Application of wheat TaWRKY69 gene in regulating resistance to wheat foot rot

Abstract

The application discloses application of a wheat TaWRKY69 gene in regulation of resistance to wheat stem base rot, and a broad-spectrum disease-resistant wheat protein TaWRKY69 is cloned from a wheat germplasm Shiyou 17 resistant to stem base rot. The TaWRKY69 gene of wheat is silenced by using a VIGS technology, and the result shows that silencing of the TaWRKY69 gene reduces the resistance of wheat to wheat stem base rot. A wheat with the TaWRKY69 gene is obtained by using an agrobacterium-mediated genetic transformation method, and the wheat with the TaWRKY69 gene enhances the resistance of wheat to stem base rot, which indicates that the TaWRKY69 gene positively regulates and improves the resistance of wheat to stem base rot. The TaWRKY69 protein can regulate the disease resistance, stem base rot resistance and false smut resistance of wheat, and can be applied to wheat breeding or assisted breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural fungal disease control technology, specifically involving the application of the wheat TaWRKY69 gene in regulating wheat stem base rot resistance. Background Technology

[0002] Wheat stem rot is a fungal disease caused by *Fusarium graminearum* or *Fusarium pseudograminearum*. It primarily affects the base of the stem and is soil-borne. Continuous return of straw to the field leads to a sustained increase in pathogen content in the soil, resulting in a rapid rise in the incidence and severity of stem rot, making it a significant threat to wheat production. Stem rot not only causes severe yield and economic losses in wheat but also produces toxins and secondary metabolites such as DON, NIV, and ZEN, which have potential adverse effects on food and feed products.

[0003] Agronomic measures are insufficient to control wheat stem rot, while chemical pesticides cause environmental pollution and increase production costs. Therefore, breeding and promoting resistant varieties is an effective, economical, and safe method for controlling stem rot. However, currently, no varieties in production have reached a level of immunity or high resistance, making the breeding of new disease-resistant varieties urgently needed.

[0004] Currently, the scarcity of easily bred and utilized wheat germplasm resources resistant to stem base rot, both domestically and internationally, along with the scarcity of resistance loci and genes, and the unclear resistance mechanisms, severely restricts research on the genetic improvement of wheat resistance to stem base rot. Therefore, it is particularly urgent to expand the scope of resistance source identification, fully utilize existing resistance sources, conduct in-depth research on resistance genetics, discover key genes for wheat resistance to stem base rot, elucidate resistance mechanisms, and utilize advanced biotechnologies such as molecular marker-assisted selection, transgenic technology, and gene editing to create new wheat germplasm resistant to stem base rot. Summary of the Invention

[0005] This application isolates and clones a broad-spectrum disease-resistant wheat protein, TaWRKY69, from the wheat germplasm Shiyou 17, which is resistant to stem base rot. The TaWRKY69 gene in wheat is silenced using VIGS technology. The TaWRKY69 gene-silenced wheat is then inoculated with Fusarium graminearum WHF220. The results show that TaWRKY69 gene silencing reduces wheat resistance to wheat stem base rot. Agrobacterium-mediated genetic transformation is used to obtain TaWRKY69-transgenic wheat. This TaWRKY69-transgenic wheat exhibits enhanced resistance to stem base rot, indicating that the TaWRKY69 gene positively regulates wheat resistance to stem base rot. The TaWRKY69 protein can regulate wheat disease resistance, stem base rot resistance, and Fusarium graminearum resistance, and can be applied to wheat breeding or assisted breeding. The specific technical solution of this invention is as follows:

[0006] The first aspect is the cloning of wheat disease resistance protein TaWRKY69 and its encoding gene. This application isolates and clones a broad-spectrum disease-resistant wheat protein from the stem rot resistant wheat germplasm Shiyou 17, names it TaWRKY69 protein, and names the gene encoding TaWRKY69 protein the TaWRKY69 gene.

[0007] Secondly, the acquisition of wheat with the VIGS-silenced TaWRKY69 gene.

