Cloning and application of wheat broad-spectrum multi-resistance gene WAI-B2

Abstract

The invention discloses cloning and application of a wheat broad-spectrum multi-resistance gene WAI-B2, and belongs to the technical field of genetic engineering breeding. The technical problem to be solved by the invention is how to improve the stress resistance of plants, for example, how to improve the disease resistance of wheat. In order to solve the technical problems, in the first aspect, the invention provides application of the protein or a substance for regulating gene expression or a substance for regulating the activity or content of the protein in regulation of plant stress resistance, the protein is coded by the gene, and the protein is WAI-B2 protein. The invention provides a biological function identification method for gene positioning, map-based cloning and disease resistance of a wheat stress resistance gene WAI-B2. The wheat stress resistance gene WAI-B2 can be widely applied to the plant fields of wheat disease-resistant genetic breeding, germplasm resource improvement, transgenosis, genome editing breeding and the like, and plays an important role in improving germplasm resources of crops such as wheat and the like.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering breeding technology, and specifically relates to the cloning and application of the wheat broad-spectrum multi-resistance gene WAI-B2. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the world's major staple crops. Breeding disease-resistant wheat varieties is a key goal of wheat breeding and the most cost-effective approach to combating wheat disease threats. Cloning disease-resistance genes and elucidating their mechanisms provide materials and theoretical foundations for wheat disease-resistance breeding. Most cloned disease-resistance genes in recent years encode nucleotide-binding and leucine-rich repeat (NLR) proteins. These NLR-type disease-resistance proteins confers resistance to a single or limited number of specific pathogenic races. However, widespread cultivation of varieties containing genes specific for a single race can lead to targeted selection, causing rare pathogenic races that are pathogenic to these genes to become dominant, resulting in a loss of disease resistance and the subsequent spread of disease. Therefore, it is necessary to identify broad-spectrum disease-resistance resources, clone multiple resistance genes with multiple effects, and synthesize multiple resistance genes to breed new, broad-spectrum, and durable disease-resistant varieties.

[0003] Wheat autoimmune (WAI) mutants exhibit characteristic histological and cytological features of disease resistance, such as elevated salicylic acid levels, a burst of reactive oxygen species, and the activation of resistance-related genes. These mutants trigger systemic disease resistance, inducing the activation of other defense mechanisms to protect against foreign pathogens. These mutants exhibit broad-spectrum resistance (BSR) to pathogens and resistance to multiple pathogens. These WAI proteins are often closely associated with cell development, apoptosis, and plant defense responses to disease and stress. This characteristic makes them advantageous for crop resistance breeding, making them important candidates for broad-spectrum disease resistance. Cloning the WAI gene, elucidating the mechanisms by which it activates immune responses to produce necrosis, and its broad-spectrum resistance to pathogens are crucial for understanding the mechanisms of programmed cell death (PCD) and for wheat disease resistance breeding. Summary of the Invention

[0004] The technical problem to be solved by this application is: how to improve the stress resistance of plants, for example, how to improve the disease resistance of wheat.

[0005] To solve the above technical problems, the present application provides the use of a protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is WAI-B2 protein;

[0006] A1) Application in regulating plant stress resistance;

[0007] A2) Application in the preparation of products for regulating plant stress resistance;

[0008] A3) Application in regulating plant powdery mildew resistance;

[0009] A4) Application in the preparation of products for regulating plant powdery mildew resistance;

[0010] A5) Application in regulating plant stem rust disease resistance;

[0011] A6) Application in the preparation of products for regulating plant stem rust resistance;

[0012] A7) Application in regulating plant stripe rust resistance;

[0013] A8) Application in the preparation of products for regulating plant stripe rust resistance;

[0014] A9) Application in regulating plant leaf rust resistance;

[0015] A10) Application in the preparation of products for regulating plant leaf rust resistance;

[0016] A11) Application in plant breeding or plant-assisted breeding;

[0017] The WAI-B2 protein may be any of the following proteins:

[0018] a1) The amino acid sequence is the protein shown in SEQ ID No. 2;

[0019] a2) a protein related to plant stress resistance, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in a1), which has more than 80% identity with the amino acid sequence shown in a1);

[0020] a3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

[0021] Furthermore, in the application, the regulation may be improvement, promotion or upregulation.

