Application of wheat TaRFS4B gene in synergistically improving wheat scab and banded sclerotial blight resistance

Abstract

The invention discloses application of a wheat TaRFS4B gene in synergistically improving wheat scab and banded sclerotial blight resistance. The nucleotide sequence of the TaRFS4B gene is as shown in SEQ ID No.1, and the nucleotide sequence of a coding region is as shown in SEQ ID No.2. The invention further discloses application of the TaRFS4B gene in synergistically improving wheat scab and banded sclerotial blight resistance. The over-expression of the TaRFS4B gene can improve the resistance of wheat to gibberellic disease and banded sclerotial blight at the same time, provides an effective technical means for prevention and control of the two diseases in production, further accelerates the breeding process of wheat disease resistance, perfects the prevention and control measures of the diseases, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to wheat. TaRFS4B Application of genes in the synergistic improvement of wheat resistance to Fusarium head blight and sheath blight. Background Technology

[0002] Fusarium head blight and sheath blight are two important fungal diseases affecting wheat production globally, often occurring in warm and humid climates. Fusarium head blight is primarily caused by the Fusarium complex (…). Fusarium graminearum The disease is caused by *Fusarium graminearum*, a species complex, whose dominant species is *Fusarium graminearum*. It infects the wheat ear tissue during the flowering stage, causing whiteheads and leading to severe yield reduction (Bai et al., 2018). During infection, *Fusarium graminearum* secretes fungal toxins, including deoxynivalenol (DON), which contaminate wheat grains. Consuming contaminated grains can cause symptoms such as vomiting and diarrhea in humans and animals (Chen et al., 2019). Wheat sheath blight, also known as sharp eyespot, is mainly caused by *Rhizoctonia graminearum* (…). Rhizoctonia cerealis The pathogen primarily infects the base of wheat stems, forming grayish-brown eye-like lesions on the leaf sheaths. This causes necrosis of the wheat stem tissue, leading to lodging, whiteheads, and other phenomena, resulting in severe wheat yield reduction (Lu et al., 2023). In recent years, influenced by factors such as climate warming, increased nitrogen fertilizer application, insufficient straw return to the field, and crop rotation systems, Fusarium head blight and sheath blight have become frequent and prevalent diseases of wheat throughout the country (Liu et al., 2023; Bai et al., 2018; Ma et al., 2019).

[0003] Currently, the control of wheat scab and sheath blight in actual production adopts comprehensive control measures, including cultivation management, planting disease-resistant varieties, application of chemical pesticides, and application of biocontrol bacteria. However, practice shows that breeding and planting disease-resistant varieties is the most economical and effective strategy to reduce the damage caused by wheat diseases. In actual production, if susceptible wheat varieties are planted, even if other control measures are implemented to the fullest extent, it is difficult to effectively curb the occurrence of diseases. Discovering new disease-resistant genes and verifying their disease-resistant functions is the key to improving the efficiency of wheat disease-resistant breeding. Wheat resistance to scab and sheath blight is a quantitative trait, controlled by as many as hundreds of quantitative trait loci, and no immune genes have been discovered yet (Liu Caiyun et al., 2023; Lv et al., 2023; Zheng et al., 2021). Although many disease-resistant quantitative trait loci have been discovered, the genes corresponding to these loci are unknown, and most loci do not have usable molecular markers; in addition, no genes that are resistant to both diseases have been discovered, which further limits the application of breeding (Ma et al., 2025). Therefore, continuously discovering new disease-resistant genes, especially those resistant to two diseases, and analyzing their disease-resistant mechanisms, and applying them to disease-resistant breeding, is a long-term strategy to deal with the threat of diseases. Summary of the Invention

[0004] The lack of disease-resistant genes that resist both Fusarium head blight and sheath blight is a major bottleneck restricting wheat disease resistance breeding. This invention provides a wheat... TaRFS4B Application of gene in synergistic improvement of wheat resistance to Fusarium head blight and sheath blight, overexpression TaRFS4B Genes can enhance wheat's resistance to Fusarium head blight and sheath blight by increasing hydrogen peroxide content.