[0008] 1. Constructing a recombinant VIGS carrier

[0009] (1) Total RNA was extracted from leaves of wheat variety Shiyou 17 and cDNA was obtained by reverse transcription;

[0010] (2) Using the TaWRKY69 gene sequence as a reference, the optimal silencing fragment was designed using SiFi software; using the cDNA in step (1) as a template, specific primers were designed at both ends of the optimal silencing fragment of the TaWRKY69 gene to amplify the fragment, and the PCR product was recovered.

[0011] (3) Digest the vector BSMV-γ and the product of step (2) with the restriction endonuclease NheⅠ, and recover the vector backbone and the digestion product of step (2);

[0012] (4) The enzyme digestion product from step (3) was ligated using T4-ligase from Takara to obtain the recombinant plasmid BSMVγ-TaWRKY69.

[0013] 2. Vector linearization and in vitro transcription

[0014] 3. Preparation of BSMV inoculation mixture

[0015] 4. Virus inoculation

[0016] 5. VIGS Silencing Efficiency Assessment

[0017] Thirdly, the sensitivity analysis of TaWRKY69 gene-silenced wheat to Fusarium graminearum.

[0018] Wheat plants with successful TaWRKY69 gene silencing were inoculated with Fusarium graminearum WHF220;

[0019] Results: The browning area at the stem base of TaWRKY69 gene-silenced wheat plants was significantly larger than that of control wheat plants; the average disease grade of TaWRKY69 gene-silenced wheat plants was significantly higher than that of the control; the relative content of Fusarium pseudograss in the stem base of TaWRKY69 gene-silenced wheat plants was significantly higher than that of the control, indicating that wheat plants with silenced TaWRKY69 gene have reduced resistance to stem base rot.

[0020] Fourthly, the acquisition of TaWRKY69 transgenic wheat.

[0021] 1. Construction of the TaWRKY69 gene vector

[0022] (1) Total RNA was extracted from leaves of wheat variety Shiyou 17 and cDNA was obtained by reverse transcription;

[0023] (2) Using the TaWRKY69 gene sequence as a reference, and the cDNA in step (1) as a template, specific primers were designed at both ends of the coding region of the TaWRKY69 gene to amplify the gene, and the PCR product was recovered.

[0024] (3) The vector pWMB110 was digested with restriction endonucleases BamHI and SacⅠ, and the vector backbone was recovered;

[0025] (4) The recovered products from steps (2) and (3) were ligated using recombinase from Takara to obtain the recombinant expression vector pWMB110-TaWRKY69.

[0026] 2. Obtaining TaWRKY69 transgenic wheat

[0027] pWMB110-TaWRKY69 was transformed into wheat Fielder embryo callus using Agrobacterium tumefaciens infection. After screening, pre-differentiation, and differentiation, wheat plants transgenic with the TaWRKY69 gene were obtained.

[0028] Fifthly, analysis of the resistance of TaWRKY69 transgenic wheat to Fusarium graminearum.

[0029] Wheat plants transgenic with the TaWRKY69 gene were inoculated with Fusarium graminearum WHF220;

[0030] Results: The average disease severity of the three TaWRKY69 transgenic wheat lines was significantly lower than that of wild-type wheat Fielder; the browning area at the stem base of the TaWRKY69 transgenic wheat was significantly smaller than that of wild-type wheat plants. The results indicate that overexpression of the TaWRKY69 gene enhances wheat resistance to wheat stem base rot, and the TaWRKY69 gene positively regulates wheat resistance.

[0031] Sixthly, the application, acquisition, and regulation of the TaWRKY69 protein.

[0032] TaWRKY69 protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. Substances that upregulate the activity or content of TaWRKY69 protein can be substances that upregulate / overexpress the encoding gene of TaWRKY69 protein and / or substances that upregulate / overexpress the expression of the encoding gene of TaWRKY69 protein.

[0033] Compared with the prior art, the beneficial effects of this application are as follows:

[0034] This application is the first to isolate and clone a broad-spectrum disease-resistant wheat protein, TaWRKY69, from wheat resistant to stem base rot. Silencing the TaWRKY69 gene reduces wheat resistance to stem base rot, while wheat transgenic with the TaWRKY69 gene enhances wheat resistance to stem base rot. This protein can be applied to wheat breeding or assisted breeding, providing a new possibility for breeding germplasm resistant to wheat stem base rot. Attached Figure Description

[0035] Figure 1 The relative expression levels of TaWRKY69 in wheat plants with BSMV:GFP (control) and BSMV:TaWRKY69 (TaWRKY69 gene silence);

[0036] Figure 2 The infection status of the stem base of wheat plants (TaWRKY69 gene silenced) inoculated with Fusarium graminearum 21 days after inoculation with BSMV:GFP (control) and BSMV:TaWRKY69;

[0037] Figure 3 The TaWRKY69 gene silencing in wheat promotes resistance to Fusarium graminearum.