[0022] Furthermore, in the application, the evaluation index of plant breeding may be plant stress resistance.

[0023] Furthermore, in the application, the purpose of the plant breeding includes cultivating stress-resistant plants.

[0024] Furthermore, the cultivation of stress-resistant plants may be the cultivation of disease-resistant plants.

[0025] Furthermore, the cultivating of disease-resistant plants may be cultivating plants with improved disease resistance.

[0026] Furthermore, the improved disease resistance may be improved powdery mildew resistance and / or improved rust resistance.

[0027] Furthermore, the rust disease is selected from: stem rust, stripe rust or leaf rust.

[0028] Furthermore, in the application, the protein is derived from wheat.

[0029] In the present application, SEQ ID No. 2 consists of 451 amino acid residues.

[0030] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0031] A protein tag is a polypeptide or protein that is fused and expressed with a target protein using in vitro DNA recombination techniques to facilitate the expression, detection, tracing, and / or purification of the target protein. Examples of protein tags include Flag, His, MBP, HA, myc, GST, GFP, and / or SUMO tags.

[0032] Furthermore, the connection in a3) may be via a peptide bond.

[0033] Furthermore, in the application, the substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material, and the biological material can be any of the following:

[0034] B1), a nucleic acid molecule encoding the WAI-B2 protein described in the above application;

[0035] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0036] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0037] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0038] 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), or a transgenic plant cell line containing the recombinant vector described in B3);

[0039] B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0040] B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2) or a transgenic plant organ containing the recombinant vector described in B3).

[0041] Furthermore, in the relevant biological materials used, the expression cassette described in B2) refers to a DNA capable of expressing the WAI-B2 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the WAI-B2 protein encoding gene, but also a terminator and / or enhancer sequence for terminating transcription of the WAI-B2 protein encoding gene.

[0042] Among the above-mentioned related biological materials, the recombinant microorganisms mentioned in B3) can specifically be yeast, bacteria, algae and fungi.

[0043] Among the above-mentioned relevant biological materials, the plant tissues described in B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0044] Among the above-mentioned relevant biological materials, the transgenic plant organs described in B7) can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.

[0045] Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation materials.

[0046] Furthermore, in the application, the nucleic acid molecule in B1) may be a DNA molecule as described in any one of g1) to g3) below:

[0047] g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3;

[0048] g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 1, 2474-3931;

[0049] g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

[0050] SEQ ID No. 1 includes 2473 bp upstream of the start codon (positions 1-2473 of SEQ ID No. 1), a full-length gene of 1458 bp (positions 2474-3931 of SEQ ID No. 1, wherein positions 2474-3055 of SEQ ID No. 1 are the first exon sequence, positions 3056-3157 are the intron sequence, and positions 3158-3931 are the second exon sequence) and 1260 bp downstream of the stop codon (positions 3932-5191).

[0051] Furthermore, in the application, the plant can be selected from monocotyledonous plants.

[0052] Furthermore, in the application, the monocotyledonous plant can be selected from the grass family.

[0053] Furthermore, in the application, the grass plant can be selected from the genus Triticum.

[0054] Furthermore, in the application, the Triticum plant can be selected from wheat (Triticum aestivum L.).

[0055] Furthermore, in the application, plant stress resistance may be plant disease resistance.

[0056] Furthermore, in the application, the disease resistance may specifically be resistance to powdery mildew and rust.

[0057] Specifically, the rust resistance may be resistance to stem rust, resistance to stripe rust and / or resistance to leaf rust.

[0058] In this application, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0059] The aforementioned 80% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0060] The 80% or greater identity may 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. The 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0061] To solve the above technical problems, the present application also provides a method for improving plant stress resistance, which comprises regulating the plant stress resistance by regulating the expression of the above WAI-B2 protein or regulating the activity or content of the WAI-B2 protein in the recipient plant.

[0062] Furthermore, in the method, the method includes increasing the expression of the WAI-B2 protein in the recipient plant or increasing the activity or content of the WAI-B2 protein to improve the stress resistance of the plant.