[0005] To achieve the above objectives, the present invention provides, in one aspect, a wheat TaRFS4B Application of genes in the synergistic improvement of wheat resistance to Fusarium head blight and sheath blight. TaRFS4B The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the nucleotide sequence of the coding region is shown in SEQ ID No. 2.

[0006] Specifically, by overexpressing in wheat TaRFS4B Genes are used to enhance wheat resistance to Fusarium head blight and sheath blight.

[0007] Furthermore, by overexpressing in wheat TaRFS4B The gene promotes the accumulation of hydrogen peroxide, thereby enhancing resistance to wheat scab and sheath blight.

[0008] TaRFS4B The kinase activity of the gene-encoded protein is upregulated in response to infection by Fusarium head blight and Rhizoctonia solani. The amino acid sequence of the encoded protein is shown in SEQ ID No. 3.

[0009] The second aspect of the present application provides a method for synergistically improving the resistance of wheat to fusarium head blight and sharp eyespot, comprising the step of overexpressing the gene in wheat. TaRFS4B The nucleotide sequence of the gene is shown as SEQ ID No. 1, and the nucleotide sequence of the coding region is shown as SEQ ID No. 2. TaRFS4B The nucleotide sequence of the gene is shown as SEQ ID No. 1, and the nucleotide sequence of the coding region is shown as SEQ ID No. 2.

[0010] Through the above technical solution, the present application achieves the following beneficial effects: overexpressing TaRFS4B The gene can simultaneously improve the resistance of wheat to fusarium head blight and sharp eyespot, providing an effective technical means for preventing and controlling the two diseases in production, thereby accelerating the breeding process of wheat resistance, perfecting the prevention and control measures of diseases, and having a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a flow chart for identifying and isolating the gene of the present application TaRFS4B and verifying the function of the gene TaRFS4B is a flow chart for identifying and isolating the gene of the present application Figure 2 is the expression amount analysis of the gene before and after Ningmai No. 9 is inoculated with fusarium head blight fungus and sharp eyespot fungus; wherein 0 hours is the sample before inoculation, and the others are the samples at the corresponding time points after inoculation TaRFS4B The expression amount of the gene in each sample is relative to the expression amount of the gene in the sample before inoculation TaRFS4B The expression amount of the gene in each sample is relative to the expression amount of the gene in the sample before inoculation TaRFS4B Each data is an average value (3 replicates) ± standard deviation, and “**” indicates that there is a significant difference compared with 0 hours (P<0.01). P <0.01). Figure 3 is a structural schematic diagram of the gene; wherein the black rectangle represents the coding region, the white rectangle represents the 5' and 3' untranslated region (untranslated region, UTR), “ATG” and “TAG” are the translation start and stop codes respectively, and the numbers represent the number of nucleotides of each structure. TaRFS4B is expressed and purified in E. coli (A), and the kinase activity analysis is carried out in vitro (B); in the figure, A is the polyacrylamide gel chart after coomassie brilliant blue staining and decolorization, B is the result of phosphorylation of TaRFS4B protein myelin basic protein (MBP), anti-pS / T is a phosphorylated serine / threonine antibody, and His:TaRFS4B is a prokaryotic expression protein of TaRFS4B fused with a histidine (His) tag. Figure 4 Figure 5 ​TaRFS4B protein, and the kinase activity of TaRFS4B was analyzed. 0 hour is the sample before inoculation, and the other samples are the samples at the corresponding time points after inoculation. MBP is myelin basic protein, anti-pS / T is a phosphoserine / threonine antibody, and anti-TaRFS4B is a specific antibody for recognizing TaRFS4B; Figure 6 is a wheat plant overexpressing the gene in a susceptible wheat variety Fielder, and the expression level of the gene in the T0 generation of the genetically transformed plant is detected; wherein TaRFS4B the expression level of the gene is relative to the expression level of the gene in the control Fielder, and each data is an average value (3 replicates) ± standard deviation. “**” indicates that the expression level of the gene in the transgenic plant is significantly (P<0.01) increased compared with the control; TaRFS4B TaRFS4B the expression level of the gene is relative to the expression level of the gene in the control Fielder, and each data is an average value (3 replicates) ± standard deviation. “**” indicates that the expression level of the gene in the transgenic plant is significantly (P<0.01) increased compared with the control; TaRFS4B TaRFS4B the expression level of the gene is significantly (P<0.01) increased compared with the control; P Figure 7 is a wheat plant overexpressing the gene, and the resistance of the T2 generation of the overexpression plant to scab is identified; wherein A is a lesion map 14 days after inoculation, and B is a statistical result of diseased spikelets. “**” indicates that the disease rate of the overexpression plant is significantly (P<0.01) reduced compared with the control Fielder; TaRFS4B TaRFS4B the disease index of the overexpression plant is significantly (P<0.01) reduced compared with the control; P Figure 8 is a wheat plant overexpressing the gene, and the resistance of the T2 generation of the overexpression plant to sheath blight is identified; wherein A is a lesion map 21 days after inoculation, and B is a calculation result of disease index. “**” indicates that the disease index of the overexpression plant is significantly (P<0.01) reduced compared with the control Fielder; TaRFS4B TaRFS4B the hydrogen peroxide content of the overexpression plant is significantly (P<0.01) increased compared with the control; P Figure 9 is a wheat plant overexpressing the gene, and the hydrogen peroxide content in the leaves, leaf sheaths and spikes of the T2 generation of the overexpression plant is analyzed; wherein A is a picture of wheat leaves after 30 days of growth in a greenhouse, B is a picture of wheat leaves after DAB staining and alcohol decolorization after 30 days of growth in a greenhouse, C and D are the hydrogen peroxide contents in the leaf sheaths of seedlings and the spike tissues of wheat at the flowering stage, respectively. “**” indicates that the hydrogen peroxide content of the overexpression plant is significantly (P<0.01) increased compared with the control Fielder; TaRFS4B TaRFS4B the hydrogen peroxide content of the overexpression plant is significantly (P<0.01) increased compared with the control; P DETAILED DESCRIPTION