[0038] A represents the statistical results of the average disease severity of wheat in the BSMV:GFP group (control) and the BSMV:TaWRKY69 group (TaWRKY69 gene silence).

[0039] B represents the relative content of Fusarium graminearum in wheat plants of the BSMV:GFP group (control) and the BSMV:TaWRKY69 group (TaWRKY69 gene silence).

[0040] Figure 4 PCR detection of wheat transgenic TaWRKY69;

[0041] Figure 5 The relative expression levels of TaWRKY69 in transgenic and recipient wheat plants;

[0042] Figure 6 The resistance of TaWRKY69 transgenic wheat to Fusarium graminearum, among which,

[0043] A represents the statistical results of the average disease severity of wild-type wheat (Fielder) and three transgenic TaWRKY69 lines (OX3, OX7, OX8).

[0044] B represents the infection status of the stem base of wild-type wheat (Fielder) and TaWRKY69 transgenic wheat 21 days after inoculation with Fusarium graminearum.

[0045] in Figure 1 , Figure 3 , Figure 5 and Figure 6 middle,

[0046] * and ** represent P<0.05 and P<0.01, respectively, indicating that the difference between the two groups is statistically significant. Detailed Implementation

[0047] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0048] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0051] The above-mentioned biological materials, namely Shi You 17 and Fielder in the following embodiments, can be obtained from the applicant. The obtained biological materials are only for repeating the experiments of this application and cannot be used for other purposes.

[0052] The grading standards for wheat stem rot were based on the method of Zhou Miaoping et al. (Zhou Miaoping, Yao Jinbao, Zhang Peng, Yu Guihong, Ma Hongxiang. Establishment of a new method for screening and identifying wheat germplasm resistant to stem rot. Journal of Plant Genetic Resources, 2016, 17(2):377-382), with slight modifications. See Table 1 for details.

[0053] Table 1. Grading of Wheat Stem Base Rot

[0054] Reagents and Materials

[0055]

[0056] instrument

[0057]

[0058]

[0059] Example 1: Cloning of wheat disease resistance protein TaWRKY69 and its encoding gene

[0060] This application isolates and clones a broad-spectrum disease-resistant wheat protein from the stem rot-resistant wheat germplasm Shiyou 17, and names it TaWRKY69 protein. Its amino acid sequence is shown in SEQ ID NO:1. The gene encoding TaWRKY69 protein is named TaWRKY69 gene.

[0061] The specific cloning method is as follows:

[0062] Total RNA was extracted from the stems of wheat variety Shiyou 17 inoculated with *Sterculia vesicatoria*. Following the procedure of the first-strand cDNA synthesis kit from Tiangen Biotech, the extracted RNA samples were reverse transcribed to synthesize first-strand cDNA, which served as a template for gene cloning. PCR amplification was performed using primers TaWRKY69-F: 5'-TCCGTCCGTGTCCGATTC-3' (SEQ ID NO:4) and TaWRKY69-R: 5'-CACTCTTGAACAGCGAAATCC-3' (SEQ ID NO:5). The amplification program was as follows: pre-denaturation at 94℃ for 3 minutes; followed by 35 cycles of 98℃ for 10 seconds, 58℃ for 30 seconds, and 68℃ for 60 seconds; extension at 68℃ for 5 minutes. The target PCR band was recovered by 1.5% agarose gel electrophoresis. The PCR product was ligated into the pMD18-T vector and sent to Biomed for sequencing.

[0063] Sequencing results showed that the DNA fragment obtained by PCR amplification using wheat cDNA of Shiyou 17 as a template was the cDNA sequence of the TaWRKY69 gene, and the nucleotide sequence of its coding frame is shown in SEQ ID NO:2.