[0063] Furthermore, in the method described above, increasing the expression of the WAI-B2 protein in the recipient plant or increasing the activity or content of the WAI-B2 protein can be achieved by introducing a nucleic acid molecule encoding the WAI-B2 protein into the recipient plant.

[0064] Furthermore, in the method described above, the nucleic acid molecule may be a DNA molecule described in any one of g1) to g3) below:

[0065] g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3;

[0066] g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 1;

[0067] g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

[0068] Furthermore, in the method, the plant can be selected from monocotyledonous plants.

[0069] Furthermore, in the method, the monocotyledonous plant can be selected from the grass family.

[0070] Furthermore, in the method described above, the grass plant can be selected from the genus Triticum.

[0071] Furthermore, in the method described above, the Triticum plant can be selected from wheat (Triticum aestivum L.).

[0072] To solve the above technical problems, the present application also provides a method for wheat breeding, which comprises increasing the expression of the WAI-B2 protein in the recipient plant or increasing the activity or content of the WAI-B2 protein to obtain a target plant with improved stress resistance.

[0073] Furthermore, the method increases the expression of the WAI-B2 protein or increases the activity or content of the WAI-B2 protein in the recipient plant by introducing the nucleic acid molecule encoding the WAI-B2 protein into the recipient plant.

[0074] Furthermore, in the method described above, the nucleic acid molecule may be a DNA molecule described in any one of g1) to g3) below:

[0075] g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3;

[0076] g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 1;

[0077] g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

[0078] Furthermore, in the present application, the plant stress resistance may be plant disease resistance.

[0079] Furthermore, in the present application, the disease resistance may specifically be resistance to powdery mildew and resistance to rust.

[0080] Specifically, in the present application, the rust resistance may be resistance to wheat stem rust, resistance to wheat stripe rust and / or resistance to wheat leaf rust.

[0081] In order to solve the above technical problems, the present application also provides the above protein and / or the above biological material.

[0082] The beneficial technical effects achieved by this application are as follows:

[0083] This application provides methods for gene localization, map-based cloning, and biological function identification of disease resistance of the wheat stress resistance gene WAI-B2. In some embodiments of this application, introducing the stress resistance gene WAI-B2 into recipient wheat can significantly improve the disease resistance of wheat. The wheat stress resistance gene WAI-B2 can be widely used in plant fields such as wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and modifying the germplasm resources of crops such as wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Mutant 8P4087 exhibits autoimmune necrosis, in which Figure 1 Middle A shows the phenotype of the F1 generation and 8P4087 obtained by hybridization of ND399 and AK58 / 8P4087 at the adult stage. The scale bar is 5 cm. Figure 1 Middle B shows the leaf phenotype of the F1 generation and 8P4087 obtained by hybridizing ND399 and AK58 / 8P4087. The scale bar is 1 cm. Figure 1 Middle C shows the results of diaminobenzidine (DAB) staining, with a scale bar of 200 μm; Figure 1 Middle D shows the result of trypan blue staining, and the scale bar is 200 μm.

[0085] Figure 2 The distribution of candidate SNPs obtained by BSR-Seq on chromosomes.

[0086] Figure 3 Fine positioning and map-based cloning of WAI-B2.

[0087] Figure 4 This figure shows the functional verification of WAI-B2 transgene. The scale bar represents 1 cm.

[0088] Figure 5 This figure shows the resistance identification of WAI-B2 transgenic positive plants to powdery mildew, stem rust, leaf rust and stripe rust. The scale bar is 1 cm. DETAILED DESCRIPTION

[0089] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0090] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0091] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0092] The plant expression vector pCAMBIA1300 in the following examples is deposited by the applicant and disclosed in the document "(Lu et al. (2020) A rare gain of function mutation in a wheat tandem kinase confersresistance to powdery mildew. Nat. Commun. 11, 680)". The public can obtain the above-mentioned biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

[0093] The powdery mildew physiological race was kindly donated by Yu Dazhao's research group at the Institute of Plant Protection and Soil and Fertilizer of Hubei Academy of Agricultural Sciences and was published in the document "Lu et al., A rare gain of function mutation in a wheat tandem kinase confersresistance to powdery mildew. Nature Communication, 2020, 11, 680". The public can obtain the above-mentioned biological materials from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

