure 3 Using the MutRNA-Seq method to determine that G4 is the candidate gene; among them, A is a schematic diagram of the MutRNA-Seq experimental method; the red dots indicate the differential SNP sites; B is the mutant analysis and alternative splicing forms of the Pm57 candidate gene G4; the boxes represent exons, the gray lines represent introns, and the three underlined regions of different colors represent the first kinase domain (Kin I), the second kinase domain (Kin II), and the VWA domain (VWA) respectively, and the red asterisk represents the stop codon.

[0057] Figure 4 Transgenic functional verification of the powdery mildew candidate gene; among them, A is to identify the powdery mildew resistance of T 0 generation transgenic plants using the detached leaf segment method; Fielder is the highly susceptible powdery mildew receptor control, + indicates that the transgenic identification is a positive plant, and - indicates that the transgenic identification is a negative plant; B is the resistance difference between some T 1 generation transgenic lines (L1, L2, and L4) and the receptor control (Fielder); Fielder is the highly susceptible powdery mildew receptor control, + indicates that the transgenic identification is a positive plant, and - indicates that the transgenic identification is a negative plant. Specific implementation manners

[0058] The present invention will be further described in detail below in combination with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0059] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. In the following examples, for quantitative tests, unless otherwise specified, three repeated experiments are set, and the results are averaged.

[0060] The powdery mildew mentioned refers to the powdery mildew of wheat caused by Blumeria graminis f. sp. tritici. Unless otherwise marked in the following text, Blumeria graminis f. sp. tritici specifically refers to the Blumeria graminis f. sp. tritici strain E09 (BgtE09) that is widely prevalent in northern China.

[0061] According to the phenotype, the powdery mildew resistance of plants adopts a grading standard of 0-4 levels. 0 means immune (the plant has no symptoms related to powdery mildew). 0; means nearly immune (showing hypersensitive necrosis reaction and chlorosis reaction). 1 means highly resistant (the hyphal layer is thinner and there are fewer lesions, and it can transmit green). 2 means moderately resistant (the hyphal layer is thicker, does not transmit green, and can produce a certain amount of spores). 3 means moderately susceptible (the hyphal layer is relatively thick and there are many lesions, with a large amount of sporulation, but the hyphae do not form a continuous sheet). 4 means highly susceptible (the hyphal layer is thick, the hyphae are dense and there are many lesions, with a large amount of sporulation, and the hyphae form a continuous sheet). Generally speaking, 0-2 is resistant, and 3-4 is susceptible.

[0062] The wheat varieties Chinese Spring (abbreviated as CS, TA3808), Chinese Spring-Aegilops searsii translocation line 89-69 (TA5109), and 89-88 are recorded in the following literature: Dong Z, Tian X, Ma C, et al. Physical mapping of Pm57, a powdery mildew resistance gene derived from Aegilops searsii. Int J Mol Sci. 2020, 21:322. In the following text, the wheat variety Chinese Spring is simply referred to as Chinese Spring. The wheat variety Fielder, simply referred to as wheat Fielder, is a powdery mildew-susceptible variety.

[0063] The pWMB110 vector is recorded in the following literature: Liu J, Chen Z, Wang Z, et al. Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains. Mol Plant. 2021, 14(9):1472-1488.

[0064] Example 1. Analysis of the Resistance of Aegilops searsii Pm57 Gene to Wheat Powdery Mildew

[0065] To preliminarily study the resistance mechanism of Aegilops searsii Pm57 gene against wheat powdery mildew, the Chinese Spring-Aegilops searsii translocation line 89-69 carrying the Pm57 gene in the genome and the susceptible control Chinese Spring were inoculated with powdery mildew. The leaves were stained with DAB 2 days after inoculation and with TPN 7 days after inoculation (7 dpi).

[0066] The results are as Figure 1 shown. The susceptible control Chinese Spring (CS) showed high susceptibility to E09, while the translocation line 89-69 (89-69) had immune-level resistance to the powdery mildew isolate E09. DAB staining revealed the accumulation of H 2 O 2 in 89-69, which was not observed in CS. TPN staining showed cell necrosis in 89-69. These results indicate that the resistance of the Pm57 gene involves the accumulation of H 2 O 2 and cell hypersensitive necrosis.

