Cloning and application of wheat broad-spectrum multi-resistance gene WAI-A1

Abstract

The invention discloses cloning and application of a wheat broad-spectrum multi-resistance gene WAI-A1, 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, on one hand, 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-A1 protein. The invention provides a biological function identification method for gene positioning, map-based cloning and disease resistance of the wheat stress resistance gene WAI-A1. The wheat stress resistance gene WAI-A1 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 invention belongs to the technical field of genetic engineering breeding, and specifically relates to the cloning and application of a wheat broad-spectrum multi-resistance gene WAI-A1. Background Art

[0002] Wheat powdery mildew and rust are the main fungal diseases that affect wheat production in my country. Once they break out, they will seriously reduce the yield and quality of wheat and cause huge economic losses. At present, the prevention and control of wheat powdery mildew and rust mostly adopts the method of spraying pesticides and fungicides. Compared with the large-scale use of pesticides and fungicides, breeding persistent and resistant wheat varieties is the most economical, safe and effective measure to prevent and control powdery mildew and stripe rust. The key to breeding disease-resistant varieties lies in the excavation and effective use of disease-resistant genes.

[0003] In recent years, with the rapid development of next-generation sequencing technology, abundant genomic resources and research tools have accelerated the molecular localization and cloning of wheat disease resistance genes. Most cloned rust and powdery mildew resistance genes encode nucleotide binding and leucine-rich repeat domain (NLR) proteins. NLR-type disease resistance proteins are only resistant to specific subspecies of a single or a few pathogens. After a few years of large-scale use in production, they are very likely to lose disease resistance and cause a pandemic. Due to the large number of pathogens that cause wheat diseases and limit yields, a single gene that provides resistance to multiple pathogens is of great value in crop improvement. Summary of the invention

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

[0005] In order to solve the above technical problems, in a first aspect, the present invention provides the use of a protein or a substance regulating gene expression or a substance regulating the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is WAI-A1 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 stripe rust resistance;

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

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

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

[0014] A9) Application in plant breeding or plant assisted breeding;

[0015] The WAI-A1 protein may be any of the following proteins:

[0016] a1), the amino acid sequence is the protein shown in SEQ ID No.2;

[0017] a2), a protein related to plant stress resistance, which has more than 80% identity with the amino acid sequence shown in a1) and is obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in a1);

[0018] a3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

[0019] Furthermore, in the application, the regulation may be enhancement, promotion or positive regulation.

[0020] Furthermore, in the application, the purpose of plant breeding includes cultivating stress-resistant plants.

[0021] In particular, plants may be bred for disease resistance, for example resistance to powdery mildew and / or rust.

[0022] Furthermore, in the application, the protein is derived from wheat.

[0023] In the present invention, SEQ ID No. 2 consists of 488 amino acid residues.

[0024] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0025] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0026] Furthermore, in the application, the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material, and the biological material can be any of the following:

[0027] B1), a nucleic acid molecule encoding the WAI-A1 protein described in the above application;

[0028] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0029] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0030] 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);

[0031] 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);

[0032] 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);

[0033] 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).

[0034] Furthermore, in the relevant biological materials used, the expression cassette described in B2) refers to a DNA capable of expressing the WAI-A1 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the WAI-A1 protein encoding gene, but also a terminator and / or enhancer sequence for terminating transcription of the WAI-A1 protein encoding gene.

[0035] Among the above-mentioned related biological materials, the recombinant microorganisms described in B3) can specifically be yeast, bacteria, algae and fungi.

[0036] Among the above-mentioned related biological materials, the plant tissue described in B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0037] Among the above-mentioned related biological materials, the transgenic plant organs described in B7) can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.

[0038] 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.

[0039] Furthermore, in the application, the nucleic acid molecule described in B1) may be a DNA molecule described in any one of g1) to g3) below:

[0040] g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 3;

[0041] g2), the nucleotide sequence of the coding strand is the DNA molecule of SEQ ID No. 1, 4634-6211;

[0042] g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.