[0094] The powdery mildew-susceptible variety Xue Zao and the transgenic receptor material Fielder are preserved in this laboratory and disclosed in the document "Lu et al., A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nature Communication, 2020, 11, 680". The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

[0095] The physiological species of stem rust fungus was kindly donated by Chen Shisheng's research group at the Modern Agriculture Research Institute of Peking University and was published in the document "Li et al., Mapping and Characterization of a Wheat Stem Rust Resistance Gene inDurum Wheat "Kronos". Front Plant Sci. 2021Oct 15; 12: 751398." The public can obtain the above-mentioned biological materials from the Modern Agriculture Research Institute of Peking University. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

[0096] The stripe rust physiological race was kindly donated by Du Jiuyuan's research group at the Wheat Research Institute of Gansu Academy of Agricultural Sciences and is published in the document "Bai Bin et al., Current status and strategies of utilization of stripe rust resistance genes in winter wheat breeding in the Northwest stripe rust source area. Chinese Agricultural Sciences, 2024, DOI: 10.3864 / j.issn.0578-1752.2024.01.002". The public can obtain the above-mentioned biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.

[0097] The leaf rust physiological race was kindly donated by Li Zaifeng's research group at Hebei Agricultural University and is disclosed in the document "Zhang, et al., QTL mapping of adult-plant resistance to leaf and stripe rust in wheat cross SW8588 / Thatcher using the wheat 55K SNP array. Plant Disease, 2019, 103: 3041-3049". The public can obtain the above-mentioned biological material from the applicant. The obtained biological material is only used for repeating the experiments of this application and cannot be used for other purposes.

[0098] The powdery mildew-susceptible variety Xue Zao and the transgenic receptor material Fielder are preserved in this laboratory and published in the document "Lu et al., A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nature Communication, 2020, 11, 680". The public can obtain the above-mentioned biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

[0099] The highly susceptible stem rust fungus variety Rusty was kindly donated by Chen Shisheng's research group at the Modern Agriculture Research Institute of Peking University and was published in the document "Li et al., Mapping and Characterization of a Wheat Stem Rust Resistance Gene in Durum Wheat "Kronos". Front Plant Sci. 2021Oct 15; 12: 751398." The public can obtain the above-mentioned biological materials from the Modern Agriculture Research Institute of Peking University. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

[0100] The highly susceptible stripe rust cultivar Mingxian 169 is maintained in our laboratory and published in the paper "Mapping stripe rust resistance gene YrZH22 in Chinese wheat cultivar Zhoumai 22 by bulked segregant RNA-Seq (BSR-Seq) and comparative genomics analyses. Theoretical and Applied Genetics, 2017, 2191-2201." The public can obtain the aforementioned biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological materials are for use only in repeating the experiments described in this application and may not be used for other purposes.

[0101] The highly susceptible leaf rust variety Zhengzhou 5389 was kindly donated by Li Zaifeng's research group at Hebei Agricultural University and is disclosed in the document "Zhang, et al., QTL mapping of adult-plant resistance to leaf and stripe rustin wheat cross SW 8588 / Thatcher using the wheat 55K SNP array. Plant Disease, 2019, 103: 3041-3049". The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.

[0102] Example 1: WAI-B2 gene localization and functional verification

[0103] 1.1. Phenotypic analysis of the autoimmune mutant 8P4087

[0104] The wheat autoimmune mutant 8P4087 was obtained by EMS mutagenesis of the common wheat Nongda 399 (ND399). Under natural conditions, compared with the wild type ND399, the leaves of 8P4087 showed an autoimmune necrosis phenotype during the seedling stage. The necrotic spots first appeared at the tip of the leaf and then expanded to the middle and base of the leaf. As the wheat grew and developed, the size and number of necrotic spots gradually increased. The F1 generation was obtained by hybridizing AK58 with normal leaves with 8P4087. The number and degree of necrotic spots on the leaves of the F1 generation of hybrids were weaker than those of 8P4087. The results of trypan blue and diaminobenzidine (DAB) staining showed that cell death occurred in the tissues at the necrotic spot site and was accompanied by the accumulation of peroxides ( Figure 1 In the laboratory, low temperature (16°C) is required to produce necrotic spots.

