Application of wheat taRFS4B gene in synergistically improving resistance to fusarium head blight and sharp eyespot

Abstract

The application discloses a kind of wheat TaRFS4B The application discloses application of a 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 gene can simultaneously improve the resistance of wheat to fusarium head blight and sheath blight, provides an effective technical means for preventing and controlling the two diseases in production, further speeds up the wheat breeding process, perfects the prevention and control measures of diseases, and has 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 can be 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] A second aspect of the present invention provides a method for synergistically enhancing resistance to Fusarium head blight and sheath blight in wheat, comprising overexpressing 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.

[0010] Through the above technical solution, the present invention achieves the following beneficial effects: Overexpression TaRFS4B The gene can simultaneously enhance wheat's resistance to Fusarium head blight and sheath blight, providing an effective technical means for controlling these two diseases in production, thereby accelerating the process of wheat disease-resistant breeding and improving disease control measures, and has broad application prospects. Attached Figure Description

[0011] Figure 1 This invention identifies and separates... TaRFS4B Genes and verification TaRFS4B Flowchart of gene function; Figure 2 These are Ningmai 9 wheat varieties before and after inoculation with Fusarium head blight and Sheath blight pathogens, respectively. TaRFS4B Gene expression level analysis; where 0 hours represents samples before inoculation, and the others represent samples at corresponding time points after inoculation. The expression levels in each sample... TaRFS4B Gene expression levels are relative to the levels in the sample before inoculation. TaRFS4B Gene expression levels, each data point is the mean (3 replicates) ± standard deviation, "**" indicates a significant difference compared to 0 hours. P <0.01); Figure 3 yes TaRFS4B A schematic diagram of a gene structure; where black rectangles represent coding regions, white rectangles represent 5′ and 3′ untranslated regions (UTRs), “ATG” and “TAG” are the start and stop codons, respectively, and numbers indicate the number of nucleotides in each structure; Figure 4 The TaRFS4B protein was expressed and purified in E. coli (A), and its kinase activity was analyzed in vitro (B). In the figure, A is a polyacrylamide gel image after Coomassie brilliant blue staining and destaining, B is the result of TaRFS4B protein phosphorylating myelin basic protein (MBP), anti-pS / T is a phosphorylated serine / threonine antibody, and His:TaRFS4B is the prokaryotic expression protein of TaRFS4B fused with a histidine (His) tag. Figure 5This is an antibody for preparing TaRFS4B protein. TaRFS4B was immunoprecipitated from Ningmai 9 before and after inoculation with Fusarium head blight (A) and sheath blight (B), respectively, and its kinase activity was analyzed. Among them, 0 hours is the sample before inoculation, and the others are the samples at the corresponding time points after inoculation. MBP is myelin basic protein, anti-pS / T is phosphorylated serine / threonine antibody, and anti-TaRFS4B is a specific antibody that recognizes TaRFS4B. Figure 6 Overexpression in the susceptible wheat variety Fielder TaRFS4B Genes were detected in T0 generation genetically transformed plants. TaRFS4B Gene expression levels; among which TaRFS4B Gene expression levels are relative to the control Fielder. TaRFS4B Gene expression levels, each data point is the mean (3 replicates) ± standard deviation, "**" indicates that compared with the control, the expression level in transgenic plants was higher. TaRFS4B Gene expression levels were significantly higher ( P <0.01) increase; Figure 7 yes TaRFS4B Identification of Fusarium head blight resistance in plants with overexpressed genes (T2 generation); where A shows lesion images 14 days after inoculation, B shows the statistical results of diseased spikelet incidence, and "**" indicates a comparison with the control Fielder. TaRFS4B The disease spikelet rate of overexpressing plants was significantly higher. P <0.01) decreases; Figure 8 yes TaRFS4B Identification of sheath blight resistance in plants with overexpressed genes (T2 generation); where A shows lesion images 21 days after inoculation, B shows the calculated disease index, and "**" indicates a comparison with the control Fielder. TaRFS4B The disease index of overexpressing plants was significantly higher ( P <0.01) decreases; Figure 9 yes TaRFS4B Analysis of hydrogen peroxide content in leaves, leaf sheaths, and spikelets of wheat plants with overexpressed genes (T2 generation); where A is an image of wheat leaves after 30 days of greenhouse growth, B is an image of wheat leaves after 30 days of greenhouse growth stained with DAB and destained with alcohol, C and D are the hydrogen peroxide contents in the leaf sheaths of wheat seedlings and the spike tissues of wheat at the flowering stage, respectively, and "**" indicates the comparison with the control Fielder. TaRFS4B The hydrogen peroxide content in the overexpressing plants was significantly higher. P <0.01) increases. Detailed Implementation