[0043] SEQ ID No. 1 includes 4633 bp upstream of the start codon (positions 1-4633 of SEQ ID No. 1), a full gene length of 1578 bp (positions 4634-6211 of SEQ ID No. 1, wherein positions 4634-4814 of SEQ ID No. 1 are the first exon sequence, positions 4815-4925 are the intron sequence, and positions 4926-6211 are the second exon sequence) and 1873 bp downstream of the stop codon (positions 6212-8084).

[0044] Furthermore, in the application, the plant can be selected from any one of the following:

[0045] C1), monocots;

[0046] C2), Gramineae;

[0047] C3), wheat plants;

[0048] C4), wheat (Triticum aestivum L.).

[0049] Furthermore, in the above application, plant stress resistance may be plant disease resistance.

[0050] Furthermore, in the above application, the disease resistance may specifically be resistance to powdery mildew and resistance to rust.

[0051] Specifically, the rust resistance may be resistance to stripe rust and / or resistance to leaf rust.

[0052] In the above application, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0053] In the above applications, the above 80% 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.

[0054] To solve the above technical problems, in a second aspect, the present invention provides a method for improving plant stress resistance, the method comprising regulating the plant stress resistance by regulating the expression of the WAI-A1 protein or regulating the activity or content of the WAI-A1 protein in a recipient plant.

[0055] Furthermore, in the method described above, the method includes increasing the expression of the above-mentioned WAI-A1 protein in the recipient plant or increasing the activity or content of the WAI-A1 protein to improve the stress resistance of the plant.

[0056] To solve the above technical problems, in a third aspect, the present invention provides a method for wheat breeding, which comprises increasing the expression of the WAI-A1 protein in a recipient plant or increasing the activity or content of the WAI-A1 protein to obtain a target plant with improved stress resistance.

[0057] Furthermore, the above method increases the expression of the WAI-A1 protein or increases the activity or content of the WAI-A1 protein in the recipient plant by introducing the above nucleic acid molecule encoding the WAI-A1 protein into the recipient plant.

[0058] Furthermore, in the above method, the plant stress resistance may be plant disease resistance.

[0059] Furthermore, in the above method, the disease resistance may specifically be resistance to powdery mildew and resistance to rust.

[0060] Specifically, the rust resistance may be resistance to stripe rust and / or resistance to leaf rust.

[0061] Furthermore, in the above method, the plant can be selected from any one of the following:

[0062] C1), monocots;

[0063] C2), Gramineae;

[0064] C3), wheat plants;

[0065] C4), wheat (Triticum aestivum L.).

[0066] To solve the above technical problems, in a fourth aspect, the present invention provides the above protein and / or the above biomaterial.

[0067] The beneficial technical effects achieved by the present invention are as follows:

[0068] The present invention provides a method for identifying the genetic location and positional cloning of the wheat stress resistance gene WAI-A1 and the biological function of disease resistance. The wheat stress resistance gene WAI-A1 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

[0069] Figure 1 Mutant E291 exhibits autoimmune necrosis, in which Figure 1 Middle A shows the phenotype of the adult plant of F1 generation and Mo75 obtained by hybridization of E291, Mo75 / E291, and the scale bar is 5 cm; Figure 1 Middle B shows the leaf phenotypes of the F1 generation and Mo75 obtained by hybridization of E291, Mo75 / E291, and the scale bar is 1 cm; Figure 1 Middle C shows the trypan blue staining result, and the scale bar is 200 μm; Figure 1 D in the middle is the result of diaminobenzidine (DAB) staining, and the scale bar is 200 μm.

[0070] Figure 2 The distribution of candidate SNPs obtained by BSR-Seq on chromosomes.

[0071] Figure 3 Fine positioning and positional cloning of WAI-A1.

[0072] Figure 4 Verification of WAI-A1 transgenic function.

