Cloning and application of wheat broad-spectrum polyantigen gene WAI-A1

Abstract

The application discloses cloning and application of a wheat broad-spectrum polygene WAI-A1 and belongs to the technical field of genetic engineering breeding. The technical problem to be solved by the application is how to improve the stress resistance of plants, for example, how to improve the disease resistance of wheat. In order to solve the above technical problem, in the first aspect, the application provides application of a protein or a substance for regulating gene expression or a substance for regulating the activity or content of the protein in the regulation of the stress resistance of plants, wherein the gene codes the protein, and the protein is WAI-A1 protein. The application provides a gene positioning, map-based cloning and biological function identification method of the wheat stress resistance gene WAI-A1. The wheat stress resistance gene WAI-A1 can be widely applied to the fields of wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding and the like, and has an important role in improving and improving the germplasm resources of crops such as wheat.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering breeding, and particularly relates to cloning of a wheat broad-spectrum multi-resistance gene WAI-A1 and application thereof. BACKGROUND

[0002] Wheat powdery mildew and rust are the main fungal diseases affecting wheat production in China, and once outbreak, will seriously reduce the yield and quality of wheat and cause huge economic losses. At present, the prevention and treatment of wheat powdery mildew and rust mostly adopts the method of spraying pesticides and fungicides. Compared with the large use of pesticides and fungicides, cultivating durable and resistance wheat varieties is the most economical, safe and effective measure for preventing and treating powdery mildew and stripe rust, and the key to cultivating 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, rich genomic resources and research tools accelerate the molecular positioning and cloning of wheat disease-resistant genes. Most of the cloned rust and powdery mildew resistance genes encode nucleotide binding and leucine-rich repeat domain (NLR) proteins. NLR type disease-resistant proteins only have disease resistance to a single or a few specific races of pathogenic fungi, and are very easy to lose disease resistance after being used on a large scale for several years, resulting in a large outbreak of diseases. Due to the large number of pathogenic species causing wheat diseases and limiting yield, a single gene providing resistance to multiple pathogens has important value in crop improvement. SUMMARY

[0004] The technical problem to be solved by the present 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 problem, in a first aspect, the present application provides the use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein in any of the following, the protein being WAI-A1 protein;

[0006] A1) in regulating the stress resistance of a plant;

[0007] A2) in the preparation of a product for regulating the stress resistance of a plant;

[0008] A3) in regulating the powdery mildew resistance of a plant;

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

[0010] A5) in regulating the stripe rust resistance of a plant;

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

[0012] A7) use in modulating leaf rust resistance in plants;

[0013] A8) use in the manufacture of a product for modulating leaf rust resistance in plants;

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

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

[0016] a1) a protein with an amino acid sequence as shown in SEQ ID No. 2;

[0017] a2) a protein with an amino acid sequence as shown in a1) with 80% or more identity to the amino acid sequence as shown in a1) and being associated with plant stress resistance, obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence as shown in a1);

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

[0019] Further, in the application, the modulation can be enhancement or promotion or positive modulation.

[0020] Further, in the application, the purpose of the plant breeding includes breeding plants with stress resistance.

[0021] Specifically, it can be breeding plants with disease resistance, such as powdery mildew and / or rust resistance.

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

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

[0024] The above-mentioned protein can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.

[0025] The protein tag refers to a polypeptide or protein fused and expressed with the target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking 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] Further, in the application, the substance for modulating gene expression or the substance for modulating the activity or content of the protein is a biological material, which can be any one of the following:

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

[0028] B2) an expression cassette comprising the nucleic acid molecule of B1);

[0029] B3) a recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2);

[0030] B4) a recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

[0031] B5) a transgenic plant cell line comprising the nucleic acid molecule of B1), or a transgenic plant cell line comprising the expression cassette of B2), or a transgenic plant cell line comprising the recombinant vector of B3);

[0032] B6) a transgenic plant tissue comprising the nucleic acid molecule of B1), or a transgenic plant tissue comprising the expression cassette of B2), or a transgenic plant tissue comprising the recombinant vector of B3);

[0033] B7) a transgenic plant organ comprising the nucleic acid molecule of B1), or a transgenic plant organ comprising the expression cassette of B2), or a transgenic plant organ comprising the recombinant vector of B3).

