Application of wheat cell wall related kinase TaWAK-4A in cultivating gibberellic disease resistant plants

Abstract

The invention provides application of a TaWAK-4A gene in cultivation of a gibberellic disease resistant plant or application of the TaWAK-4A gene in regulation and control of gibberellic disease resistance of the plant, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the TaWAK-4A gene is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2; by using a virus-induced gene silencing method, the expression of the gene is knocked down in the Sumai No.3, so that the resistance of the gibberellic disease can be obviously reduced; the TaWAK-4A gene is over-expressed in arabidopsis thaliana by utilizing agrobacterium tumefaciens-mediated genetic transformation, so that the disease resistance to gibberellic disease bacteria can be remarkably improved; cell wall related kinase protein coded by the gene interacts with LRR receptor kinase to positively regulate the cell death reaction of plants and the bursting reaction of active oxygen. The gene is derived from wheat, and overexpression does not cause food safety problems, so that the gene can be widely applied to cultivation of gibberellic disease resistant plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of wheat cell wall-related kinase TaWAK-4A in cultivating Fusarium head blight-resistant plants. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world and is the staple food for about 40% of the global population. With the growth of the population, the demand for wheat has increased significantly. However, the production of wheat is affected by abiotic and biotic stresses. Wheat Fusarium head blight (FHB) is a fungal disease mainly caused by Fusarium graminearum Schwabe. The occurrence of Fusarium head blight not only causes a serious reduction in wheat yield but also accumulates fungal toxins such as deoxynivalenol in the grains, thereby affecting the quality of wheat. Recently, with the changes in climate and planting systems, wheat Fusarium head blight has begun to spread from warm and humid wheat-growing areas to the whole wheat area.

[0003] The Fusarium head blight resistance of wheat is a quantitative trait controlled by multiple genes, and its resistance composition is affected by the interaction of multiple quantitative trait loci (QTLs). So far, nearly 500 FHB resistance loci have been reported on the 21 chromosomes of wheat. Since the effects of the vast majority of loci are small, the major resistance genes that can be used in breeding are extremely rare. Currently, there are a total of 9 QTLs with strong effects and officially named, and only two loci, Fhb1 and Fhb7, have been successfully cloned. Therefore, finding new resistance resources and exploring new genes for wheat Fusarium head blight resistance is the key to breaking the slow progress of current wheat Fusarium head blight resistance breeding.

[0004] Due to continuous exposure to the threat of countless microbial pathogens, plants have evolved complex innate immune mechanisms. The plant cell wall is the initial physical barrier against invasion, and pattern recognition receptors (PRRs) are deployed on the cell surface to perceive various immunogenic signals as an immune barrier and activate pattern-triggered immune responses (PTI). Wall-associated kinases (WAKs) are an important independent subgroup in the large family of receptor-like kinases and can participate in the recognition of pathogen infection signals in plants. Currently, the research on WAKs related to immunity in wheat is still in its infancy, especially in the research on WAKs related to Fusarium head blight resistance. Therefore, it is urgent to provide new gene resources for wheat Fusarium head blight resistance breeding through the research on wall-associated kinases. Summary of the Invention

[0005] The object of the present invention is to provide the application of wheat TaWAK-4A gene in cultivating plants resistant to Fusarium head blight and in regulating the resistance to Fusarium head blight.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] A wheat gene TaWAK-4A, which is as follows in (1) or (2):

[0008] (1) having the nucleotide sequence shown in SEQ ID NO.1;

[0009] (2) a nucleotide sequence having at least 60% homology or more compared with SEQ ID NO.1; preferably, a nucleotide sequence having at least 70% homology or more compared with SEQ ID NO.1; more preferably, a nucleotide sequence having at least 80% homology or more compared with SEQ ID NO.1; even more preferably, a nucleotide sequence having at least 90% homology or more compared with SEQ ID NO.1; most preferably, a nucleotide sequence having at least 95% homology or more compared with SEQ ID NO.4.

[0010] A protein TaWAK-4A encoded by the above-mentioned wheat gene TaWAK-4A, and the protein TaWAK-4A is as follows in (Ⅰ) or (Ⅱ):

[0011] (Ⅰ) a protein having the amino acid sequence shown in SEQ ID NO.2;

[0012] (Ⅱ) a protein which has the amino acid sequence shown in SEQ ID NO.2 and has the same function after substitution and / or deletion and / or addition of one or several amino acid residues.