[0064] Example 2: Obtaining wheat with VIGS-silenced TaWRKY69 gene

[0065] 1. Construction of the TaWRKY69 recombinant silencing vector VIGS

[0066] (1) Total RNA was extracted from the leaves of wheat variety Shiyou 17 and cDNA was obtained by reverse transcription. (See Example 1 for details of the procedure)

[0067] (2) Using the Chinese spring TaWRKY69 gene sequence (TraesCS3B02G324400.1) published on the Ensembl website as a reference, the optimal silencing fragment was designed using SiFi software. Then, using the cDNA from step (1) as a template, specific primers were designed at both ends of the optimal silencing fragment of the TaWRKY69 gene to amplify the fragment, and the PCR product was recovered.

[0068] The amplification primers are as follows:

[0069] BSMV-TaWRKY69-F: 5'-AAT GCTAGC CCCCACCGTGCTGCTCGT-3'(SEQ ID NO:6);

[0070] BSMV-TaWRKY69-R: 5'-CCA GCTAGC GGTCACCGTCGTGATCGCC-3' (SEQ ID NO:7).

[0071] The underlined part is the NheⅠ recognition sequence; BSMV is barley stripemosaic virus (BSMV).

[0072] (3) Digest the vector BSMV-γ and the product of step (2) with the restriction endonuclease NheⅠ, and recover the vector backbone and the digestion product of step (2).

[0073] (4) The enzyme digestion product of step (3) was ligated using T4-ligase from Takara to obtain recombinant plasmid BSMVγ-TaWRKY69. Sequencing was performed, and the structure of recombinant plasmid BSMVγ-TaWRKY69 was described as follows based on the sequencing results: a recombinant VIGS vector in which the DNA fragment with the nucleotide sequence shown in SEQ ID NO:3 was inserted in reverse at the NheⅠ restriction site of the vector BSMV-γ.

[0074] 2. Vector linearization and in vitro transcription

[0075] (1) Linearization of VIGS vectors:

[0076] The vector BSMV-α was digested with the restriction endonuclease MluⅠ, and the vector backbone was recovered.

[0077] The vector BSMV-β was digested with the restriction endonuclease SpeⅠ, and the vector backbone was recovered.

[0078] The vector BSMV-γ-GFP was digested with the restriction endonuclease MluⅠ, and the vector backbone was recovered.

[0079] The vector BSMVγ-TaWRKY69 was digested with the restriction endonuclease MluⅠ, and the vector backbone was recovered.

[0080] (2) Using Promega's RiboMAX TMThe Large Scale RNA Production Systems-T7 kit performs in vitro transcription on the vectors recovered in step (1) to obtain α, β, and γ-GFP / γ-TaWRKY69 transcription reaction solutions.

[0081] 3. Preparation of BSMV inoculation mixture

[0082] Take a 1.5 mL centrifuge tube, add 10 μL each of α, β, and γ-GFP / γ-TaWRKY69 transcription reaction solution, mix well, then add 60 μL of RNase-free ddH2O, and then add 90 μL of GKP solution (solvent is water, containing 50 mM glycine, 30 mM K2HPO4, 1% Bentonite and 1% Celite, pH 9.2), mix, and obtain BSMV:TaWRKY69 virus inoculation mixture (α+β+γ-TaWRKY69) or BSMV:GFP (α+β+γ-GFP) virus inoculation mixture.

[0083] 4. Virus inoculation

[0084] When wheat seedlings of CI12633 reached the two-leaf-one-heart stage, 10 μL of either the BSMV:TaWRKY69 virus inoculation mixture or the BSMV:GFP virus inoculation mixture was applied to a clean glove and rubbed onto the second leaf of the seedling 6-8 times, controlling the pressure during rubbing. After rubbing, a small amount of RNase-free ddH2O was sprayed from top to bottom to maintain humidity. Clean gloves were changed for each treatment. After virus inoculation, the seedlings were incubated at 23±2℃ in the dark for 24 hours, and then the incubation period was adjusted to 16h / 8h light / dark cycle. Phenotypic changes were observed and recorded regularly.