[0034] Further, in the biological material related to the above application, the expression cassette of B2) refers to a DNA capable of expressing the WAI-A1 protein in a host cell, which can include not only a promoter for initiating the transcription of the WAI-A1 protein coding gene, but also a terminator or / and enhancer sequence for terminating the transcription of the WAI-A1 protein coding gene.

[0035] In the above biological material, the recombinant microorganism of B3) can be yeast, bacteria, algae and fungi.

[0036] In the above biological material, the plant tissue of B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0037] In the above biological material, the transgenic plant organ of B7) can be roots, stems, leaves, flowers, fruits and seeds of a transgenic plant.

[0038] In the above biological material, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ can or can not include propagation material.

[0039] Further, in the above application, the nucleic acid molecule of B1) can be a DNA molecule as described in any one of g1) to g3):

[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 a DNA molecule of SEQ ID No. 1 from 4634 to 6211;

[0042] g3) the DNA molecule has more than 80% identity to the DNA molecule of g1) or g2), and regulates plant stress resistance.

[0043] wherein SEQ ID No. 1 comprises 4633 bp upstream of the start codon (positions 1-4633 of SEQ ID No. 1), the full length of the gene of 1578 bp (positions 4634-6211 of SEQ ID No. 1, wherein positions 4634-4814 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] Further, in the use, the plant can be selected from any one of the following:

[0045] C1) monocotyledonous plants;

[0046] C2) plants of the family Poaceae;

[0047] C3) plants of the genus Triticum;

[0048] C4) Triticum aestivum L.

[0049] Further, in the use, the plant stress resistance can be plant disease resistance.

[0050] Further, in the use, the disease resistance can be specifically resistance to powdery mildew and rust.

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

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

[0053] In the above use, the identity of 80% or more 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%.

[0054] To solve the above technical problem, in a second aspect, the present application provides a method for improving the stress resistance of a plant, which comprises modulating the stress resistance of a plant by modulating the expression of the above WAI-A1 protein or modulating the activity or content of the above WAI-A1 protein in a recipient plant.

[0055] Further, in the above method, the method comprises improving the stress resistance of a plant by improving the expression of the above WAI-A1 protein or improving the activity or content of the above WAI-A1 protein in a recipient plant.

[0056] To solve the above technical problem, in a third aspect, the present application provides a method for breeding wheat, which comprises improving the stress resistance of a plant by improving the expression of the above WAI-A1 protein or improving the activity or content of the above WAI-A1 protein in a recipient plant.

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

[0058] Further, in the above method, the stress resistance of a plant can be the disease resistance of a plant.

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

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

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

[0062] C1), a monocotyledonous plant;

[0063] C2), a grass plant;

[0064] C3), a wheat plant;

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

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

[0067] The application has the following beneficial technical effects:

[0068] The present application provides a method for genetic mapping, map-based cloning and biological function identification of wheat resistance gene WAI-A1. The wheat resistance gene WAI-A1 can be widely used in wheat disease resistance breeding, germplasm improvement, transgenic and genome editing breeding and other plant fields, and has an important role in improving and improving the germplasm resources of wheat and other crops. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 To show autoimmune necrosis of mutant E291, wherein Figure 1 A is E291, F1 generation obtained by crossing Mo75 / E291, and the adult phenotype of Mo75, with a scale of 5 centimeters; Figure 1 B is E291, F1 generation obtained by crossing Mo75 / E291, and the leaf phenotype of Mo75, with a scale of 1 centimeter; Figure 1 C is the result of trypan blue staining, with a scale of 200 microns; Figure 1 D is the result of diaminobenzidine (DAB) staining, with a scale of 200 microns.

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

[0071] Figure 3 Fine mapping and map-based cloning of WAI-A1.

[0072] Figure 4 Transgenic function verification of WAI-A1.

[0073] Figure 5 Resistance identification of WAI-A1 transgenic positive plants to powdery mildew, leaf rust and stripe rust. DETAILED DESCRIPTION

[0074] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

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

[0076] In the quantitative test in the following examples, three replicates were set, and the results were averaged, unless otherwise specified.

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

[0078] The Erysiphe necator physiological race was donated by the research group of Yu Dazhao of the Plant Protection and Soil Fertilizer Institute of Hubei Academy of Agricultural Sciences, and was disclosed in the literature “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 above-mentioned biological materials can be obtained by the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the above-mentioned biological materials obtained can only be used for repeating the experiments of the application and cannot be used for other purposes.