[0013] A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the above TaWAK-4A gene.

[0014] The application of the protein TaWAK-4A encoded by the above TaWAK-4A gene or the above recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in improving the resistance of crops to Fusarium head blight, controlling Fusarium head blight of crops or cultivating new germplasms of crops with resistance to Fusarium head blight.

[0015] In the above application, using the wheat TaWAK-4A gene as a target gene, by genetic engineering methods, any alternative splice variant of the TaWAK-4A gene is overexpressed to cultivate new germplasms of crops with significantly improved resistance to Fusarium head blight and apply them in production.

[0016] Furthermore, the crop is wheat.

[0017] The expression vector containing the TaWAK-4A gene provided by the present invention can be used to transform various plant hosts including wheat, and cultivate new materials with resistance to Fusarium head blight.

[0018] The present invention also provides a method for improving the resistance of wheat to Fusarium head blight, which constructs an overexpression vector of the wheat cell wall-associated kinase gene TaWAK-4A to obtain plants with enhanced resistance to Fusarium head blight; the nucleotide sequence of the wheat cell wall-associated kinase gene TaWAK-4A is as shown in SEQ ID No.1.

[0019] Furthermore, the overexpression vector of the wheat cell wall-associated kinase gene TaWAK-4A is obtained by subcloning the CDS sequence of the TaWAK-4A gene into the pBInGFP4 expression vector.

[0020] Furthermore, the primer sequences used for constructing the overexpression vector of the wheat cell wall-associated kinase gene TaWAK-4A are as shown in SEQ ID No.13 and SEQ ID No.14.

[0021] The present invention also provides the application of the TaRLP1-3A gene in improving the resistance of crops to Fusarium head blight, controlling Fusarium head blight of crops or cultivating new germplasms of crops with resistance to Fusarium head blight, and the nucleotide sequence of the TaRLP1-3A gene is as shown in SEQ ID No.5.

[0022] The present invention also provides the application of the TaWAK-4A gene and the TaRLP1-3A gene in improving the resistance of crops to Fusarium head blight, controlling Fusarium head blight of crops or cultivating new germplasms of crops with resistance to Fusarium head blight. The sequence of the TaWAK-4A gene is as shown in SEQ ID No.1, and the nucleotide sequence of the TaRLP1-3A gene is as shown in SEQ ID No.5.

[0023] An application of the molecular mechanism of TaWAK-4A regulating Fusarium head blight resistance, and this molecular mechanism has the following characteristics or descriptions:

[0024] (1) Improve plant disease resistance by regulating the plant's innate immune pathway or enhancing the innate immune response;

[0025] (2) Interact with LRRs receptor-like proteins to regulate the plant's cell death response and reactive oxygen species burst response;

[0026] (3) Directly participate in the recognition of immunogenic signals, including but not limited to small molecules such as chitin, flag22, extracellular polypeptides, etc.;

[0027] (4) Interact with one or more proteins to enhance the affinity of cell receptors for binding immunogenic molecules.

[0028] Beneficial effects

[0029] The wall receptor kinase TaWAK-4A related to Fusarium head blight (FHB) resistance was identified through genome-wide association study (GWAS) of a natural wheat population, and then isolated, cloned, functionally verified and applied. By genetic transformation, TaWAK-4A can be applied to cultivate new materials with FHB resistance in various plants, and can also be used as a marker gene for plants with FHB resistance.

[0030] TaWAK-4A interacts with the LRRs receptor-like protein TaRLP1-3A to regulate immune responses. The discovery and utilization of TaWAK-4A will contribute to the cultivation of wheat varieties with FHB resistance and solve the problems in wheat production. Through in-depth study of the molecular mechanism of TaWAK-4A in FHB resistance, it has important theoretical significance for wheat breeders to select wheat varieties with high FHB resistance. Description of the Drawings

[0031] Figure 1 Genome-wide association combined with VIGS screening was used to identify the FHB-resistant gene TaWAK-4A. (A) A significant association locus was detected on the long arm of chromosome 4A (471.4–476.1 Mb) based on GWAS analysis in four environments (Liuhe, Nanjing, 2018–2021); (B) The expression pattern of TaWAK-4A induced by Fg; (C) RNA-seq analysis of the expression pattern of TaWAK-4A induced by Fg in Fhb1-containing or -free wheat; (D) Two haplotypes of TaWAK-4A were detected in the natural population; (E) The FHB disease phenotypes of Sumai 3 wheat with control and TaWAK-4A silencing inoculated with strain F0609 after 14 days; (F) The investigated diseased spikelet rate; (G) The investigation of fungal biomass of Fg.