[0073] Figure 5 To identify the resistance of WAI-A1 transgenic positive plants to powdery mildew, leaf rust and stripe rust. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0075] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0076] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0077] The plant expression vector pCAMBIA1300 (Lu et al. (2020) A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 11, 680) in the following examples can be obtained by the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0078] 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 above-mentioned biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0079] The physiological subspecies of stripe rust were kindly donated by Du Jiuyuan's research group at the Wheat Research Institute of Gansu Academy of Agricultural Sciences and 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 Science, 2023, DOI: 10.3864 / j.issn.0578-1752.220611". The public can obtain the above biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0080] The physiological subspecies of leaf rust 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 biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0081] 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 above-mentioned biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0082] The highly susceptible variety Mingxian 169 to stripe rust is preserved by our laboratory and published in the document “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 above biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0083] The highly susceptible leaf rust variety Zhengzhou 5389 was donated by Li Zaifeng's research group at Hebei Agricultural University and was 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 material from the applicant. The obtained biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0084] Example 1: WAI-A1 gene localization and functional verification

[0085] 1.1. Phenotypic analysis of the autoimmune mutant E291

[0086] The wheat autoimmune mutant E291 was obtained by hybridizing wild emmer wheat IW172 and durum wheat Mo75 and inducing EMS mutagenesis in the stable progeny line 13-16 after multiple generations of self-pollination. Under natural conditions, compared with the wild type 13-16, E291 leaves showed autoimmune necrosis phenotypes at the seedling stage. Necrotic spots first appeared at the tip of the leaves and then expanded to the middle and base of the leaves. As wheat grew and developed, the size and number of necrotic spots gradually increased. The F1 generation was obtained by hybridizing Mo75 with normal leaves with E291. The number and degree of necrotic spots on the leaves of the F1 generation of hybrids were weaker than those of E291. 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 ).

[0087] 1.2. WAI-A1 positional cloning

[0088] The F1 generation obtained in 1.1 was self-pollinated to obtain a mapping population containing 6720 F2 plants. The F2 plants were further self-pollinated to obtain 6492 F3 families. All experimental materials were sown in the original seed farm of Gaoyi County, Shijiazhuang City, Hebei Province, and phenotypic investigation and analysis were carried out during the adult stage. The survey results showed that among the 6492 F3 families, 1642 families had normal leaves, 3274 families had segregated leaves, and 1576 families had immune necrosis spots. The chi-square test showed that the ratio was consistent with the 1:2:1 segregation ratio controlled by a single gene (Table 1).

[0089] Table 1 Genetic analysis of the wheat broad-spectrum multi-resistance gene WAI-A1

[0090]

[0091] χ2 0.05 =5.991,df=2

[0092] The above genetic analysis showed that the immune autoactivation phenotype in mutant E291 was controlled by an incomplete dominant single gene, temporarily named WAI-A1. To locate the gene, leaves of 35 homozygous normal plants and 35 homozygous necrotic plants in the F2 population were used to construct mixed pools Bulk-normal-5A and Bulk-autoimmunity-5A, respectively. Transcriptome sequencing was performed on the Illumina HiSeq4000 platform. 46,597,269 and 43,515,843 original read pairs were obtained from the two mixed pools, respectively. After analysis, 218 candidate SNPs related to the target trait were finally obtained (allele frequency difference (AFD)>0.8and Fisher's exact test P-value<1e-10), all of which were located on the long arm of chromosome 5A ( Figure 2 ).