[0067] Example 2. Identification and Functional Verification of Wheat Powdery Mildew Resistance Gene Pm57

[0068] I. Map-based Cloning of Wheat Powdery Mildew Resistance Gene Pm57

[0069] Using a partial sequence of the Aegilops searsii 2S s genome (X67593-X62492 in Figure 2 ), 16 pairs of 2S s chromosome-specific molecular markers (L1-L16 in Figure 2 ) were developed. Using these 16 pairs of molecular markers, a new round of screening for recombinant single plants was carried out on the recombinant inbred lines previously screened in the study (Dong Z, Tian X, Ma C, et al. Physical mapping of Pm57, a powdery mildew resistance gene derived from Aegilops searsii. Int J Mol Sci 2020, 21:322.). Sixty-four recombinant single plants with exchanges between molecular markers L1 and L16 were identified from 1155 inbred progeny plants, and they were divided into 6 recombinant genotypes (types I-VI in Figure 2 ). Combining the inoculation identification with the powdery mildew physiological race E09, the Pm57 gene was mapped between molecular markers L10 and L13. The physical distance between the two molecular markers on the Aegilops searsii 2S s chromosome is 0.74 Mb, and there are 15 annotated coding genes among them.Figure 2 Among G1 - G15), it includes two genes encoding disease - resistant related proteins with tandem kinase domains (tandem S_TKc domains).

[0070] II. Using the MutRNA - Seq method to determine G4 as a candidate gene

[0071] Perform MutRNA - Seq on 5 susceptible mutants (51 - 3, 60 - 3, 141 - 3, 209 - 1, 216 - 2, which are recorded in Ma Chao, Dong Zhenjie, Tian Xiubin, etc. Screening and Identification of Mutants with Loss of Resistance to Powdery Mildew Gene Pm57 from Aegilops searsii. Journal of Plant Genetic Resources, 2020, 21(2):8.) previously generated by EMS mutagenesis. Figure 3 In which A is a schematic diagram of the MutRNA - Seq experimental method.

[0072] Plant the control (WT) and 5 Pm57 - susceptible mutants (51 - 3, 60 - 3, 141 - 3, 209 - 1, 216 - 2) in a plant incubator. When they grow to the one - leaf - one - heart stage, inoculate the seedling leaves with Blumeria graminis f. sp. tritici E09. After the disease appears, take the middle leaf segments of the first leaf, extract total RNA respectively, and send them to Novogene Bioinformatics Technology Co., Ltd. for high - throughput transcriptome sequencing. The sequencing platform used is HiSeq 4000, PE150. After the sequencing is completed, combined with bioinformatics methods, using the Aegilops searsii genomic sequence in the mapped interval as a reference, perform SNP analysis on the expressed genes within the mapped interval in the mutants to find candidate genes with more mutations on the candidate genes in different mutants. This method avoids the problem that it is difficult to design genomic specific primers when usually amplifying the candidate gene sequence by PCR, and can quickly obtain the sequence information of candidate genes in different mutants.

[0073] The results are as Figure 3 shown in B of, indicating that among the 15 genes within the fine - mapped interval (i.e., between molecular markers L10 and L13), only 8 genes are expressed (G2, G4, G6, G7, G8, G9, G10, and G15). Further analyze the SNP information of these 8 expressed genes and find that G4 has missense mutations in 3 susceptible mutants (51 - 3, 141 - 3, 209 - 1). Subsequently, use the cDNA of the mutant to clone G4 for sequencing verification and find that the missense mutation really exists. And in the 216 - 2 susceptible mutant, a 6587bp mutation is found at the exon - intron junction of the G4 gene, resulting in the insertion of an 88bp intron and premature termination. Among the 5 identified susceptible mutants, the G4 gene sequences of 4 susceptible mutants have mutations. This result indicates that the candidate gene G4 is very likely to be the powdery mildew resistance gene Pm57 in Aegilops searsii. In addition, it is also found that there are 7 alternative splicing forms of the G4 gene (Figure 3 Among BIF1 - IF7, the full - length alternative splicing form IF1 accounts for 59.8%.