[0105] 1.2. WAI-B2 positional cloning

[0106] The F1 generation obtained in 1.1 was self-pollinated to generate a mapping population consisting of 809 F2 plants. All experimental materials were sown at the original seed farm in Gaoyi County, Shijiazhuang City, Hebei Province, and phenotypic analysis was performed at the mature stage. The results showed that among the 809 F2 families, 201 had normal leaves, 420 showed segregation, and 188 showed immune necrotic spots. The chi-square test showed that the segregation ratio of these families was consistent with a 1:2:1 segregation ratio, consistent with single-gene control (Table 1).

[0107] Table 1 Genetic analysis of the wheat broad-spectrum multi-resistance gene WAI-B2

[0108]

[0109] χ 2 0.05 =5.991,df=2

[0110] The above genetic analysis showed that the immune autoactivation phenotype in mutant 8P4087 is controlled by an incompletely dominant single gene, temporarily named WAI-B2. To locate this gene, leaves from 30 homozygous normal plants and 30 homozygous necrotic plants in the F2 population were used to construct bulk pools Bulk-normal-4B and Bulk-autoimmunity-4B, respectively. Transcriptome sequencing was performed on the Illumina HiSeq4000 platform. After analysis, 22 candidate SNPs associated with the target trait were finally obtained (allele frequency difference (AFD)>0.8 and Fisher's exact test P-value<1e-10), 19 of which were located on the long arm of chromosome 4B and 3 on the long arm of chromosome 2B ( Figure 2 ).

[0111] To further develop molecular markers, 117 SSR or EST primer pairs were developed using the target interval Chinese Spring reference sequence (http: / / www.wheatgenome.org / ). Screening revealed that five primer pairs (BW1-BW5) were polymorphic between the parents and the two DNA pools. Genotyping was performed using the polymorphic primers in an F2 population of 809 individuals. Ultimately, WAI-B2 was mapped to a genetic interval of 0.012 cM between molecular markers BW2 and BW5, corresponding to a physical interval of 205 kb on the chromosome arm of the Chinese Spring RefSeq v1.04BL. Figure 3 ). Gene annotation results showed that a total of 8 high-confidence genes were annotated in this interval. Expression analysis and sequence amplification comparison revealed that only one high-confidence gene, TraesCS4B01G392100, was different between the parents. The gene coding region was 1356 bp long and contained 2 exons. The gene had a transmembrane domain and a single transmembrane domain. Figure 3 ). Sequences of wild-type ND399 and mutant 8P4087 were amplified using primers BDY4, BDYg2-1, and BDYH19. Sequence comparison analysis revealed that compared with the wild-type, TraesCS4B01G392100 had a C-to-T SNP mutation in the exon of mutant 8P4087, resulting in a change of amino acid 425 from leucine to phenylalanine ( Figure 3 ).

[0112] Table 2 Primer sequences used in this study

[0113]

[0114] 1.3 WAI-B2 Functional Verification

[0115] Sequence amplification was performed using the adapter-added primer BDY-COM and a high-fidelity DNA polymerase. After sequencing verification, a 5191 bp DNA fragment ProWAI-B2:WAI-B2 (SEQ ID No. 1) was obtained from the mutant 8P4087, wherein SEQ ID No. 1 includes 2473 bp upstream of the start codon (SEQ ID No. 1 positions 1-2473), a full gene length of 1458 bp (SEQ ID No. 1 positions 2474-3931, wherein SEQ ID No. 1 positions 2474-3055 are the first exon sequence, positions 3056-3157 are the intron sequence, and positions 3158-3931 are the second exon sequence), and 1260 bp downstream of the stop codon (positions 3932-5191). The pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit was used to replace the fragment between the BamH I and Hind III restriction enzyme recognition sites of the pCAMBIA1300 vector with the DNA fragment ProWAI-B2:WAI-B2, while keeping the other sequences of the pCAMBIA1300 vector unchanged to obtain the recombinant expression vector pCAMBIA1300-ProWAI-B2:WAI-B2. After the recombinant plasmid was transformed into Escherichia coli, a single clone was picked for sequencing verification. The recombinant expression vector pCAMBIA1300-ProWAI-B2:WAI-B2 expresses the WAI-B2 protein with an amino acid sequence of SEQ ID No. 2, and the open reading frame (ORF) of the WAI-B2 gene is an RNA molecule with a nucleotide sequence of SEQ ID No. 3 (sequence shown in Table 3).