[0093] In order to further develop molecular markers, a total of 92 pairs of SSR or EST primers were developed using the Chinese Spring reference sequence (http: / / www.wheatgenome.org / ) of the target interval as a template. After screening, 9 pairs of primers (CW1-CW9) were found to be polymorphic between the parents and the two DNA pools. Genotyping was performed in the F2 population containing 6492 individuals using polymorphic primers, and WAI-A1 was finally located in the genetic interval of 0.015cM between molecular markers CW6 and CW7, corresponding to the physical interval of 73.32kb of the chromosome arm of the Chinese Spring RefSeq v1.05AL ( Figure 3 ). Gene annotation results showed that this interval contained only one high-confidence gene, ANK-TM, with a coding region length of 1467 bp and two exons. The N-terminus of the gene contained six ankyrin (ANK) repeats and the C-terminus contained five continuous transmembrane (TM) helices ( Figure 3 ). Primers Ln14, Ln3 and Ln10 were used to amplify the wild type 13-16 and mutant E291. Sequence comparison analysis revealed that compared with the wild type, ANK-TM had a C-to-T SNP mutation in the exon of mutant E291, which caused the amino acid at position 282 to change from alanine to valine ( Figure 3 ).

[0094] Table 2 Primer sequences used in this study

[0095]

[0096] 1.3 WAI-A1 Functional Verification

[0097] Sequence amplification was performed using the primer Ln-COM with a linker and a high-fidelity DNA polymerase. After sequencing verification, a 8084bp DNA fragment ProWAI-A1:WAI-A1 (SEQ ID No.1) was obtained from the mutant E291, wherein SEQ ID No.1 includes 4633bp upstream of the start codon (positions 1-4633 of SEQ ID No.1), a full gene length of 1578bp (positions 4634-6211 of SEQ ID No.1, wherein positions 4634-4814 of SEQ ID No.1 are the first exon sequence, positions 4815-4925 are the intron sequence, and positions 4926-6211 are the second exon sequence) and 1873bp downstream of the stop codon (positions 6212-8084). The fragment between the BamH I and Hind III restriction enzyme recognition sites of the pCAMBIA1300 vector was replaced with the DNA fragment ProWAI-A1: WAI-A1 using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit, and the other sequences of the pCAMBIA1300 vector were kept unchanged to obtain the recombinant expression vector pCAMBIA1300-ProWAI-A1: WAI-A1. After the recombinant plasmid was transformed into Escherichia coli, a single clone was picked for sequencing verification. The recombinant expression vector pCAMBIA1300-ProWAI-A1: WAI-A1 expressed the WAI-A1 protein with an amino acid sequence of SEQ ID No.2, and the nucleotide sequence of the open reading frame (ORF) of the WAI-A1 gene was SEQ ID No.3 (sequence shown in Table 3).

[0098] Table 3 Nucleotide and amino acid sequences of WAI-A1

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] After verification, the recombinant expression vector pCAMBIA1300-ProWAI-A1:WAI-A1 was introduced into the callus of the recipient wheat variety Fielder using the Agrobacterium-mediated genetic transformation method. The seedlings differentiated from the callus were transferred to the growth screening medium and cultured under light at 22-24°C. The regenerated wheat plants transformed with pCAMBIA1300-ProWAI-A1:WAI-A1 (referred to as the T0 generation transgenic plants with the WAI-A1 gene) were obtained. The genomic DNA of the transgenic wheat to be tested was extracted, and the genomic DNA was used as a template. PCR amplification was performed using primers COM1 and COM3, and the recombinant expression plasmid pCAMBIA1300-ProWAI-A1:WAI-A1 was used as a positive control, and the genome of wheat Fielder was used as a negative control. PCR amplification was performed, and the expected amplified product fragment size was about 330bp. The PCR reaction procedure was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 58℃ renaturation for 30s, 72℃ extension for 30s, 35 cycles; 72℃ extension for 10min. The PCR amplification products were detected by 1% agarose gel electrophoresis, UV photography was performed, and the results were recorded. A total of three T0 generation transgenic positive plants were obtained by PCR detection, which were named WAI-A1-L1, WAI-A1-L2 and WAI-A1-L3. Compared with the control Fileder, the leaves of the transgenic positive plants showed necrotic spots in the adult stage ( Figure 4 ). T0 generation transgenic positive plants were self-pollinated to obtain T1 generation transgenic positive plants, and the genotype and phenotype of the transgenic T1 generation were further identified. 24 plants were selected for each transgenic line for genotype and phenotype identification. The results showed that the leaves of all transgenic positive plants showed immune necrosis spots in the adult stage, while the leaves of negative plants showed normal appearance (Table 4).