[0032] Figure 2 Silencing TaWAK-4A weakened the FHB resistance of Sumai 3. (A–B) Relative expression levels of defense-related genes in control and TaWAK-4A-silenced plants; (C) Determination of salicylic acid (SA) content; (D) Chitin-induced reactive oxygen species (ROS) burst responses in control and TaWAK-4A-silenced plants.

[0033] Figure 3 Overexpression of TaWAK-4A in Arabidopsis thaliana increased the resistance to Fusarium graminearum. (A) Protein expression level of TaWAK-4A in transgenic Arabidopsis thaliana; (B) Transcriptional level of TaWAK-4A in transgenic Arabidopsis thaliana; (C) Lesion size of Fusarium graminearum infection in transgenic Arabidopsis thaliana; (D) Fungal biomass of Fg in control and transgenic plants; (E) Disease severity in control and transgenic plants; (F) Expression levels of pathogenesis-related genes (PR); (G) Chitin-induced ROS responses in control and TaWAK-4A-transgenic plants.

[0034] Figure 4 TaWAK-4A interacts with TaRLP1-3A in vivo and in vitro. (A) Yeast two-hybrid assay was used to verify the interaction between TaWAK-4A and TaRLP1-3A; (B) Dual-luciferase complementation assay was used to verify the in vivo interaction between TaWAK-4A and TaRLP1-3A; (C) Bimolecular fluorescence complementation assay was used to verify the interaction between TaWAK-4A and TaRLP1-3A in tobacco plants; (D) Co-immunoprecipitation assay was used to verify the in vivo interaction between TaWAK-4A and TaRLP1-3A; (E) GST-Pull down assay was used to verify the in vitro interaction between TaWAK-4A and TaRLP1-3A.

[0035] Figure 5 Cell death responses regulated by TaWAK-4A. (A) Subcellular localization of TaWAK-4A; (B) Neither of the two haplotypes of TaWAK4A directly triggers cell death in tobacco leaves; (C) Hap.1-TaWAK-4A inhibits the cell death response induced by BAX; (D-F) The cell death inhibited by Hap.1-TaWAK-4A depends on NbRLP1 (a homologous gene of TaRLP1-3A), rather than NbBAK1.

[0036] Figure 6 TaRLP1-3A is related to Fusarium head blight resistance in wheat and Arabidopsis. (A, B, C, D, E, F) Silencing TaRLP1-3A in Sumai 3 significantly reduces the resistance to Fusarium head blight; (G, H, I, J, K) Overexpressing TaRLP1-3A in Arabidopsis enhances the resistance to Fusarium graminearum.

[0037] Figure 7 The interaction between TaWAK-4A and TaRLP1-3A requires the EDR4-N domain and the doubleRPT1 motif. (A) Prediction of the protein domains of TaWAK-4A; (B) Yeast two-hybrid screening for the key regions of the interaction between TaWAK-4A and TaRLP1-3A; (C) GST-Pull down and (D) Co-immunoprecipitation assays to confirm the key regions of the interaction.

[0038] Figure 8 Working model for the interaction between TaWAK-4A and TaRLP1-3A regulating Fusarium head blight resistance in wheat. Detailed implementation methods

[0039] The following examples define the present invention and describe methods for isolating and cloning a complete coding region DNA fragment containing the TaWAK-4A gene and verifying the function of the TaWAK-4A gene in the present invention, as well as the molecular mechanism by which TaWAK-4A regulates Fusarium head blight resistance. Based on the following description and these examples, those skilled in the art can determine the basic features of the present invention and, without departing from the spirit and scope of the present invention, can make various changes and modifications to adapt it to different uses and conditions. Unless otherwise specified in the examples, all are conventional methods and experimental reagents used in the art.