[0074] The G4 nucleotide sequence is as shown in SEQ ID No.1, the amino acid sequence of the G4 protein encoded by it is as shown in SEQ ID No.3, and its CDS is as shown in SEQ ID No.2.

[0075] Partial analysis of the mutants of the Pm57 candidate gene G4 is as Figure 3 shown. In the susceptible mutant 141 - 3, the glycine at the 177th position in the G4 protein amino acid sequence is mutated to glutamate (G177E). In the susceptible mutant 51 - 3, the glycine at the 193rd position in the G4 protein amino acid sequence is mutated to arginine (G193R). In the susceptible mutant 216 - 2, the nucleotide G at the 6587th position in the G4 protein genomic sequence is mutated to A (G6587A). In the susceptible mutant 209 - 1, the glycine at the 903rd position in the G4 protein amino acid sequence is mutated to aspartic acid (G903D).

[0076] III. Transgenic functional verification of powdery mildew candidate genes

[0077] To determine that the resistance of Chinese Spring - Aegilops searsii translocation line 89 - 69 to powdery mildew is determined by the candidate gene G4, transgenic functional verification of the candidate gene was carried out.

[0078] 1. Construction of expression vector

[0079] The coding sequence of the G4 gene (shown in SEQ ID No.2) as shown in sequence 1 of the sequence list was amplified using primers (amplified with G4 - TF / G4 - TR), and then the candidate gene was recombined into linearized pWMB110 according to the method of the Seamless Cloning and Assembly Kit kit of Beijing TransGen Biotech Co., Ltd., and the plant expression vector of the G4 gene was successfully constructed. The plant expression vector of the G4 gene is a vector obtained by inserting a DNA fragment as shown in SEQ ID No.2 after the BamH I restriction site of pWMB110.

[0080] G4 - TF: AGGTCGACTCTAGA GGATCC ATGGCGTCTCCTCGCG

[0081] G4 - TR: AGCTCGGTACCCGG GGATCC CTATTGTGCTGGGTCGTC

[0082] 2. Obtaining of T0 - generation transgenic plants

[0083] The plant expression vector of the G4 gene obtained in Step 1 was introduced into Agrobacterium tumefaciens EHA105. Using this recombinant Agrobacterium, the embryogenic callus of wheat Fielder was genetically transformed, and then cultured to obtain T 0 generation plants. Through PCR transgenic verification, it was found that there were 10 stable overexpressing transgenic lines of G4 among them.

[0084] PCR transgenic verification method: Extract the genomic DNA of the plant leaves. Using the genomic DNA as a template, perform PCR amplification with the primer pair composed of G4-3F and G4-2R. If an amplification product of 344 bp is obtained, the plant is a transgenic plant.

[0085] G4-3F: 5’-AAGTACCGGAATGCCTGAAG-3’;

[0086] G4-2R: 5’-CGGGAAACTCGTTACATGTC-3’.

[0087] III. Identification of powdery mildew resistance of transgenic plants

[0088] The powdery mildew resistance of transgenic plants was identified by using the detached leaf segment method and the method of artificial inoculation with powdery mildew. The highly susceptible powdery mildew wheat Fielder was used as a control. Identify the powdery mildew resistance of the above-mentioned obtained T 0 generation plants and the derived T 1 generation families. Cultivate the seeds of the overexpressing transgenic lines in the T 0 generation plants to obtain plants, which are the T 1 generation families.

[0089] Detached leaf segment method: Take fully expanded wheat leaves, cut them into leaf segments about 2 cm long, place them face up on the filter paper surface in a petri dish (two layers of filter paper are placed in the petri dish, and 50 mg / L benzimidazole is added as a green-keeping agent). After inoculating with powdery mildew, put it into a light incubator (light intensity 2000 lx, temperature 18 °C), with 16 h of light per day. After the susceptible wheat Fielder is fully infected, the powdery mildew resistance of the tested wheat can be recorded.

