Application of fg05119 gene in prevention and control of wheat scab

Abstract

The application belongs to the technical field of plant disease prevention and treatment, and discloses Fg05119 application of a gene in prevention and control of wheat scab. The gene is involved in regulation of pathogenicity and DON toxin biosynthesis of a wheat scab pathogen Fusarium graminearum. Fg05119 The nucleotide sequence of the gene is shown as SEQ ID NO:1. The gene is involved in regulation of pathogenicity and DON toxin biosynthesis of a wheat scab pathogen Fusarium graminearum. Fg05119 The gene has application potential as a double-function molecular target for prevention and control of wheat scab and control of DON toxin pollution, and has wide application prospect. Fg05119 The gene has application potential as a double-function molecular target for prevention and control of wheat scab and control of DON toxin pollution, and has wide application prospect. Fg05119 The gene has application potential as a double-function molecular target for prevention and control of wheat scab and control of DON toxin pollution, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, and relates to... Fg05119 Application of genes in the control of wheat scab. Background Technology

[0002] Fusarium graminearum ( Fusarium graminearum Fusarium graminearum is one of the main pathogenic fungi causing wheat scab (FHB). This pathogen is highly adaptable and has a wide spread, easily infecting plants during the wheat flowering stage, causing dual damage to wheat yield and quality. After infecting wheat plants, Fusarium graminearum disrupts the normal growth and development of wheat ears and grains, leading to reduced wheat yield. During infection, the pathogen also produces fungal toxins such as deoxynivalenol (DON). These toxins are highly stable, resistant to high temperatures and processing, and can remain in wheat grains, straw, and other tissues for extended periods. They can enter the bodies of humans and animals through the grain and feed supply chain, and can also accumulate through the food chain, continuously harming human and animal health.

[0003] Currently, the control of wheat scab mainly relies on chemical agents, which have the advantages of rapid effectiveness, convenient operation, and wide applicability. However, long-term, single, and high-frequency use of chemical agents can easily lead to the continuous accumulation and expansion of drug-resistant pathogens in the field, resulting in a gradual decline in the control effect of conventional fungicides. At the same time, chemical residues can pollute farmland soil and aquatic ecosystems, disrupt the structure of field microbial communities, and cause agricultural ecological imbalance. Furthermore, existing chemical agents have limitations in simultaneously achieving effective disease control and reducing DON toxin contamination; improper application of some fungicides can even induce the production of DON toxins, exacerbating the risk of toxin contamination.

[0004] Currently, alternative technologies such as resistance breeding and biological control are not yet mature, and there is a shortage of resistant germplasm resources, making it impossible to replace chemical control methods on a large scale. Therefore, it is necessary to discover and identify a functional gene that can regulate the pathogenicity of Fusarium graminearum and the synthesis of DON toxin, and to analyze the pathogenicity and toxin synthesis mechanism of the pathogen at the molecular level, thereby providing genetic resources and theoretical basis for the biological control of wheat scab. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides Fg05119 Application of genes in the control of wheat scab. Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:1. This gene is involved in regulating the pathogenicity of Fusarium graminearum, the pathogen of wheat scab, and the biosynthesis of DON toxin. This invention relates to the constructed... Fg05119 Functional analysis of gene deletion mutants revealed that the deletion... Fg05119Following gene deletion, the colony growth rate of the strain slightly increased, but the amount of ascospores emitted decreased significantly. Furthermore, the pathogenicity of the mutant strain to wheat and its DON toxin synthesis capacity were significantly reduced. This gene has the potential to serve as a dual-functional molecular target for the control of wheat scab and DON toxin contamination, and can potentially... Fg05119 Genes, their transcripts, or encoded proteins can be used as targets for screening or developing active substances that inhibit target expression or function, which has broad application prospects.

[0006] On the one hand, the present invention provides Fg05119 The application of genes in the control of wheat scab, the aforementioned Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:1;

[0007] The pathogen causing wheat scab is Fusarium graminearum. Fusarium graminearum ; The Fg05119 Genes positively regulate the pathogenicity of Fusarium graminearum.

[0008] Furthermore, knocking out or silencing the stated Fg05119 The gene reduces the pathogenicity of Fusarium graminearum and decreases the amount of ascospores emitted by Fusarium graminearum.

[0009] Furthermore, the reduction of Fusarium graminearum pathogenicity includes weakening its ability to spread within host tissues.