[0079] The Puccinia striiformis physiological race was donated by the research group of Du Jiuyuan of the Wheat Institute of Gansu Academy of Agricultural Sciences, and was disclosed in the literature “Bai B et al., Current status and strategies of utilization of stripe rust resistance genes in winter wheat breeding in the source area of Puccinia striiformis in Northwest China. Chinese Journal of Agricultural Sciences, 2023, DOI: 10.3864 / j.issn.0578-1752.220611”. The above-mentioned biological materials can be obtained by the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the above-mentioned biological materials obtained can only be used for repeating the experiments of the application and cannot be used for other purposes.

[0080] Leaf rust races were kindly provided by Prof. Li Zaifeng at Hebei Agricultural University, and were disclosed in the literature “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, and the above biological materials obtained are only used for repeating the experiments of the present application and cannot be used for other purposes.

[0081] Powdery mildew highly susceptible induced variety Xuezao and transgenic recipient material Fielder were preserved in our laboratory, and were disclosed in the literature “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 biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the above biological materials obtained are only used for repeating the experiments of the present application and cannot be used for other purposes.

[0082] Stripe rust highly susceptible induced variety Mingxian 169 was preserved in our laboratory, and was disclosed in the literature “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, and the above biological materials obtained are only used for repeating the experiments of the present application and cannot be used for other purposes.

[0083] Leaf rust highly susceptible induced variety Zhengzhou 5389 was gifted by Professor Li Zaifeng of Hebei Agricultural University, which was disclosed in the literature “Zhang, et al., QTL mapping of adult-plant resistance to leaf and stripe rust in wheat cross SW 8588 / Thatcher using the wheat 55K SNP array. Plant Disease, 2019, 103:3041-3049”. The above biological material can be obtained by the applicant, and the above biological material is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0084] Example 1, WAI-A1 gene mapping and function verification

[0085] 1.1, phenotype analysis of autoimmune mutant E291

[0086] Wheat autoimmune mutant E291 was obtained by crossing wild diploid wheat IW172 and Durum wheat Mo75 and selfing the stable progeny line 13-16 for multiple generations. Under natural conditions, compared with wild type 13-16, E291 leaves will appear autoimmune necrosis phenotype at the seedling stage, and the necrosis spots first appear at the leaf tip, and then extend to the middle and base of the leaf. With the growth and development of wheat, the size and number of necrosis spots gradually increase. F1 generation was obtained by crossing normal Mo75 with E291, and the number and degree of necrosis spots on the leaves of F1 generation were weaker than those of E291. The results of trypan blue and diaminobenzidine (DAB) staining showed that the tissues at the necrosis spot position had cell death and peroxide accumulation Figure 1 )。

[0087] 1.2, WAI-A1 map-based cloning

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

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

[0090]

[0091] χ2 0.05 = 5.991, df = 2

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

[0093] To further develop molecular markers, the reference sequence of Chinese Spring (http: / / www.wheatgenome.org / ) in the target interval was used as a template, and a total of 92 pairs of SSR or EST primers were developed. After screening, it was found that 9 pairs of primers (CW1-CW9) had polymorphisms between the parents and the two DNA bulk pools. Using polymorphic primers for genotyping in the F2 population containing 6492 single plants, WAI-A1 was finally located in the genetic interval of 0.015 cM between molecular markers CW6 and CW7, corresponding to the physical interval of 73.32 kb in the long arm of chromosome 5A (5AL) of Chinese Spring RefSeq v1.05 (http: / / www.wheatgenome.org / ). Figure 3 Gene annotation results showed that the interval only contained one high-confidence gene ANK-TM, with a coding region length of 1467 bp, containing 2 exons. The N-terminal of the gene contained 6 ankyrin (ANK) repeats, and the C-terminal contained 5 consecutive transmembrane (TM) helices. Figure 3 Using primers Ln14, Ln3 and Ln10 for sequence amplification of wild type 13-16 and mutant E291, sequence comparison analysis found that compared with the wild type, ANK-TM had a C-to-T SNP mutation in the exon of the mutant E291, resulting in the change of the 282th amino acid from alanine to valine. Figure 3

[0094] Table 2 Primer sequences used in the present study

[0095]