[0040] Example 1

[0041] Isolation and cloning of different alternative splice variants of the TaWAK-4A gene

[0042] 1.1 Genome-wide association analysis of TaWAK-4A and its expression analysis

[0043] Previously, the applicant conducted an association analysis on 314 wheat natural populations, as Figure 1 shown, a stable association signal was mapped on chromosome 4A. A Fusarium head blight resistance-related gene TaWAK-4A was screened from this locus through transcriptome analysis combined with virus-induced gene silencing. The following primers were used to detect the expression pattern of this gene.

[0044] qpcr-TaWAK-4A-F ATTCGGTTGATCCGTGGTCC(SEQ ID No.7) qpcr-TaWAK-4A-R ACTCCACGAGAAGCTGGAGG(SEQ ID No.8)

[0045] The results were as Figure 1 shown. After induction by Fusarium graminearum, the expression level of this gene increased significantly at 24 h and 36 h.

[0046] 1.2 Obtaining the gene sequence

[0047] The ears of Sumai 3 wheat inoculated with Fg were sampled. After extracting RNA, it was reverse transcribed into cDNA. Using the following primer pairs with cDNA as the template, the CDS sequence of the TaWAK-4A gene was amplified.

[0048] TaWAK-4A-F ATGTCCATGGAAGCTCAGAA(SEQ ID No.9) TaWAK-4A-R CATGTTCTTTCTTCTGGCCA(SEQ ID No.10)

[0049] Example 2

[0050] Using virus-mediated gene silencing technology (VIGS) to study the function of the TaWAK-4A gene

[0051] 2.1 Silencing of the TaWAK-4A gene in wheat

[0052] According to the primer design principle of VIGS, the following primers were used to amplify the silencing fragment using the above cDNA as a template, which was then constructed into the pCaBS-γ vector. It was transferred into Agrobacterium, mixed with pCaBS-α and pCaBS-β, and then used to infect tobacco to propagate the barley stripe BSMV virus, which was then transferred to the expanded flag leaves of wheat. When the albino phenotype appeared after the treatment with pCaBS-TaPDS virus, qRT-PCR was used to detect the silencing efficiency of the TaWAK-4A gene in wheat ears.

[0053] VIGS-TaWAK-4A-F accaccaccgCGAGTTTATGATCGAAGAGG(SEQ ID No.11) VIGS-TaWAK-4A-R ggaagggcccTTTGTCTCAGCTGCCTCATG(SEQ ID No.12)

[0054] 2.2 Identification of disease resistance of silenced plants

[0055] After silencing the candidate gene, 10 μl of a spore suspension of the Fusarium graminearum strain F0609 with a concentration of 1×10 5 spores ml -1 was inoculated using the single-flower drip method. As Figure 1 shown, the plants with silenced TaWAK-4A were more susceptible to disease than the control plants. The diseased spikelet rate increased by about 2 times compared to the control, and the fungal biomass of Fg increased by about 1.8 times. In addition, as Figure 2 shown, after silencing TaWAK-4A, the intensity of the immune response induced by Fg in wheat ears decreased.

[0056] Example 3

[0057] Obtaining and disease resistance identification of TaWAK-4A transgenic Arabidopsis thaliana

[0058] 3.1 Obtaining TaWAK-4A transgenic Arabidopsis thaliana

[0059] Using the following primers, the CDS sequence of the TaWAK-4A gene was subcloned into the pBInGFP4 expression vector. After transferring the constructed vector into Agrobacterium, the floral dip method was used for genetic transformation to obtain TaWAK-4A transgenic Arabidopsis thaliana lines.

[0060] pBInGFP-TaWAK-4A-F tttacgaacgatagggtaccATGTCCATGGAAGCTCAGAA(SEQ ID No.13) pBInGFP-TaWAK-4A-R cccttgctcaccatggatccCATGTTCTTTCTTCTGGCCA(SEQ ID No.14)

[0061] The stable transgenic lines were screened through the following steps: The seeds of Arabidopsis thaliana in the T0 generation of genetic transformation were spread on a 1 / 2 MS medium with a resistance of 100 μg mL -1 kanamycin. The resistant seedlings that had grown for about 10 days were transplanted into the soil. After another 20 days, the young rosette leaves were taken to extract DNA, and primers designed based on the conserved region of the CDS of the TaWAK-4A gene were used for amplification to detect positive ones. The positive offspring were harvested individually and spread on the kanamycin-resistant medium, and the lines with a segregation ratio of 3:1 in the offspring were selected and self-crossed for one generation to harvest the stably inherited transgenic Arabidopsis thaliana T 3Overexpression system.