[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 for Fusarium head blight was performed using the single-flower drip method (Jiang et al., 2020). The Fusarium head blight strain used in this invention was the international reference strain, *Fusarium graminearum* strain NRRL31084 (PH-1). The strain was cultured on potato dextrose agar (PDA) at 25°C for 3 days until the mycelium covered the surface of the medium. A small amount of mycelium was then placed in mung bean soup liquid medium and cultured on a shaker at 25°C for 3 days (180 r / min) to induce conidia production, and then stored at 4°C. Before inoculation, 10 μL of the bacterial solution was examined under a microscope to count the number of spores. The spore concentration was then diluted to 1 × 10⁻⁶ using mung bean soup liquid medium. 5 spores / mL. When wheat reaches the flowering stage, 10 μL of spore solution is extracted and injected into newly flowering spikelets. Spikelets are collected before inoculation, 10 hours after inoculation, and 24 hours after inoculation, and frozen in liquid nitrogen for subsequent analysis.

[0016] Inoculation for sheath blight was performed using soil-borne inoculation with diseased wheat grains (Ren et al., 2020). The sheath blight strain used in this invention is the highly pathogenic race R0301 of Rhizoctonia graminearum (a gift from Researcher Chen Huaigu of the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences). The strain was cultured in a PDA culture medium at 25°C for 3 days until the mycelium covered the surface of the culture medium, and then stored at 4°C for later use. Plump wheat seeds were prepared, rinsed with tap water, soaked overnight, then cooked (without hardening or cracking), dried on filter paper, and transferred to Erlenmeyer flasks, filling the flasks to approximately one-quarter of their total volume. The flasks were then autoclaved for 45 minutes. After cooling, mycelial blocks from the PDA culture medium were placed on top of the wheat grains in the Erlenmeyer flasks for further cultivation. The flasks were incubated at 25°C in the dark for one week, with the wheat grains shaken daily to disperse the growing mycelial blocks. Mix 0.8 g of diseased wheat grains with 170 g of sterilized soil and place the mixture into a planting box. Then, plant wheat seedlings that have germinated for 7 days into the box and place them in an incubator. Take tissue samples from the base of the wheat seedlings before inoculation, 1 day after inoculation, and 2 days after inoculation, and freeze them in liquid nitrogen for subsequent analysis.

[0017] Total RNA was extracted from the samples using the TransZol standard RNA extraction kit (Beijing TransGen Biotech Co., Ltd.) following the instructions provided with the kit. 5 μg of total RNA was added to DNase I (Invitrogen, USA) to remove genomic DNA, and then used as a template for oligo(dT) extraction. 18 Total cDNA was obtained by reverse transcription using oligomeric 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 substance] remained significantly higher than before inoculation; after inoculation with *Rhizoctonia solani*, [the 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] Protein kinase activity was analyzed using an in vitro phosphorylation method, with commercially available myelin basic protein extracted from guinea pig brains (Merck, USA) as the phosphorylation substrate. The specific steps were as follows: 0.4 µg of TaRFS4B protein and 4 µg of myelin basic protein were incubated at room temperature for 1 hour in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 1 mM ATP. Then, 5×SDS-PAGE loading buffer was added, and the mixture was incubated in a 95°C metal bath for 5 minutes. After cooling to room temperature, the mixture was separated using a 12% polyacrylamide gel, and the protein on the gel was electrotransferred to a PVDF membrane (Nanjing Novizan Biotechnology Co., Ltd.). The PVDF membrane was incubated with 5% skim milk solution at room temperature for 1 hour. A commercially available phosphorylated serine / threonine antibody (ECM Biosciences, USA) was added, and the membrane was incubated at room temperature for 2 hours. Finally, the membrane was incubated with an IgG antibody conjugated with horseradish peroxidase (Aibotek Biotechnology Co., Ltd.) at room temperature for 1 hour. Horseradish peroxidase substrate was evenly spread on a PVDF membrane, and the membrane was placed in a fully automated chemiluminescence imaging analyzer (Shanghai Tianneng Technology Co., Ltd.) for observation and photography.

[0024] Protein purification results showed that the TaRFS4B protein size was approximately 36 kDa, consistent with the predicted size (see [link to study]). Figure 4 (See Figure A in the diagram). In vitro kinase activity assays showed that TaRFS4B protein possesses autophosphorylation activity and can phosphorylate myelin basic proteins (see Figure A in the diagram). Figure 4 (See Figure B in the diagram). These results indicate that the TaRFS4B protein possesses the ability to autophosphorylate and phosphorylate substrates, making it a typical protein kinase.