[0012] ​​​​​​​​​The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0013] like Figure 1 As shown, the technical solution of the present invention is as follows: Based on the sequence information of the sequenced wheat variety TraesCS4B02G239400 at the sequencing site, primers are designed to detect the effects of wheat inoculation with Fusarium head blight and sheath blight before and after inoculation. TaRFS4B Changes in gene expression levels; obtained from the disease-resistant wheat variety Ningmai 9 using RT-PCR technology. TaRFS4B The coding region sequence of the gene was determined, and the protein encoded by this gene was expressed in *E. coli*. The kinase activity of the protein was verified by in vitro phosphorylation experiments. Preparation... TaRFS4B Antibodies were obtained from wheat before and after inoculation with Fusarium head blight and sheath blight using immunoprecipitation technology. TaRFS4B The encoded protein was analyzed, and its kinase activity was examined before and after inoculation. Agrobacterium-mediated genetic transformation was used to overexpress the protein in the wheat variety Fielder, which is susceptible to Fusarium head blight and sheath blight. TaRFS4B Genes were used to determine hydrogen peroxide content in genetically transformed plants and to analyze resistance to Fusarium head blight and sheath blight, demonstrating overexpression. TaRFS4B The gene can enhance wheat's resistance to Fusarium head blight and sheath blight by increasing hydrogen peroxide content. See the following examples for details.

[0014] Example 1: Cloning from wheat variety Ningmai 9 TaRFS4B Gene 1. After treatment with Fusarium head blight and Sheath blight pathogens, TaRFS4B Gene expression pattern analysis To verify protein kinase genes TaRFS4B ( Triticum aestivum R esistance to F usariumhead blight and S harp eyespot on chromosome 4B Whether it participates in regulating the wheat response to Fusarium head blight and Sheath blight was analyzed using quantitative reverse transcription-PCR (qRT-PCR) technology. TaRFS4B The expression patterns of genes in wheat variety Ningmai 9 (moderately resistant to Fusarium head blight, moderately resistant to moderate sheath blight) before and after inoculation with Fusarium head blight and sheath blight, respectively.