[0106] Table 4 Genotype and phenotype identification results of WAI-A1 transgenic families

[0107]

[0108] 1.4. Identification of resistance of WAI-A1 to powdery mildew and rust

[0109] The T2 transgenic positive homozygous lines (WAI1 5A -L1, WAI1 5A -L2 and WAI1 5A -L3) for disease resistance identification. Fifteen plants were randomly selected from each transgenic line for identification of three types of physiological races (powdery mildew physiological races, stripe rust physiological races and leaf rust physiological races), and the recipient material Fielder was used as the susceptible control.

[0110] Among them, the T0 generation transgenic positive plants WAI-A1-L1, WAI-A1-L2 and WAI-A1-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.

[0111] 1.4.1 Powdery mildew inoculation and identification methods

[0112] Xue Zao, a highly susceptible wheat powdery mildew material, was used as a control and material for the propagation of powdery mildew. Before the identification material was planted, the induced material Xue Zao was planted in a 10 cm diameter seedling pot as a propagation pot. The same number of Xue Zao was inoculated with five powdery mildew physiological races (E09, E21, HB-24, 3-53 and 5-83) donated by Yu Dazhao's research group at the Institute of Plant Protection and Soil and Fertilizer of Hubei Academy of Agricultural Sciences to make it fully diseased. During the period, continuous planting and inoculation were carried out to ensure the supply of powdery mildew. Three plastic flower pots with a diameter of 32 cm were used for each transgenic line and control variety Fielder, with 6 plants planted in each pot, and flower pots of the same specifications with the highly susceptible induced variety Xue Zao were placed around. During the jointing stage of the transgenic line and the induced variety Xue Zao, the powdery mildew spores in the same number of propagation pots were shaken off onto the inoculated leaves of the induced variety. After the induced varieties became diseased, the transgenic lines were inoculated with fungi by artificial dusting and natural propagation. When the induced variety Xue Zao was fully diseased, the severity of the disease in the transgenic lines and the susceptible control Fielder was investigated, and the percentage of the powdery mildew spore pile area on the flag leaf to the total leaf area was recorded using a grading standard of 1-100%, where 0 means no spores and 100% means the leaves are covered with spores.

[0113] 1.4.2. Stripe rust inoculation and identification methods

[0114] Each transgenic line and control variety Fielder were placed in three plastic flower pots with a diameter of 32 cm, and six plants were planted in each pot. Pots of the same size with the highly susceptible induced variety Mingxian 169 were placed around them. The stripe rust physiological races CYR32, CYR33, CYR34, ZS and Gui22-1 donated by Du Jiuyuan's research group at the Wheat Research Institute of Gansu Academy of Agricultural Sciences were mixed in equal amounts to prepare a mixed stripe rust inoculation solution, and the induced variety Mingxian 169 was inoculated during the jointing stage of wheat. The preparation method of the mixed stripe rust inoculation solution is as follows: dissolve the equal amounts of mixed fresh stripe rust spores in water, drop 0.001% Tween solution (Tween8), mix well and evenly spray on the leaves of the induced material Mingxian 169, and keep it moist in the dark for 24 hours. After the induced variety became ill, the transgenic line and the control Fielder were inoculated by artificial dusting and natural propagation. Disease resistance identification was carried out in the adult stage when the induced variety was fully ill. The leaf rust phenotype of adult plants was identified based on the surface area of ​​the flag leaf occupied by spores or lesions, which was recorded using a 0-100% grading scale, where 0 means no spores and 100% means the leaf is covered with spores.