[0062] 3.2 Determination of the transcriptional level and protein expression level of TaWAK-4A in transgenic Arabidopsis

[0063] Extract the RNA of transgenic Arabidopsis plants. As Figure 3 shown, use qRT-PCR to detect the transcriptional level of the TaWAK-4A gene. Extract the total protein of transgenic plants and use Western blot to detect the protein expression level.

[0064] 3.3 Identification of disease resistance of TaWAK-4A transgenic Arabidopsis

[0065] Use the Fusarium graminearum F0609 and the in vitro leaf infection method to identify the resistance of TaWAK-4A transgenic Arabidopsis to Fg. As Figure 3 shown, TaWAK-4A transgenic Arabidopsis shows higher resistance than the empty vector. The lesion expansion area is smaller than that of the control, the fungal biomass of Fg is reduced by about 75% compared with the wild type, and the disease severity is reduced by about 60%.

[0066] Example 4

[0067] Molecular mechanism application of TaWAK-4A regulating Fusarium head blight resistance

[0068] 4.1 Screening of interacting proteins of TaWAK-4A

[0069] Use the following primers to construct the Y2H bait vector of TaWAK-4A and use BD-TaWAK-4A to screen the cDNA library after wheat is inoculated with Fg.

[0070] BD-TaWAK-4A-F atggccatggaggccgaattcATGTCCATGGAAGCTCAGAA(SEQ ID No.15) BD-TaWAK-4A-R atgcggccgctgcaggtcgacCATGTTCTTTCTTCTGGCCA(SEQ ID No.16)

[0071] After Y2H screening, as Figure 4 shown, TaWAK-4A interacts with an LRRs receptor-like protein TaRLP1-3A. Subsequently, this interaction was verified using dual-luciferase complementation (SLC), bimolecular fluorescence complementation (BiFC), GST-Pull down, and co-immunoprecipitation (Co-IP) experiments.

[0072] 4.2 Subcellular localization of TaWAK-4A protein and cell death response involved

[0073] In tobacco, transiently express the fusion protein of TaWAK-4A-GFP. As Figure 5 shown, TaWAK-4A-GFP is localized on the plasma membrane. Subclone the CDS of TaWAK-4A into the tobacco expression vector pGR107. As Figure 5As shown, TaWAK-4A does not directly induce a cell death response, but depends on NbRLP1 to inhibit the cell death response induced by BAX. Among them, NbRLP1 is a homolog of TaRLP1-3A.

[0074] 4.3 TaRLP1-3A is involved in plant resistance to Fusarium head blight

[0075] Silence TaRLP1-3A in wheat. As Figure 6 shown, the resistance of TaRLP1-3A-silenced wheat to Fusarium head blight was significantly reduced compared with the control. The diseased spikelet rate increased by about 1.9 times compared with the control, and the fungal biomass of Fg increased by about 2.2 times. Overexpression of TaRLP1-3A in Arabidopsis thaliana and inoculation with Fg experiments showed that the resistance of transgenic plants to Fusarium graminearum was significantly improved. Compared with the disease severity of the wild type, it was reduced by about 60% on average, and the fungal biomass of Fg was reduced by about 75% on average.

[0076] 4.4 The interaction mode between TaWAK-4A and TaRLP1-3A

[0077] Use yeast two-hybrid to screen the key regions of the interaction between TaWAK-4A and TaRLP1-3A. As Figure 7 shown, the interaction between TaWAK-4A and TaRLP1-3A requires the EDR4-N domain and the region where the doubleRPT1 motif is located. Subsequently, GST-Pull down and Co-IP experiments were used to verify the key regions of the interaction.

[0078] 4.5 The working mechanism of the interaction between TaWAK-4A and TaRLP1-3A in regulating wheat resistance to Fusarium head blight

[0079] To sum up, as Figure 8 shown, when pathogens invade, TaWAK-4A and TaRLP1-3A of the resistant genotype form a pattern recognition complex on the plasma membrane, recognize chitin or other PAMPs released during Fg infection with potential co-receptors, thereby further activating a strong plant immune response. On the contrary, in the susceptible genotype, the TaWAK-4A allele results in Hap.1-TaWAK-4A encoding a protein lacking a zinc-ribbon domain. Hap.1-TaWAK-4A inhibits the function of TaRLP1-3A, blocks the recognition of chitin or other PAMPs, resulting in a weak immune response. The research of this application reveals the role of TaWAK-4A in sensing immune-related signals and provides genetic resources for improving the ability of wheat to resist Fg.