[0015] Inoculation of scab was performed by single flower injection method (Jiang et al., 2020). The scab strain used in the present application was the international reference strain Fusarium graminearum strain NRRL31084 (PH-1). The strain was cultured on potato dextrose agar medium (PDA) at 25°C for 3 days until the mycelium covered the surface of the medium. Then a little mycelium was picked and placed in green bean broth liquid medium and cultured at 25°C in a shaker for 3 days (at a speed of 180 r / min) to produce conidia and stored in a refrigerator at 4°C. Before inoculation, 10 μL of the bacterial solution was taken and examined under a microscope to calculate the number of spores, and the spore solution was diluted to 1×10 5 When the wheat grew to the flowering stage, 10 μL of the spore solution was injected into the newly opened spike, and the spikes before inoculation, 10 hours after inoculation, and 24 hours after inoculation were cut and frozen in liquid nitrogen for subsequent analysis.

[0016] Inoculation of sharp eyespot was performed by diseased wheat grain soil transmission inoculation method (Ren et al., 2020). The sharp eyespot strain used in the present application was the high pathogenic race R0301 of Rhizoctonia cerealis (donated by Professor Chen Huai'gu of Jiangsu Academy of Agricultural Sciences Institute of Plant Protection). The strain was cultured on PDA at 25°C for 3 days until the mycelium covered the surface of the medium and was stored in a refrigerator at 4°C. Full-grain wheat seeds were prepared, washed with tap water, soaked overnight, then boiled (without hard kernels and without bursting), placed on filter paper to dry the surface moisture, then transferred to a conical flask so that the volume was about one fourth of the total volume of the conical flask, and autoclaved at high temperature and high pressure for 45 minutes. After cooling, the mycelial block on the PDA medium was placed on the wheat grains in the conical flask for culture. The conical flask was cultured at 25°C in the dark, and the growing mycelial block was shaken once a day to scatter. 0.8 g of diseased wheat grains was mixed with 170 g of sterilized soil, and planted in a square box, then the 7-day-old wheat seedlings were planted in the square box and cultured in an incubator. The base tissues of the wheat seedlings before inoculation, 1 day after inoculation, and 2 days after inoculation were cut and frozen in liquid nitrogen for subsequent analysis.

[0017] The total RNA of the above-mentioned samples was extracted by using TransZol Universal RNA Extraction Kit (Beijing Zomanbio Technology Co., Ltd.) according to the method provided in the instruction manual attached to the kit. 5 μg of total RNA was taken, DNase I (Invitrogen, USA) was added to remove genomic DNA, and then used as a template for reverse transcription to obtain total cDNA by using oligo(dT) 18 oligomer primers and M-MLV reverse transcriptase (Invitrogen, USA).

[0018] The whole genome of the wheat variety *Chinese Spring* has been sequenced. Based on the sequence information of the TraesCS4B02G239400 site in this variety, a pair of qRT-PCR primers, TaRFS4B-RT-1F (5′-TGTGGAGCCTCGGTATGG-3′) and TaRFS4B-RT-1R (5′-GCAGATGGCGCACATGA-3′), were designed for detection. TaRFS4B Gene expression levels. The relative expression levels of genes were measured using the expression level of the wheat endogenous actin gene and the RNA content of the sample was normalized. The detection primers were TaAct-RT-1F (5′-GACAGGATGAGCAAGGAGAT-3′) and TaAct-RT-1R (5′-CCGATCCACACACTGTACTT-3′).

[0019] The experimental results showed that 10 hours after inoculation with Fusarium graminearum, TaRFS4B The expression level of [the substance] increased significantly 24 hours after vaccination. TaRFS4B The expression level of [the pathogen] remained significantly higher than before inoculation; after inoculation with *Rhizoctonia solani*, [the pathogen's] expression level was still significantly higher than before inoculation. TaRFS4B The expression level gradually increased, significantly higher than before vaccination. Figure 2 This result suggests that... TaRFS4B The gene may be involved in regulating wheat's resistance to both Fusarium head blight and sheath blight, and enhancing the expression of this gene may synergistically improve wheat's resistance to these two diseases.