[0085] 5. VIGS Silencing Efficiency Assessment

[0086] Fifteen days after inoculation, the gene silencing efficiency of wheat plants was determined. Total RNA was extracted from wheat leaves infected with BSMV virus and reverse transcribed to obtain cDNA. qRT-PCR was performed using the cDNA template, with the Actin gene as an internal control gene, to detect the relative expression level of the TaWRKY69 gene.

[0087] The primers used to detect the TaWRKY69 gene are as follows:

[0088] RT-TaWRKY69-F: 5'-TGGCACCGGACGGCGCTATA-3' (SEQ ID NO: 8);

[0089] RT-TaWRKY69-R: 5'-ACGATGGCGCACTCCGGGAG-3' (SEQ ID NO: 9).

[0090] The primers used to detect the Actin gene are as follows:

[0091] RT-Actin-F: 5'-CCTCTCTGCGCCAATCGT-3' (SEQ ID NO: 10);

[0092] RT-Actin-R: 5'-TCAGCCGAGCGGGAAATTGT-3' (SEQ ID NO: 11).

[0093] The results are as follows Figure 1 As shown, the relative expression level of the TaWRKY69 gene was detected by qPCR, with Actin selected as the internal control gene. Each data point represents the average (±SD) of three parallel experiments. Compared with wheat plants infected with BSMV:GFP, the transcript level of wheat plants infected with BSMV:TaWRKY69 was significantly reduced, indicating that the TaWRKY69 gene was successfully silenced.

[0094] Example 3: Sensitivity analysis of TaWRKY69 gene-silenced wheat to Fusarium oxysporum.

[0095] Wheat plants from which the TaWRKY69 gene was successfully silenced in Example 2 were inoculated with Fusarium graminearum WHF220. Samples were taken at 0, 1, 3, 5, and 7 days post-infection for histological observation and RNA isolation. The gene silencing efficiency was analyzed using real-time reverse transcription PCR (qRT-PCR) (see Example 2 for details). The infection status of the plants was photographed 21 days post-infection.

[0096] The vaccination method is as follows:

[0097] 1. Strain propagation:

[0098] Mycelia were picked from preserved *Fusarium graminearum* blocks (WHF220) and inoculated onto sterilized PDA plates. The plates were incubated at 25°C until the mycelia completely covered the PDA plates. Uniformly sized wheat grains were selected, boiled in boiling water for 15 minutes, rinsed thoroughly with cold water, and spread evenly on clean filter paper to air dry in a ventilated area for 8-10 hours. After drying, the grains were dispensed into 1000mL Erlenmeyer flasks, the caps were sealed, and sterilized at 121°C for 15 minutes. The PDA plates with fully colonized mycelia were cut into 1cm × 1cm blocks and inoculated into the aforementioned Erlenmeyer flasks containing wheat grains. The blocks were incubated at 25°C for 7 days for material inoculation.

[0099] 2. Wheat plants inoculated with fungi

[0100] 10g of bacterial wheat grains (1, prepared by strain propagation) were inoculated into a box containing wheat (i.e., wheat plants from which the TaWRKY69 gene was successfully silenced in Example 2), and evenly spread over the box (on top of the soil substrate). Water was sprayed every 2 days. A BSMV:GFP control wheat was also included in the experiment.

[0101] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 The results showed that 21 days after infection with Fusarium graminearum, the browning area at the stem base of wheat plants with BSMV:TaWRKY69 silenced was significantly larger than that of control wheat plants, indicating that wheat plants with silenced TaWRKY69 genes had reduced resistance to stem base rot.

[0102] Figure 3 The mean disease severity of wheat plants with BSMV:TaWRKY69 was significantly higher than that of the BSMV:GFP control plants (P < 0.05).

[0103] Figure 3 The results from the B assay showed that the relative content of *Fusarium graminearum* at the stem base of wheat plants infected with BSMV:TaWRKY69 was significantly higher than that in the BSMV:GFP control plants (P < 0.01). Figure 3 Each data point represents a specific value from the experiment. The above results indicate that silencing the TaWRKY69 gene reduces wheat resistance to wheat stem rot. This suggests that the TaWRKY69 gene positively regulates wheat resistance to stem rot.

[0104] Example 4: Obtaining and Identifying TaWRKY69 Genetically Modified Wheat

[0105] 1. Construction of the TaWRKY69 gene vector

[0106] (1) Total RNA was extracted from wheat leaves of Shiyou 17 and cDNA was obtained by reverse transcription.