[0116] Table 3 Nucleotide and amino acid sequences of WAI-B2

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] After verification, the recombinant expression vector pCAMBIA1300-ProWAI-B2:WAI-B2 was introduced into callus tissue of the recipient wheat variety Fielder using Agrobacterium-mediated genetic transformation. Seedlings differentiated from the callus were transferred to growth screening medium and cultured at 22-24°C under light conditions. Regenerated wheat plants transfected with pCAMBIA1300-ProWAI-B2:WAI-B2 (referred to as T0-generation transgenic plants carrying the WAI-B2 gene) were obtained. Genomic DNA was extracted from the transgenic wheat to be tested and used as a template for PCR amplification using primers BDYCOM1 and BDYCOM2. The recombinant expression plasmid pCAMBIA1300-ProWAI-B2:WAI-B2 was used as a positive control, and the genome of wheat Fielder was used as a negative control. The expected amplified product fragment size was approximately 550 bp. The PCR reaction procedure was as follows: 94°C pre-denaturation for 5 minutes; 35 cycles of 94°C denaturation for 30 seconds, 58°C annealing for 30 seconds, and 72°C extension for 30 seconds; and 72°C extension for 10 minutes. PCR amplification products were detected by 1% agarose gel electrophoresis, and UV-photographed and recorded. Three T0-generation transgenic-positive plants were obtained by PCR testing and named WAI-B2-L1, WAI-B2-L2, and WAI-B2-L3. Compared with the control Fielder, the leaves of the transgenic-positive plants showed necrotic spots at the seedling stage when cultured at 16°C ( Figure 4 The T0 generation transgenic positive plants were self-pollinated to obtain T1 generation transgenic positive plants. The genotype and phenotype of the transgenic T1 generation were further identified. Twelve individual plants were selected for genotypic and phenotypic identification for each transgenic line. The results showed that the leaves of all transgenic positive plants showed immune necrotic spots at the seedling stage when cultured at 16°C, while the leaves of negative plants showed normal appearance (Table 4).

[0123] Table 4 Genotype and phenotype identification results of WAI-B2 transgenic families

[0124]

[0125] 1.4. Identification of WAI-B2 resistance to powdery mildew and rust

[0126] Disease resistance was assessed in the T2 generation of transgenic homozygous lines using a mixture of powdery mildew, stem rust, stripe rust, and leaf rust races in a greenhouse. Individual wheat plants from each transgenic line were randomly selected for resistance to the four races (powdery mildew, stem rust, stripe rust, and leaf rust). The recipient material, Fielder, served as a susceptible control.

[0127] Among them, the T0 generation transgenic positive plants WAI-B2-L1, WAI-B2-L2 and WAI-B2-L3 were self-pollinated to obtain the T1 generation transgenic positive plants, and the T1 generation transgenic positive plants were self-pollinated to obtain the T2 generation transgenic positive plants WAI-B2-L1, WAI-B2-L2 and WAI-B2-L3.

[0128] 1.4.1 Powdery mildew inoculation and identification methods

[0129] Xuezao, a wheat material highly susceptible to powdery mildew, was used as a control and as a material for propagating powdery mildew. Prior to planting the identified materials, the induced material, Xuezao, was planted in 10-cm-diameter seedling pots, which served as propagation pots. Five powdery mildew races (E09, E21, HB-24, 3-53, and 5-83) kindly donated by Yu Dazhao's research group at the Institute of Plant Protection and Soil and Fertilizer, Hubei Academy of Agricultural Sciences, were inoculated with equal numbers of Xuezao plants to fully infect the disease. Planting and inoculation were continued throughout the process to ensure a constant supply of powdery mildew.

[0130] The experiment was divided into an experimental group and a Fielder control group. The wheat tested in the experimental group was the T2 generation transgenic positive plants WAI-B2-L1, WAI-B2-L2 and WAI-B2-L3, while the wheat tested in the Fielder control group was Fielder wheat.