[0020] 2. TaRFS4B Gene cloning and structural analysis Using the total cDNA of Ningmai 9 obtained above as a template, a pair of PCR primers TaRFS4B-CDS-1F (5′-CAGCAAATGGGTCGC) were used. GGATCC ATGGCTCTCGTCCGGCAG-3′, underlined Bam HI restriction site), TaRFS4B-CDS-1R (5′-GTGGTGGTGGTGGTG CTCGAG CTACTGGTCGCCCAGCTCC-3′, underlined Xho (I restriction site) amplified to obtain TaRFS4B The coding region cDNA of the gene was obtained. Using Ningmai 9 DNA as a template, amplification was performed using a pair of PCR primers TaRFS4B-genome-1F (5′-ACCACTCCATATATACCAGCAGCGA -3′) and TaRFS4B-genome-1R (5′-ATGAAGAAGGTATCAATTCTCAAAACTG -3′). TaRFS4B DNA of genes.

[0021] The PCR products were separated by 1% agarose gel electrophoresis, and the amplified products were recovered using an agarose gel DNA recovery kit (Tiangen Biotech (Beijing) Co., Ltd.). TaRFS4B The gene's DNA was ligated to the TA cloning vector pGEM-T (Promega, USA). TaRFS4B The coding region cDNA of the gene was ligated into the prokaryotic expression vector pET-28a (Novagen, Germany). Positive clones were sequenced and compared. TaRFS4B The genome sequence and coding region cDNA sequence were determined. TaRFS4B The gene consists of 1307 nucleotides (SEQ ID No. 1), has only one exon, and no introns; its coding region consists of 996 nucleotides (SEQ ID No. 2). Figure 3 ), which encodes 331 amino acids (SEQ ID No. 3).

[0022] Example 2: TaRFS4B kinase activity analysis of gene-encoded proteins 1. In vitro kinase activity analysis of TaRFS4B prokaryotic expression protein TaRFS4B The gene encodes a protein kinase. To verify whether the TaRFS4B protein possesses kinase activity, the protein containing the enzyme constructed in Example 1 above was used. TaRFS4B The prokaryotic expression vector pET-28a, containing the cDNA coding region, was transformed into *E. coli* expression strain BL21(DE3) (Shenzhen Kangti Life Science Technology Co., Ltd.) to express the TaRFS4B protein in *E. coli*. Protein expression was performed according to the instructions provided with the pET-28a vector. Protein purification was performed using a 6×His fusion protein purification kit (Qiagen, Germany), following the manufacturer's instructions. The purified protein was added to 5×SDS-PAGE loading buffer (Shanghai Sangon Biotech Co., Ltd.), incubated at 95°C for 5 minutes, separated by 10% polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue, and photographed.

[0023] The protein kinase activity was analyzed by in vitro phosphorylation method, using commercial myelin basic protein extracted from guinea pig brain (Merck, USA) as the phosphorylation substrate. The specific steps were as follows: 0.4 μg of TaRFS4B protein was incubated with 4 μg of myelin basic protein in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP at room temperature for 1 hour, then 5x SDS-PAGE loading buffer was added, 95°C metal bath for 5 minutes, after cooling to room temperature, the proteins were separated by 12% polyacrylamide gel, and then transferred to PVDF membrane (Nanjing Novibio Biotech Co., Ltd.). The PVDF membrane was incubated with 5% skim milk solution at room temperature for 1 hour, then commercial phosphorylated serine / threonine antibody (ECM Biosciences, USA) was added and incubated at room temperature for 2 hours, and then IgG antibody conjugated with horseradish peroxidase (Abmole Biotechnology Co., Ltd.) was added and incubated at room temperature for 1 hour. The horseradish peroxidase substrate was evenly spread on the PVDF membrane, and the membrane was placed in a full-automatic chemiluminescence image analyzer (Shanghai Tianheng Technology Co., Ltd.) for observation and photography.