[0080] Sequence Listing:

[0081] The nucleotide sequence of the TaWAK-4A gene of the present invention is shown in SEQ ID NO.1:

[0082] SEQ ID NO.1

[0083]

[0084] The amino acid sequence of the TaWAK-4A protein of the present invention is shown in SEQ ID NO.2:

[0085] SEQ ID NO.2

[0086]

[0087] The nucleotide sequence of the Hap.1-TaWAK-4A gene of the present invention is shown in SEQ ID NO.3:

[0088] SEQ ID NO.3

[0089] ATGTCCATGGAAGCTCAGAATCTTCGGTTCGTCAGGTGCCCCAAATGCCTCCAGCTTCTCGTGGAGTACCCGTCCATTCCGGTTTACCAGTGTGGTGGCTGCGGCACCATTCTTAGAGCGAAAAATCGAGGTGCGCAAGTAACTCAGCC TGATTCGGTATCCGATGAACAGAACAATTTTCCACACAGCTTGGAAGGGTCCCCTCAGACCAGCAAGTCTATTTGTTCCGATGAACTGAAAGTTGTCTCTGCTGATATGCAGCCTAGTGAAAATTTGGTTGAGGGAAATATTTCCTCTGTCGGCAAGCATGCCATTTCCGGTGAGAATCTTAACACAGAAAGGACTATGTCTGTTGGAGAGAGTGCAGCATCTGGCGAGGTTGACGGTGAGGAGAATTGTTCCCTAAGTGTTGGCAATGCCCGAGACCCCGAGTTTATGATCGAAGAGGCAGATGACAAAGGTGCCACAGTTAATTCTAGTATGAAACTAATAGAAAATGTACAGAGTGTTGAAATAAGTGAAGATGCAGATGGGGAAAAGGGTTGTATTATGGATGATGCAAACGATGCTAGTGTTGCCAGCGAAGCTGAAACTGTCCATAGCATCGCAGGTGAGGAACTGGGAGATGATTCCGGTAAAAATGTGATAGGAGAAATAGAGAGCATGTCTGAGCAAAAAAAATTCTGCTGGCAATAA;

[0090] The amino acid sequence of the Hap.1-TaWAK-4A protein of the present invention is shown in SEQ ID NO.4:

[0091] SEQ ID NO.4

[0092] MSMEAQNLRFVRCPKCLQLLVEYPSIPVYQCGGCGTILRAKNRGAQVTQPDSVSDEQNNFPHSLEGSPQTSKSICSDELKVVSADMQPSENLVEGNISSVGKHAISGENLNTERTMSVGESAASGEVDGEENCSLSVGNARDPEFMIEEADDKGATVNSSMKLIENVQSVEISEDADGEKGCIMDDANDASVASEAETVHSIAGEELGDDSGKNVIGEIESMSEQKKFCWQ*。

[0093] The nucleotide sequence of the TaRLP1-3A gene of the present invention is shown in SEQ ID NO.5:

[0094] SEQ ID NO.5

[0095]

[0096] The amino acid sequence of the TaRLP1-3A protein of the present invention is shown in SEQ ID NO.6:

[0097] SEQ ID NO.6

[0098] MASIWQAKLLFLALLLAFATTSYAHGGGSLELRCHPDQAATLLQLKKSFFFDEAATSLSSWKEGTDCCRWEGVGCGGSSGRVTALSLSRLGLMRSKGLNPVICNLTCLKFLDLSMTDFSEYNMSAVGLERLTFLTHLNLSNSNLEGEIPAGIGKLKNLVSLDLSSEDPSDDESDIHVVSLPNSLWISDFQALVGNLSNLRELRLDDISDCGFLGRMPSSIGNLVNLRSLSLHSSYFSGKIPSTIGNLTNLRRLDISDCGFIGPLPTAIGNLADLRSLTIYSSGFSGPIPCTIGFLTNLESLRFFNSKFTGTIPYTSGNLSSLQSMDFTLCKLSGPIPHEVGKLKELTALALGVNTFSRGIPYSIGVIPASLFNVPLQ