[0107] (2) Using the Chinese spring TaWRKY69 gene sequence (TraesCS3B02G324400.1) published on the Ensembl website as a reference, and using the cDNA in step (1) as a template, specific primers were designed at both ends of the coding region of the TaWRKY69 gene to amplify the gene, and the PCR product was recovered.

[0108] The amplification primers are as follows:

[0109] OE-TaWRKY69-F:

[0110] 5'- CGACTCTAGAGGATCC ATGTCGGACTGCAGCACG-3' (SEQ ID NO: 12);

[0111] OE-TaWRKY69-R:

[0112] 5'- ATCGGGGAAATTCGAGCTC TCAGGAGCAGGGCGCGGT-3' (SEQ ID NO: 13).

[0113] The underlined part is the carrier connector sequence.

[0114] (3) The vector pWMB110 was digested with restriction endonucleases BamHⅠ and SacⅠ, and the vector backbone was recovered.

[0115] (4) The recovered products from steps (2) and (3) were ligated using recombinase from Takara to obtain a recombinant expression vector containing the wheat TaWRKY69 gene, named pWMB110-TaWRKY69.

[0116] 2. Obtaining TaWRKY69 transgenic wheat

[0117] pWMB110-TaWRKY69 was transformed into wheat Fielder embryo callus using Agrobacterium tumefaciens infection. After screening, pre-differentiation, and differentiation, wheat plants transgenic with the TaWRKY69 gene were obtained.

[0118] 3. PCR identification of genetically modified wheat

[0119] Three positive transgenic plants were obtained. After multiple generations, T2 generation homozygous transgenic lines OE#3, OE#7, and OE#8 were obtained. Figure 4 ).

[0120] 4. Expression identification of transgenic wheat

[0121] Using Fielder as a reference, 2 -△△CT The expression level of gene TaWRKY69 in leaves of T2 generation positive transgenic lines was detected by a method, and the results are as follows: Figure 5 As shown, the expression level of TaWRKY69 in the leaves of T2 generation positive transgenic lines (OE#3, OE#7 and OE#8) was significantly higher than that of the control, at 21.71, 22.32 and 32.45 times that of the control, respectively.

[0122] Example 5: Resistance analysis of TaWRKY69 transgenic wheat to Fusarium graminearum.

[0123] The wheat plants transgenic with the TaWRKY69 gene obtained in Example 4 were inoculated with Fusarium graminearum WHF220, and the infection status of the plants was photographed 21 days after infection.

[0124] For the inoculation method, please refer to Example 3.

[0125] The results are as follows Figure 6 As shown, after infection with *Fusarium graminearum*, compared with wild-type wheat (Fielder), the average disease severity of the TaWRKY69-overexpressing transgenic lines OX3, OX7, and OX8 was significantly lower (P < 0.01). The browning area at the stem base of the TaWRKY69-overexpressing transgenic wheat was significantly smaller than that of wild-type wheat plants, indicating enhanced resistance. These results suggest that overexpression of the TaWRKY69 gene enhances wheat resistance to wheat stem base rot, implying that the TaWRKY69 gene improves wheat's resistance to this disease.

[0126] In summary, silencing the TaWRKY69 gene significantly reduced plant disease resistance, while overexpression of TaWRKY69 significantly enhanced disease resistance. This indicates that this gene is a positive regulator in the response to wheat stem rot.

[0127] Example 6: Application and Acquisition of TaWRKY69 Protein

[0128] 1. Application of TaWRKY69 protein

[0129] As shown in Examples 1, 2, 3, 4, and 5, the TaWRKY69 protein can be used for:

[0130] (1) Regulating wheat disease resistance;

[0131] (2) Prepare products that regulate wheat disease resistance;

[0132] (3) Regulating resistance to wheat stem base rot;

[0133] (4) Prepare products that regulate wheat resistance to stem rot;

[0134] (5) Regulating wheat resistance to Fusarium graminearum;

[0135] (6) Prepare products that regulate wheat resistance to Fusarium graminearum;

[0136] (7) Applied to wheat breeding or to assist wheat breeding;

[0137] Upregulating TaWRKY69 protein activity or content can increase wheat resistance to Fusarium graminearum and increase wheat stem rot resistance; downregulating TaWRKY69 protein activity or content can decrease wheat resistance to Fusarium graminearum and increase wheat stem rot resistance.