[0024] The protein purification results showed that the size of TaRFS4B protein was about 36 kDa, which was consistent with the predicted size (see Figure A in Figure 4 The in vitro kinase activity test results showed that TaRFS4B protein had autophosphorylation activity and could phosphorylate myelin basic protein (see Figure B in Figure 4 These results showed that TaRFS4B protein had the ability of autophosphorylation and phosphorylation of substrate, and was a typical protein kinase.

[0025] 2. Analysis of TaRFS4B kinase activity in wheat after treatment with Gibberella and Rhizoctonia In order to verify whether the kinase activity of TaRFS4B protein in wheat responds to the invasion of Gibberella and Rhizoctonia, the specific antibody of TaRFS4B protein was purified from rabbit serum immunized with the above-mentioned prokaryotic expression protein of TaRFS4B.

[0026] The total protein was extracted from the wheat tissue treated with Gibberella and Rhizoctonia in Example 1. The method was as follows: the wheat tissue was ground into powder with liquid nitrogen and a mortar, then transferred to a centrifuge tube, and extraction buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 5 mM EGTA (pH 7.0), 5 mM Na3VO 4，10 mM NaF, 50 mM β-glycerophosphate, 10% glycerol, and a complete EDTA-free protease inhibitor cocktail were placed on a centrifuge and incubated at 4°C for 20 minutes. After sonication, the mixture was lysed twice and centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was then transferred to a new centrifuge tube, which yielded the total protein from wheat.

[0027] Antibody against TaRFS4B protein was added to the extracted total wheat protein, and the mixture was incubated at 4°C for 10 hours using a centrifuge. Then, protein A magnetic beads (Shanghai Taoshu Biotechnology Co., Ltd.) were added and incubated for another 4 hours. The protein A beads were adsorbed using a magnet, and the supernatant was discarded. The beads were washed five times with the protein extraction buffer described above. The beads were then resuspended in kinase buffer (25 mM Tris-HCl (pH 7.5), 1 mM EGTA, 12 mM MgCl2, 0.1 mM Na3VO4, 0.3 mM ATP). The adsorbed protein on the beads was the TaRFS4B protein from wheat. 5 µg of myelin basic protein was added to the resuspended solution as a phosphorylation substrate, and protein kinase activity was analyzed using the method described in Example 1.

[0028] The results showed that after wheat was inoculated with Fusarium head blight and sheath blight, respectively, the kinase activity of TaRFS4B protein was significantly increased. Figure 5 These results indicate that the kinase activity of the TaRFS4B protein can respond to infection by Fusarium head blight and Sheath blight.

[0029] Example 3: TaRFS4B Application of genes in improving resistance to wheat scab and sheath blight 1. TaRFS4B Obtaining overexpressing plants and analyzing expression levels Will TaRFS4B The coding region of the gene was linked to the plant expression vector pWMB110 (the plant expression vector pWMB110 was donated by Researcher He Yi of Jiangsu Academy of Agricultural Sciences, and is mentioned in the literature "Wang K, Shi L, Liang X, et al. The gene..."). TaWOX5overcomes genotype dependency in wheat genetic transformation. NatPlants. 2022, 8:110-117” and the paper “Cloning and functional analysis of several genes related to Agrobacterium transformation and tissue culture regeneration in wheat” disclosed that the public can obtain the above biological materials from the applicant, and the above biological materials are only used for repeating the experiments of the present application and cannot be used for other purposes), which carries a maize ubiquitin promoter with constitutive and overexpression characteristics. The successfully constructed vector is introduced into the susceptible wheat variety Fielder using Agrobacterium-mediated genetic transformation method (Zhou et al., 2024).

[0030] The present application obtains a total of 6 independently transformed plants. The plants are planted in a greenhouse, and the leaves of the transformed plants are taken at the booting stage, the RNA is extracted, and the expression amount of the TaRFS4B gene in the transformed plants is detected by qRT-PCR technology in Example 1. The results show that the expression amount of the TaRFS4B gene in 5 of the genetically transformed plants is significantly increased compared with the control Fielder P <0.01) Figure 6 .