[0090] Identification by artificial inoculation with powdery mildew: Harvest the seeds of the T 0 generation transgenic lines, plant them in nutrient soil, and culture them in a light incubator at 18 °C, humidity 50%, and photoperiod 16 h / 8 h. When the first leaf of the wheat is fully expanded, inoculate the wheat powdery mildew strain E09 by the method of artificial shaking of powdery mildew spores. After the susceptible wheat Fielder is fully infected, the powdery mildew resistance of the tested wheat can be recorded.

[0091] The results are as Figure 4 shown. Among them, A is the identification of T 0Powdery mildew resistance of the first-generation transgenic plants; Fielder is a highly susceptible powdery mildew receptor control, + indicates that the transgenic identification is a positive plant (i.e., the overexpression transgenic line of G4), - indicates that the transgenic identification is a negative plant; B is the powdery mildew resistance identification of the T 1 -generation transgenic lines; Harvest the seeds of the T 0 -generation transgenic lines, and cultivate to obtain the T 1 -generation families. Conduct disease resistance identification on 10 T 1 -generation families. Among the 10 T 1 -generation families, there are disease-resistant and susceptible single plants in L4. The DNA sequence of G4 was detected in all disease-resistant single plants, while the DNA sequence of G4 was not detected in all susceptible single plants. Fielder is a highly susceptible powdery mildew receptor control, + indicates that the PCR transgenic verification is a positive plant, - indicates that the PCR transgenic verification is a negative plant.

[0092] The results showed that all 10 T 0 -generation overexpression transgenic plants of G4 and their derived positive T 1 -generation family plants are all disease-resistant, while Fielder and negative plants are significantly susceptible, proving that the candidate gene G4 is the powdery mildew resistance gene in Aegilops searsii.

[0093] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A protein, characterized in that: the protein is any one of the following proteins: (a1) A protein with an amino acid sequence of SEQ ID No. 3; (a2) A fusion protein obtained by connecting a tag to the end of the protein defined in (a1).

2. Related biological materials of the protein according to claim 1, characterized in that: the related biological materials are any one of the following: c1) A nucleic acid molecule encoding the protein according to claim 1; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

3. The related biological materials according to claim 2, characterized in that: c1) The nucleic acid molecule is any one of the following DNA molecules: d1) A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1; d2) A DNA molecule with a coding sequence shown as SEQ ID NO.

2.

4. The application of the protein according to claim 1, characterized in that: the application is any one of the following: (1) Improving the powdery mildew resistance of plants; (2) Cultivating transgenic plants with enhanced powdery mildew resistance; the plant is a plant of the genus Triticum.

5. The application of the related biological materials according to claim 2 or 3, characterized in that: the application is any one of the following: (1) Improving the powdery mildew resistance of plants; (2) Cultivating transgenic plants with enhanced powdery mildew resistance; the plant is a plant of the genus Triticum.

6. The application according to claim 4 or 5, characterized in that: the plant of the genus Triticum is Triticum Fielder.

7. A method for plant breeding, characterized in that: it includes enhancing and / or increasing the expression and / or the content and / or the activity of the coding gene of the protein according to claim 1 in the starting plant to obtain a plant with the following characteristics: compared with the starting plant, the powdery mildew resistance of the plant is improved; the plant is a plant of the genus Triticum.

8. The method according to claim 7, characterized in that: the plant of the genus Triticum is Triticum Fielder.

9. A method for preparing a transgenic plant, characterized in that: it includes the step of introducing a substance that enhances and / or increases the expression and / or the content and / or the activity of the coding gene of the protein according to claim 1 into the starting plant to obtain a transgenic plant; compared with the starting plant, the transgenic plant has the following characteristics: compared with the starting plant, the powdery mildew resistance of the transgenic plant is improved; the plant is a plant of the genus Triticum.

10. The preparation method according to claim 9, characterized in that: the substance that enhances and / or increases the expression and / or the content and / or the activity of the coding gene of the protein according to claim 1 is any one of the biological materials in c1)-c4) of claim 2.

11. The preparation method according to claim 10, characterized in that: the wheat plant is wheat Fielder.

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