[0138] 2. Obtaining TaWRKY69 protein

[0139] TaWRKY69 protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0140] TaWRKY69 protein can be any of the following proteins:

[0141] (1) A protein with an amino acid sequence as shown in SEQ ID NO:1;

[0142] (2) Proteins with more than 80% identity to the amino acid sequence shown in SEQ ID NO:1 obtained by substituting and / or deleting and / or adding amino acid residues, and which are related to plant disease resistance.

[0143] (3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a protein with an amino acid sequence as shown in SEQ ID NO:1.

[0144] The connection in (3) above can be a peptide bond link.

[0145] Example 7: Substances that upregulate the activity or content of TaWRKY69 protein

[0146] Substances that upregulate the activity or content of TaWRKY69 protein may be substances that upregulate / overexpress the gene encoding TaWRKY69 protein and / or substances that upregulate / overexpress the gene encoding TaWRKY69 protein.

[0147] 1. Methods for upregulating TaWRKY69 gene expression

[0148] Substances that upregulate TaWRKY69 gene expression can be regulated by at least one of the following six methods:

[0149] (1) Regulation at the transcriptional level of the TaWRKY69 gene;

[0150] (2) Regulation after transcription of the TaWRKY69 gene (that is, regulation of splicing or processing of the primary transcript of the TaWRKY69 gene).

[0151] (3) Regulation of RNA transport of TaWRKY69 gene (that is, regulation of the transport of TaWRKY69 gene mRNA from the nucleus to the cytoplasm).

[0152] (4) Regulation of the translation of the TaWRKY69 gene;

[0153] (5) Regulation of the degradation of TaWRKY69 gene mRNA;

[0154] (6) Post-translational regulation of the TaWRKY69 gene (that is, regulation of the activity of the protein translated from the TaWRKY69 gene).

[0155] 2. The substance that upregulates TaWRKY69 protein expression is a biological material.

[0156] The substance that upregulates the expression of the TaWRKY69 protein-coding gene or regulates the activity or content of the TaWRKY69 protein is a biological material, and the biological material may be any of the following:

[0157] (1) Nucleic acid molecules that upregulate / overexpress the expression of the gene encoding TaWRKY69 protein or upregulate / overexpress the content or activity of the TaWRKY69 protein;

[0158] The nucleic acid molecule may be an RNA encoding a gene that targets the TaWRKY69 protein or a DNA encoding the RNA, and the nucleotide sequence of the target sequence of the RNA is positions 366-627 of SEQ ID NO:2.

[0159] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0160] This expression cassette can be used to insert a DNA molecule containing the target sequence into a proprietary expression cassette of a BSMV:γ (BSMV:GFP) vector.

[0161] (3) A recombinant vector containing the nucleic acid molecule described in (2), or a recombinant vector containing the expression cassette described in (2);

[0162] The recombinant expression vector may be a recombinant BSMV:γ vector. In some embodiments of this application, the structure of the recombinant BSMV:γ vector is as follows: a DNA molecule with a nucleotide sequence as shown in positions 366-627 of SEQ ID NO:2 is inserted in reverse at the NheⅠ restriction site of the vector BSMV:γ.

[0163] (4) Recombinant microorganisms containing the nucleic acid molecules described in (3), or recombinant microorganisms containing the expression cassette described in (2), or recombinant microorganisms containing the recombinant vector described in (3);

[0164] The recombinant microorganism could be recombinant barley stripe mosaic virus (BSMV).

[0165] Recombinant barley stripe mosaic virus can be obtained through in vitro transcription.

[0166] Recombinant barley stripe mosaic virus can cause post-transcriptional gene silencing of the gene encoding the TaWRKY69 protein, that is, gene inactivation at the post-transcriptional level by specifically inhibiting the target RNA.

[0167] (5) A transgenic plant cell line containing the nucleic acid molecule described in (1) or a transgenic plant cell line containing the expression cassette described in (2) or a transgenic plant cell line containing the recombinant vector described in (3);

[0168] Transgenic plant cell lines may or may not include propagation material.