[0096] 1.3, WAI-A1 function verification​​

[0097] An 8084 bp DNA fragment ProWAI-A1:WAI-A1 (SEQ ID No. 1) was obtained from mutant E291 by using the primer Ln-COM with an adaptor, high-fidelity DNA polymerase for sequence amplification, and sequencing verification, wherein SEQ ID No. 1 comprises 4633 bp upstream of the start codon (1-4633 of SEQ ID No. 1), the full length of 1578 bp (4634-6211 of SEQ ID No. 1, wherein 4634-4814 of SEQ ID No. 1 is the first exon sequence, 4815-4925 is the intron sequence, and 4926-6211 is the second exon sequence), and 1873 bp downstream of the stop codon (6212-8084). The fragment ProWAI-A1:WAI-A1 was used to replace the fragment between the BamH I and Hind III enzyme digestion recognition sites of the pCAMBIA1300 vector by using a 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 a recombinant expression vector pCAMBIA1300-ProWAI-A1:WAI-A1. After the recombinant plasmid was transformed into E. coli, a single colony was picked for sequencing verification. The recombinant expression vector pCAMBIA1300-ProWAI-A1:WAI-A1 expressed the WAI-A1 protein 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 (see Table 3 for the sequence).

[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 by Agrobacterium-mediated genetic transformation. The seedlings differentiated from the callus were transferred to the growth screening medium and cultured at 22-24°C under light. The regenerated wheat plants transformed with pCAMBIA1300-ProWAI-A1:WAI-A1 (referred to as T0 generation transgenic plants transformed with 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 for PCR amplification using primers COM1 and COM3, with the recombinant expression plasmid pCAMBIA1300-ProWAI-A1:WAI-A1 as a positive control and the genome of wheat Fielder as a negative control. The expected size of the PCR amplification product was about 330 bp. The PCR reaction program was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 10 min. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the results were recorded by ultraviolet photography. Three T0 generation transgenic positive plants were obtained by PCR detection, which were named WAI-A1-L1, WAI-A1-L2 and WAI-A1-L3, respectively. Compared with the control Fileder, the leaves of the transgenic positive plants showed necrotic spots during the seedling stage. Figure 4 The T0 generation transgenic positive plants were selfed to obtain T1 generation transgenic positive plants, and the genotype and phenotype of the transgenic T1 generation were continuously identified. The results showed that the leaves of all transgenic positive plants showed immune necrotic spots during the seedling stage, while the leaves of the negative plants showed normal (Table 4).

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

[0107]

[0108] 1.4, Resistance identification of WAI-A1 to powdery mildew and rust

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

[0110] T0 generation transgenic positive plants WAI-A1-L1, WAI-A1-L2 and WAI-A1-L3 were selfed to obtain T1 generation transgenic positive plants, and the T1 generation transgenic positive plants were selfed to obtain T2 generation transgenic positive plants.

[0111] 1.4.1, inoculation and identification method of powdery mildew

[0112] The high-susceptible wheat powdery mildew material Xuezao was used as a disease susceptible control and a material for propagating powdery mildew. Before planting the identification material, the inducement material Xuezao was planted in a 10 cm diameter seedling pot as a propagation pot, and 5 physiological races of powdery mildew (E09, E21, HB-24, 3-53 and 5-83) provided by Yu Dazhao of the Plant Protection and Soil Fertilizer Institute of Hubei Academy of Agricultural Sciences were inoculated on the same amount of Xuezao to make it fully diseased, and the Xuezao was continuously planted and inoculated to ensure the supply of powdery mildew. Each transgenic line and the control variety Fielder were planted in 3 plastic pots with a diameter of 32 cm, 6 plants per pot, and the same size pots with high-susceptible inducement variety Xuezao were placed around. At the jointing stage of the transgenic lines and the inducement variety Xuezao, the same amount of powdery mildew spores in the propagation pots were shaken off and inoculated on the leaves of the inducement variety. After the inducement variety Xuezao was fully diseased, the transgenic lines were inoculated by artificial brushing and natural transmission, and the disease severity of the transgenic lines and the disease susceptible control Fielder was investigated when the inducement variety Xuezao was fully diseased. The area of powdery mildew spore heap on the flag leaf accounted for the percentage of the total leaf area, which was recorded using a 1-100% grading standard, wherein 0 represents no spores, and 100% represents that the leaf is covered with spores.