[0131] Each group was equipped with three 32-cm-diameter plastic pots, each containing six test wheat plants. The experimental and Fielder control wheat plants were surrounded by pots of the same size planted with the highly susceptible cultivar Xue Zao. The plants were incubated at 16°C until the jointing stage, after which they were returned to suitable conditions for inoculation. Powdery mildew spores were shaken from an equal number of inoculation pots onto the inoculated leaves of the cultivar in the susceptible control group. After the cultivar became diseased, the wheat plants in the experimental and Fielder control groups were inoculated with the fungus through manual dusting and natural spread. When the cultivar Xue Zao became fully diseased, the severity of the disease in the experimental and Fielder control wheat plants was assessed. The percentage of powdery mildew spore area on the flag leaf to the total leaf area was recorded using a 1-100% scale, with 0 indicating no spores and 100% indicating a complete leaf coverage.

[0132] 1.4.2. Stem rust inoculation and identification methods

[0133] The experiment was divided into two groups: an experimental group and a Fielder control group. The wheat tested in the experimental group was the T2 generation transgenic positive plants WAI-B2-L1, WAI-B2-L2 and WAI-B2-L3, while the wheat tested in the Fielder control group was Fielder wheat.

[0134] The two groups planted three 32-cm-diameter plastic pots, with six test wheat plants in each pot. Pots of the same size with the highly susceptible cultivar Rusty were placed around them and cultured at 16°C until the jointing stage, after which they were returned to conditions suitable for inoculation. The conditions in the artificial climate chamber were a 16-hour photoperiod, 22°C during the day, 20°C at night, and 80-90% humidity. During the jointing stage of wheat, fresh spores of the physiological subspecies 34C3RTGQM of the stem rust fungus were inoculated onto the cultivar Rusty using the manual sweeping method. After inoculation, the plants were kept in the dark and moisturized for 24 hours. 12 days after the cultivar Rusty was inoculated, the wheat in the experimental group and the Fielder control group were inoculated by manual dusting and natural propagation. The severity of disease in the experimental and Fielder control wheat plants was investigated when the induced cultivar Rusty was fully diseased. The leaf rust phenotype of the adult plant was determined by the surface area of the flag leaf covered with spores or lesions, and this was recorded using a 0-100% grading scale, where 0 indicates immunity and no spores, and 100% indicates a leaf covered with spores.

[0135] 1.4.3 Stripe rust inoculation and identification methods

[0136] The experiment was divided into two groups: an experimental group and a Fielder control group. The wheat tested in the experimental group was the T2 generation transgenic positive plants WAI-B2-L1, WAI-B2-L2 and WAI-B2-L3, while the wheat tested in the Fielder control group was Fielder wheat.

[0137] The two groups were each planted in three 32-cm-diameter plastic pots, each with six test wheat plants. Pots of the same size containing the highly susceptible cultivar Mingxian 169 were placed around the plants and incubated at 16°C until the jointing stage, after which they were returned to suitable conditions for inoculation. A mixed stripe rust inoculum was prepared by mixing equal amounts of stripe rust races CYR32, CYR33, CYR34, ZS, and Gui22-1, kindly donated by Du Jiuyuan's research group at the Wheat Research Institute of the Gansu Academy of Agricultural Sciences. The mixed stripe rust inoculum was inoculated into the cultivar Mingxian 169 at the jointing stage. The mixed stripe rust inoculum was prepared by dissolving equal amounts (by mass) of fresh stripe rust spores in water, adding a drop of 0.001% Tween 8, and spraying evenly onto the leaves of the cultivar Mingxian 169. The mixture was then kept in the dark and moisturized for 24 hours. After the cultivar became diseased, the wheat in the experimental and Fielder control groups was inoculated using both manual dusting and natural propagation. Disease resistance in the experimental and Fielder control wheat plants was assessed at the adult stage when the induced varieties were fully diseased. Leaf rust phenotypes in adult plants were determined by the surface area of the flag leaf covered with spores or lesions, using a 0-100% scale, where 0 indicates immunity without spores and 100% indicates a complete spore mass on the leaf.

[0138] 1.4.4 Leaf rust inoculation and identification methods

[0139] The experiment was divided into two groups: an experimental group and a Fielder control group. The wheat tested in the experimental group was the T2 generation transgenic positive plants WAI-B2-L1, WAI-B2-L2 and WAI-B2-L3, while the wheat tested in the Fielder control group was Fielder wheat.