[0025] 2. Analysis of TaRFS4B kinase activity in wheat after treatment with Fusarium head blight and Sheath blight pathogens. To verify whether the kinase activity of TaRFS4B protein in wheat responds to infection by Fusarium head blight and Sheath blight, rabbits were immunized with the prokaryotic expression protein of TaRFS4B as an antigen, and specific antibodies against TaRFS4B protein were purified from rabbit serum.

[0026] Total protein was extracted from wheat tissue treated with Fusarium head blight and Sheath blight pathogens as described in Example 1. The method was as follows: Wheat tissue was ground into powder using liquid nitrogen and a mortar, then transferred to centrifuge tubes. Extraction buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 5 mM EGTA (pH 7.0), 5 mM Na3VO4) was added. 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..."). TaWOX5The invention overcomes genotype dependency in wheat genetic transformation. NatPlants. 2022, 8:110-117. The paper "Cloning and functional analysis of several genes in wheat related to Agrobacterium-mediated transformation and tissue culture regeneration" discloses that the public can obtain the above-mentioned biological materials from the applicant (the obtained biological materials are only for repeating the experiments of this invention and cannot be used for other purposes). The vector carries a maize ubiquitin promoter with constitutive and overexpression characteristics. The successfully constructed vector was introduced into the susceptible wheat variety Fielder using Agrobacterium-mediated genetic transformation (Zhou et al., 2024).

[0030] This invention yielded a total of 6 independently transformed plants. The plants were grown in a greenhouse, and when they reached the heading stage, leaves were harvested, RNA was extracted, and the RNA was detected in the transformed plants using the qRT-PCR technique described in Example 1. TaRFS4B Gene expression levels. The results showed that, compared to the control Fielder, five genetically transformed plants showed higher expression levels. TaRFS4B Gene expression levels were significantly increased ( P <0.01)( Figure 6 ).

[0031] 2. TaRFS4B Disease resistance analysis of overexpression plants Select T0 generation TaRFS4B Genetically transformed plants numbered 1 and 5, whose gene expression levels were elevated, were propagated, and resistance to Fusarium head blight and sheath blight was analyzed in the T2 generation.

[0032] Fusarium graminearum spores were injected into wheat ears during the wheat flowering stage. Two weeks after inoculation, resistance was assessed using the diseased spikelet percentage (number of diseased spikelets / total number of spikelets × 100%). Results showed that, compared to the control Fielder, TaRFS4B Genetically transformed plants with increased gene expression levels showed significantly enhanced resistance to Fusarium head blight. P <0.01)( Figure 7 ).

[0033] Resistance to sheath blight was assessed during the seedling stage. Following the method described in Example 1, diseased wheat grains were mixed with sterilized soil, and sterilized wheat seeds were sown in the mixture and cultured in an incubator. After three weeks of culture, disease resistance was assessed using a disease index, as reported in previous methods (Ren et al., 2020). Results showed that, compared to the control Fielder, resistance to sheath blight was significantly lower. TaRFS4B Genetically transformed plants with increased gene expression levels showed significantly enhanced resistance to sheath blight. P <0.01)( Figure 8 ).

[0034] 3. TaRFS4B Analysis of hydrogen peroxide content in overexpressing plants TaRFS4B After three weeks of growth in a greenhouse, overexpressing plants exhibited severe cell death in their leaves, a phenomenon not observed in the control group Fielder (see [link to relevant documentation]). Figure 9 (See Figure A in the image). To verify whether cell death was caused by hydrogen peroxide accumulation, the leaves were stained with DAB stain (Beijing Cooler Master Technology Co., Ltd.), and then destained with 95% alcohol. TaRFS4B Brownish patches appeared on the leaves of the overexpressing plants (see...) Figure 9 Figure B in the diagram shows that a large amount of hydrogen peroxide has accumulated, suggesting... TaRFS4B Overexpression of [a substance] can lead to the accumulation of hydrogen peroxide.

[0035] Hydrogen peroxide accumulation is an important mechanism by which plants fight pathogens. To verify... TaRFS4B Whether the disease resistance of overexpressing plants is caused by hydrogen peroxide accumulation was determined using a commercially available hydrogen peroxide detection kit (Shanghai Sangon Biotech Co., Ltd.). TaRFS4B The hydrogen peroxide content in the leaf sheath and spikelets of the plant was overexpressed. Results showed that, compared to the control Fielder, TaRFS4B The hydrogen peroxide content in the leaf sheaths and spikelets of the gene-overexpressing plants was significantly increased. P <0.01) (see Figure 9 (Figures C and D in the diagram).

[0036] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0037] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0038] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

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. This was demonstrated by overexpression in wheat. TaRFS4B Genes can be used to enhance wheat resistance to Fusarium head blight and sheath blight.

2. The application according to claim 1, 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.

3. 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.

4. A method for synergistically enhancing resistance to wheat scab and sheath blight, 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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