[0113] 1.4.2, inoculation and identification method of stripe rust

[0114] Each transgenic line and control variety Fielder was planted in 3 plastic pots with a diameter of 32 cm, 6 plants in each pot, and the same size pots with susceptible inducing variety Mingxian 169 were placed around. The mixed inoculum of Puccinia striiformis f. sp. tritici CYR32, CYR33, CYR34, ZS and Gui22-1 was prepared by mixing equal amounts of fresh spores in water and then adding 0.001% Tween 8 solution. The mixture was uniformly sprayed on the leaves of the inducing material Mingxian 169 and kept in the dark for 24 hours. After the inducing variety was infected, the transgenic lines and control Fielder were inoculated by artificial brushing and natural transmission. The disease resistance of the transgenic lines and control Fielder was identified when the inducing variety was fully infected. The spore or lesion area on the flag leaf was used for phenotypic identification, and a 0-100% grading standard was used for recording, where 0 represents immunity without spores, and 100% represents the leaf covered with spore piles.

[0115] 1.4.3, Puccinia recondita inoculation and identification method

[0116] Each transgenic line and control variety Fielder was planted in 3 plastic pots with a diameter of 32 cm, 6 plants in each pot, and the same size pots with susceptible inducing variety Zhengzhou 5389 were placed around. The mixed inoculum of Puccinia striiformis f. sp. tritici THJS, PGTS, THND and PHTT was prepared by mixing equal amounts of fresh spores in water and then adding 2 ml Tween 20. The spore suspension was prepared by adding water to a concentration of about 2-3 mg / ml. Inoculation was generally carried out at about 4 pm, the prepared spore suspension was uniformly sprayed on the inducing row with a spray bottle, and immediately covered with plastic film for incubation and humidification. The plastic film was removed at about 10 am the next day. After the inducing variety was infected, the transgenic lines and control Fielder were inoculated by artificial brushing and natural transmission. The disease resistance of the transgenic lines and control Fielder was identified when the inducing variety was fully infected. The spore or lesion area on the flag leaf was used for phenotypic identification, and a 0-100% grading standard was used for recording, where 0 represents immunity without spores, and 100% represents the leaf covered with spore piles.

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

[0118] The above detailed the present application. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application.​

Claims

1. The use of a protein or a substance that enhances or promotes gene expression or a substance that increases the content of said protein in any of the following: said gene encodes said protein, said protein being WAI-A1 protein; A1) Application in improving plant resistance to powdery mildew; A2) Application in the preparation of products that enhance plant resistance to powdery mildew; A3) Application in improving plant resistance to stripe rust; A4) Application in the preparation of products that enhance plant resistance to stripe rust; A5) Application in improving plant resistance to leaf rust; A6) Application in the preparation of products that enhance plant resistance to leaf rust; A7) Application in plant breeding or plant-assisted breeding, wherein the purpose of the plant breeding is to cultivate disease-resistant plants, wherein the disease resistance is resistance to powdery mildew and / or resistance to rust; The WAI-A1 protein is a protein with the amino acid sequence shown in SEQ ID No. 2; The substance that enhances or promotes gene expression or increases protein content is a biological material, and the biological material is any one of the following: B1) The nucleic acid molecule encoding the WAI-A1 protein; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules 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) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3); The rust resistance refers to resistance to stripe rust and / or leaf rust; The plant in question is wheat.

2. The application according to claim 1, characterized in that: B1) The nucleic acid molecule is a DNA molecule whose coding sequence is SEQ ID No.

3.

3. A method for improving plant disease resistance, characterized in that: The method includes increasing the expression of WAI-A1 protein or increasing the content of WAI-A1 protein in recipient plants to improve plant stress resistance; The WAI-A1 protein is a protein with the amino acid sequence shown in SEQ ID No. 2; The disease resistance is resistance to powdery mildew and / or resistance to rust; The rust resistance refers to resistance to stripe rust and / or leaf rust; The plant in question is wheat.

4. A method for wheat breeding, characterized in that: The method includes increasing the expression of WAI-A1 protein or increasing the content of WAI-A1 protein in recipient plants to obtain target plants with improved disease resistance; The WAI-A1 protein is a protein with the amino acid sequence shown in SEQ ID No. 2; The disease resistance is resistance to powdery mildew and / or resistance to rust; The rust resistance refers to resistance to stripe rust and / or leaf rust; The plant in question is wheat.

5. The method according to claim 3 or 4, characterized in that: The method involves introducing a nucleic acid molecule encoding the WAI-A1 protein into a recipient plant to increase the expression or content of the WAI-A1 protein in the recipient plant.

6. The protein and / or the biomaterial in the application of claim 1 or 2.

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