[0099] YLDLSGNQLFGPIKEFNVVSSDLEYLSLERNKLNGKIPRSFFELTNLVSMFVG

[0100] WNNLVGKVELTSFQRLTKLHDLRLSHNKLSIVDGEGNNNSSSSYLSRLSLLGL

[0101] ASCKITKFSSILTRLGDVGDLDLIPMHALDLSSNGLQGQIPMPYSSAVFLDYSD

[0102] NNFSSVLPNFTLYLGTTTYLRMSNNSINGHIPQSVCNFRLDFLDLSYNNFSGG

[0103] LPSCLIEYASLTVLNLRENHFEGTLPSDITSECTFGTVDLHGNKIEGQLPRTLSN

[0104] CKELEVFDIGNNLIVDTFPSWLGELPNLYVLILRSNHFYGYINYSAGNHQSKD

[0105] YFSSLQIIDLASNNFSGTLNTEWFGHTGPTVLAQNISTGPGGLYQDSTKITYKG

[0106] SSVTFEKILTTLTAIDFSNNKLEGTIPESVGWLVSLHVLNMSHNAFTGKIPSQL

[0107] GGMTDLESMDLSCNQLSGIIPQELANLTFLGMLNLSDNQLVGKIPQSRQFSTF

[0108] DNSSFKDNLRLCGPPLSNPCVVSPAPPSLVQVEDSSHVDVILFLFFGLGYGVGF

[0109] AAAILMRWGRIGEWLVKSARALRT*。

Claims

1. A wheat cell wall-associated kinase protein TaWAK-4A, characterized in that: The wheat cell wall-associated kinase protein TaWAK-4A comprises the amino acid sequence described in any one of the following (1) and (2): (1) the amino acid sequence shown in SEQ ID NO.2; (2) A sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO.

2.

2. A wheat cell wall-related kinase gene TaWAK-4A, characterized in that: Encoding the wheat cell wall-associated kinase protein TaWAK-4A according to claim 1, wherein the wheat cell wall-associated kinase gene is the following (1) or (2): (1) having the nucleotide sequence shown in SEQ ID NO.1; (2) A nucleotide sequence having at least 60% homology with SEQ ID NO.1; preferably, a nucleotide sequence having at least 70% homology with SEQ ID NO.1; further preferably, a nucleotide sequence having at least 80% homology with SEQ ID NO.1; further preferably, a nucleotide sequence having at least 90% homology with SEQ ID NO.1; most preferably, a nucleotide sequence having at least 95% homology with SEQ ID NO.

4.

3. A recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the gene according to claim 2.

4. Use of the protein according to claim 1 or the gene according to claim 2 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria according to claim 3 in improving crop resistance to ergot disease, preventing and controlling crop ergot disease or cultivating new crop germplasm with ergot disease resistance.

5. The use according to claim 4, characterized in that: The crop is wheat.

6. A method for improving wheat scab resistance, characterized in that: An overexpression vector of wheat cell wall-related kinase gene TaWAK-4A was constructed to obtain plants with enhanced resistance to fusarium rust. The nucleotide sequence of the wheat cell wall-related kinase gene TaWAK-4A is shown in SEQ ID No.

1.

7. The method for improving wheat head blight resistance according to claim 6, characterized in that: The wheat cell wall-related kinase gene TaWAK-4A overexpression vector is obtained by subcloning the CDS sequence of the TaWAK-4A gene into the pBInGFP4 expression vector.

8. The method for improving wheat head blight resistance according to claim 6, characterized in that: The primer sequences used to construct the wheat cell wall-associated kinase gene TaWAK-4A overexpression vector are shown in SEQ ID No.13 and SEQ ID No.

14.

9. The use of TaRLP1-3A gene in improving crop resistance to fusarium head blight, preventing and controlling crop fusarium head blight, or cultivating new crop germplasm with fusarium head blight resistance, characterized in that: The nucleotide sequence of the TaRLP1-3A gene is shown in SEQ ID No.

5.

10. Application of TaWAK-4A gene and TaRLP1-3A gene in improving crop resistance to fusarium head blight, preventing and controlling crop fusarium head blight, or cultivating new crop germplasm with fusarium head blight resistance, characterized in that: The sequence of the TaWAK-4A gene is shown in SEQ ID No.1, and the nucleotide sequence of the TaRLP1-3A gene is shown in SEQ ID No.5.

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