[0140] Two groups planted six test wheat plants in three 32-cm-diameter plastic pots, each surrounded by pots of the highly susceptible wheat variety Zhengzhou 5389. The plants were incubated at 16°C until the jointing stage, after which they were returned to suitable conditions for inoculation. A mixed inoculum of THJS, PGTS, THND, and PHTT, donated by Li Zaifeng's research group at Hebei Agricultural University, was prepared. The wheat plants were inoculated with the induced wheat variety Zhengzhou 5389 at the jointing stage. The inoculum was prepared by mixing equal amounts (by mass) of the THJS races and 2 ml of Tween 20, then adding water to create a spore suspension at a concentration of approximately 2-3 mg / ml. Inoculation was typically performed around 4:00 PM. The suspension was evenly sprayed onto the induced wheat plants using a spray bottle and immediately covered with plastic film to maintain moisture. The film was removed around 10:00 AM the following day. After the induced cultivar became diseased, wheat samples in the experimental and Fielder control groups were inoculated using both manual dusting and natural spread. Resistance to the disease in the adult plant was assessed when the induced cultivar reached full disease. Leaf rust phenotypes in the adult plant were determined by the surface area of the flag leaf covered by spores or lesions, using a 0-100% scale, where 0 indicates immunity without spores and 100% indicates a complete spore coverage.

[0141] The results of the severity survey and analysis showed that after inoculation with mixed races of powdery mildew, stem rust, stripe rust, and leaf rust, the average severity of the WAI-B2 transgenic line was 20%, 10%, 10%, and 15%, respectively, while the average severity of the wild-type wheat Fielder was 80%, 80%, 90%, and 80%, respectively. The results showed that the WAI-B2 transgenic positive plants had good resistance to mixed races of powdery mildew, stem rust, stripe rust, and leaf rust ( Figure 5 ).

[0142] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is WAI-B2 protein; A1) Application in regulating plant stress resistance; A2) Application in the preparation of products for regulating plant stress resistance; A3) Application in regulating plant powdery mildew resistance; A4) Application in the preparation of products for regulating plant powdery mildew resistance; A5) Application in regulating plant stem rust disease resistance; A6) Application in the preparation of products for regulating plant stem rust resistance; A7) Application in regulating plant stripe rust resistance; A8) Application in the preparation of products for regulating plant stripe rust resistance; A9) Application in regulating plant leaf rust resistance; A10) Application in the preparation of products for regulating plant leaf rust resistance; A11) Application in plant breeding or plant-assisted breeding; The WAI-B2 protein is any of the following proteins: a1) The amino acid sequence is the protein shown in SEQ ID No. 2; a2) a protein related to plant stress resistance, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in a1), which has more than 80% identity with the amino acid sequence shown in a1); a3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

2. The use according to claim 1, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any one of the following: B1), a nucleic acid molecule encoding the WAI-B2 protein in the application of 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), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2) or a transgenic plant organ containing the recombinant vector described in B3).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is a DNA molecule as described in any one of g1) to g3) below: g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3; g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 1, 2474-3931; g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is selected from monocotyledonous plants.

5. A method for improving plant stress resistance, characterized in that: The method comprises introducing the nucleic acid molecule encoding the WAI-B2 protein as claimed in claim 2 into a recipient plant to improve the stress resistance of the recipient plant.

6. The method according to claim 5, characterized in that: The nucleic acid molecule is a DNA molecule as described in any one of g1) to g3) below: g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3; g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 1, 2474-3931; g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

7. A method for wheat breeding, characterized in that: The method comprises introducing the nucleic acid molecule encoding the WAI-B2 protein as claimed in claim 2 into a recipient plant, thereby obtaining a target plant having improved stress resistance compared to the recipient plant.

8. The method according to claim 7, wherein: The nucleic acid molecule is a DNA molecule as described in any one of g1) to g3) below: g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3; g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 1, 2474-3931; g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

9. The method according to any one of claims 5 to 8, characterized in that: The plant stress resistance is plant disease resistance.

10. The protein and / or the biomaterial for use according to any one of claims 1 to 4.

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