[0169] (6) A transgenic plant tissue containing the nucleic acid molecule described in (1) or a transgenic plant tissue containing the expression cassette described in (2) or a transgenic plant tissue containing the recombinant vector described in (3);

[0170] The aforementioned plant tissues can originate from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0171] Transgenic plant tissues may or may not include propagation material.

[0172] (7) A transgenic plant organ containing the nucleic acid molecule described in (1) or a transgenic plant organ containing the expression cassette described in (2) or a transgenic plant organ containing the recombinant vector described in (3);

[0173] The aforementioned transgenic plant organs can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0174] Transgenic plant organs may or may not include propagation material.

[0175] (8) The nucleic acid molecule encoding the TaWRKY69 protein described above;

[0176] This nucleic acid molecule can be a DNA molecule of either A or B as follows:

[0177] A. The coding sequence of the coding strand is the DNA molecule with SEQ ID NO:2;

[0178] B is a DNA molecule that shares more than 80% identity with the DNA molecule in A and regulates plant disease resistance.

[0179] (9) Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic plant cell lines containing the nucleic acid molecules described in (8).

[0180] In this application, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained.

[0181] The aforementioned 80% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0182] The 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0183] The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0184] The 95% or higher level of identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0185] Example 8: Substances that downregulate the activity or content of TaWRKY69 protein

[0186] Substances that downregulate the activity or content of TaWRKY69 protein may be substances that downregulate / reduce / knock out the gene encoding TaWRKY69 protein and / or substances that downregulate / reduce / knock out the expression of the gene encoding TaWRKY69 protein.

[0187] Downregulation / reduction / knockout of TaWRKY69 gene expression can be achieved through gene knockout or gene silencing.

[0188] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is achieved by altering the DNA sequence to inactivate a specific target gene.

[0189] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, quelling, RNA interference (RNAi), and microRNA-mediated translational repression.

[0190] 1. Methods for downregulating TaWRKY69 gene expression

[0191] Same as Example 2.

[0192] 2. The substance that downregulates TaWRKY69 protein expression is a biological material.

[0193] The substance that downregulates the expression of the TaWRKY69 protein-coding gene or regulates the activity or content of the TaWRKY69 protein is a biological material, and the biological material may be any of the following:

[0194] Nucleic acid molecules that downregulate, reduce, or knock out the expression of the gene encoding TaWRKY69 protein, or inhibit or reduce the content or activity of TaWRKY69 protein;

[0195] The nucleic acid molecule may be an RNA encoding a gene that targets the TaWRKY69 protein or a DNA encoding the RNA, wherein the nucleotide sequence of the target sequence of the DNA is positions 366-627 of SEQ ID NO:2.

[0196] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

[0197] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of increasing the content and / or activity of TaWRKY69 protein in target wheat for the prevention and control of wheat stem rot, characterized in that, The amino acid sequence of the TaWRKY69 protein is shown in SEQ ID No.

1.

2. The application as described in claim 1, characterized in that, The TaWRKY69 protein is encoded by the TaWRKY69 gene, and the nucleotide sequence of the cDNA coding frame of the TaWRKY69 gene is shown in SEQ ID No.

2.

3. A method for preventing and controlling wheat stem base rot, characterized in that, The method includes the following steps: increasing the content and / or activity of TaWRKY69 protein in target wheat, wherein the amino acid sequence of TaWRKY69 protein is shown in SEQ ID No.

1.

4. The application of overexpression of the TaWRKY69 gene in wheat for the prevention and control of wheat stem rot, characterized in that, The nucleotide sequence of the cDNA coding frame of the TaWRKY69 gene is shown in SEQ ID No.

2.

5. A method for preventing and controlling wheat stem base rot, characterized in that, The procedure includes the following steps: overexpressing the TaWRKY69 gene in target wheat, wherein the nucleotide sequence of the cDNA coding frame of the TaWRKY69 gene is shown in SEQ ID No.

2.

6. The application of overexpression of the TaWRKY69 gene in wheat for breeding wheat varieties / lines resistant to stem rot, characterized in that, The nucleotide sequence of the cDNA coding frame of the TaWRKY69 gene is shown in SEQ ID No. 2.

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