[0031] 2. TaRFS4B Analysis of disease resistance of overexpression plants The T0 generation TaRFS4B of the genetically transformed plants with increased expression of the gene No. 1 and No. 5 are selected for breeding, and the resistance analysis of scab and sharp eyespot is carried out at the T2 generation.

[0032] The scab spore solution is injected into the wheat ear at the flowering stage of wheat, and the investigation is carried out after two weeks of inoculation, and the resistance is measured by the rate of diseased spike (number of diseased spike / total number of spike x 100%). The results show that the scab resistance of the genetically transformed plants with increased expression of the TaRFS4B gene is significantly enhanced compared with the control Fielder P <0.01) Figure 7 .

[0033] The identification of sharp eyespot resistance is carried out at the seedling stage. Using the method in Example 1, the diseased wheat grains are mixed with sterilized soil, and the sterilized wheat seeds are planted in it and placed in an incubator for culture. After three weeks of culture, the disease identification is carried out according to the reported method (Ren et al., 2020), and the resistance is measured by the disease index. The results show that the sharp eyespot resistance of the genetically transformed plants with increased expression of the TaRFS4B gene is significantly enhanced compared with the control Fielder P <0.01) Figure 8 .

[0034] 3. TaRFS4B Analysis of hydrogen peroxide content of overexpression plants TaRFS4B After three weeks of growth in the greenhouse, severe cell death was observed on the leaves of the overexpression plants, but not on the control Fielder (see Fig. A of Figure 9 To verify whether the cell death was caused by hydrogen peroxide accumulation, the leaves were stained with DAB solution (Kuailaibo Technology Co., Ltd., Beijing), and then decolorized with 95% alcohol, TaRFS4B Brown patches appeared on the leaves of the overexpression plants (see Fig. B of Figure 9 , indicating that they accumulated a large amount of hydrogen peroxide, suggesting that the overexpression of TaRFS4B caused hydrogen peroxide accumulation.

[0035] Hydrogen peroxide accumulation is an important means for plants to resist pathogens. To verify whether the disease resistance of the overexpression plants was caused by hydrogen peroxide accumulation, the hydrogen peroxide content in the leaf sheaths and spikelets of the overexpression plants was detected using a commercial hydrogen peroxide detection kit (Shanghai Shenguo Biological Engineering Co., Ltd.). TaRFS4B The results showed that, compared with the control Fielder, the hydrogen peroxide content in the leaf sheaths and spikelets of the overexpression plants was significantly increased (P<0.01) (see Figs. C and D of TaRFS4B TaRFS4B P <0.01) (see Figs. C and D of Figure 9 ).

[0036] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0037] In addition, it should be noted that, in the above specific embodiments, each specific technical feature described can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

[0038] In addition, various different embodiments of the present application can also be combined in any manner, as long as they do not deviate from the technical concept of the present application, and they should also be considered as disclosed by the present application.​​

Claims

1. A type of wheat TaRFS4B The application of genes in the synergistic improvement of wheat resistance to Fusarium head blight and sheath blight is characterized by, TaRFS4B The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the nucleotide sequence of the coding region is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, By overexpressing in wheat TaRFS4B Genes can be used to enhance wheat resistance to Fusarium head blight and sheath blight.

3. The application according to claim 2, characterized in that, By overexpressing in wheat TaRFS4B The gene promotes the accumulation of hydrogen peroxide, thereby enhancing resistance to wheat scab and sheath blight.

4. The application according to claim 1, characterized in that, TaRFS4B The kinase activity of the gene-encoded protein is upregulated in response to infection by Fusarium head blight and Rhizoctonia solani. The amino acid sequence of the encoded protein is shown in SEQ ID No.

3.

5. A method for synergistically enhancing resistance to Fusarium head blight and sheath blight in wheat, characterized in that, Including overexpression in wheat TaRFS4B The steps of gene generation; TaRFS4B The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the nucleotide sequence of the coding region is shown in SEQ ID No. 2.

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