[0115] 1.4.3 Leaf rust inoculation and identification methods

[0116] Each transgenic line and control variety Fielder were planted in three plastic flower pots with a diameter of 32 cm, with 6 plants planted in each pot, and flower pots of the same size with the highly susceptible induced variety Zhengzhou 5389 were placed around. The leaf rust physiological species THJS, PGTS, THND and PHTT donated by Li Zaifeng's research group at Hebei Agricultural University were mixed in equal amounts to prepare a leaf rust mixed inoculum, and the induced variety Zhengzhou 5389 was inoculated during the jointing stage of wheat. The preparation method of the leaf rust mixed inoculum is as follows: the leaf rust physiological species and 2ml Tween 20 were mixed in equal amounts, and water was added to prepare a spore suspension, and the concentration of the suspension was about 2-3mg / ml. Inoculation was generally carried out at around 4 pm, and the prepared bacterial suspension was evenly sprayed on the induced line with a spray bottle, and immediately covered with a plastic film for heat preservation and moisture retention, and the plastic film was uncovered at around 10 am the next day. After the induced variety became ill, the transgenic line and the control Fielder were inoculated by artificial dusting and natural propagation. When the induced variety is fully diseased, the transgenic line and the susceptible control Fielder are tested for resistance in the adult stage. The leaf rust phenotype of the adult plant is identified by the surface area of ​​the flag leaf occupied by spores or lesions, which is recorded using a 0-100% grading scale, where 0 means immunity without spores and 100% means the leaf is covered with spores.

[0117] The results of the severity survey and analysis showed that the average severity of WAI-A1 transgenic lines after inoculation with mixed physiological races of powdery mildew, stripe rust and leaf rust was 10%, 20% and 20% respectively, while the average severity of the transgenic recipient variety Fielder was 80%, 90% and 80% respectively. The results showed that WAI-A1 transgenic positive plants have good resistance to mixed physiological races of powdery mildew, stripe rust and leaf rust ( Figure 5 ).

[0118] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance regulating gene expression or a substance regulating the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is WAI-A1 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 stripe rust resistance; A6) Application in the preparation of products for regulating plant stripe rust resistance; A7) Application in regulating plant leaf rust resistance; A8) Application in the preparation of products for regulating plant leaf rust resistance; A9) Application in plant breeding or plant assisted breeding; The WAI-A1 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, which has more than 80% identity with the amino acid sequence shown in a1) and is obtained by replacing and / or deleting and / or adding amino acid residues of 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 regulating gene expression or the substance regulating protein activity or content is a biological material, and the biological material is any one of the following: B1), a nucleic acid molecule encoding the WAI-A1 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 the DNA molecule of SEQ ID No. 1, 4634-6211; 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 any one of the following: C1), monocots; C2), Gramineae; C3), wheat plants; C4), wheat (Triticum aestivum L.).

5. A method for improving plant stress resistance, characterized in that: The method comprises regulating the stress resistance of plants by regulating the expression of the WAI-A1 protein in the application of claim 1 or regulating the activity or content of the WAI-A1 protein in a recipient plant.

6. The method according to claim 5, characterized in that: The method comprises increasing the expression of the WAI-A1 protein in the application of claim 1 in a recipient plant or increasing the activity or content of the WAI-A1 protein to improve plant stress resistance.

7. A method for wheat breeding, characterized in that: The method comprises increasing the expression of the WAI-A1 protein in the application of claim 1 in a recipient plant or increasing the activity or content of the WAI-A1 protein to obtain a target plant with improved stress resistance.

8. The method according to any one of claims 5 to 7, characterized in that: The method increases the expression of the WAI-A1 protein in the application of claim 1 or increases the activity or content of the WAI-A1 protein in the recipient plant by introducing the nucleic acid molecule encoding the WAI-A1 protein in claim 2 into the recipient plant.

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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