[0010] Specifically, the present invention found through experiments that, compared with the control strain, the amount of ascospores emitted by the mutant strain ΔFg05119 was significantly reduced.

[0011] Specifically, this invention, through experiments, found that the wild-type PH-1 strain can induce typical Fusarium head blight lesions in wheat, which spread along the spikelet axis; the mutant strain ΔFg05119 only forms mild lesions at the inoculated spikelet, and the disease cannot spread to adjacent spikelets, resulting in a significant reduction in the wheat spike disease index; the pathogenicity of the replacement strain ΔFg05119-C is restored to the level of the wild-type strain. Semi-thin section results show that the mutant strain ΔFg05119 exhibits significantly reduced mycelial infection in the lemma, and no mycelial infection is observed in the spikelet axis; the replacement strain shows abundant mycelial infection in both the lemma and spikelet axis.

[0012] Furthermore, the reduction of pathogenicity of Fusarium graminearum also includes at least one of inhibiting the synthesis of DON toxin and reducing the number of toxin-producing bodies.

[0013] Specifically, this invention found through experiments that the DON toxin content in wheat grains of the mutant strain ΔFg05119 was more than 4 times lower than that of the wild-type strain PH-1. Its toxin production culture results showed that the DON toxin yield of the mutant strain ΔFg05119 was more than 2 times lower than that of the wild type, and the replenished strain restored it to near-wild-type levels.

[0014] Specifically, this invention found through experiments that wild-type strains can form a large number of ring-shaped or crescent-shaped toxin-producing bodies, while the mutant strain ΔFg05119 only forms short linear toxin-producing bodies, indicating that its formation is abnormal. Western blot results further showed that the expression level of Tri1 protein in the mutant strain ΔFg05119 was significantly reduced.

[0015] On the other hand, this invention claims protection for a method for reducing the pathogenicity of Fusarium graminearum, comprising knocking out or silencing Fusarium graminearum. Fg05119 Genes, the ones mentioned Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0016] Furthermore, this invention claims protection for its use as a screening target in screening active ingredients capable of inhibiting the pathogenicity and / or DON toxin synthesis of Fusarium graminearum, wherein the screening target is... Fg05119 Gene, Fg05119 Gene transcription products, Fg05119 One of the proteins encoded by a gene; Furthermore, the aforementioned Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The Fg05119 The nucleotide sequence of the gene transcription product is shown in SEQ ID NO:2; The Fg05119 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:3.

[0017] The active ingredients include, but are not limited to, double-stranded RNA, siRNA, antisense oligonucleotides, protein function inhibitors, and small molecule compounds.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention was discovered through research. Fg05119 The gene is closely related to the pathogenicity of Fusarium graminearum and the synthesis of DON toxin, and is crucial for the construction of... Fg05119 Functional analysis of gene deletion mutants revealed that the deletion... Fg05119After gene inoculation, the colony growth rate of the strain slightly increased, but the amount of ascospores emitted decreased significantly, and the pathogenicity of the mutant strain to wheat and its DON toxin synthesis ability were significantly reduced. Inoculation with the mutant strain ΔFg05119 resulted in only mild lesions at the inoculated spikelet, preventing the disease from spreading to adjacent spikelets, and significantly reducing the wheat spike disease index. The pathogenicity of the complement strain ΔFg05119-C recovered to the level of the wild-type strain. Semi-thin section results showed that the mutant strain ΔFg05119 exhibited significantly reduced mycelial infection in the lemma and no mycelial infection in the spikelet rachis; the complement strain showed abundant mycelial infection in both the lemma and spikelet rachis. Fg05119 The gene has the potential to be used as a molecular target with dual functions in the control of wheat scab and DON toxin contamination, and can... Fg05119 Genes, their transcripts, or encoded proteins can be used as targets for screening or developing active substances that inhibit target expression or function, which has broad application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 for Fg05119 A schematic diagram of gene knockout mutants.

[0021] Figure 2 for Fg05119 The PCR detection results of the gene deletion mutant ΔFg05119 are shown in the figure.

[0022] Figure 3 for Fg05119 The effect of gene deletion on the colony morphology and growth rate of Fusarium graminearum. Figure 3 In the diagram, A represents the colony morphology. Figure 3 B in the graph represents the colony growth rate.

[0023] Figure 4 for Fg05119 The effect of gene deletion on the sexual reproduction of Fusarium graminearum.

[0024] Figure 5 for Fg05119 The effect of gene deletion on the pathogenicity of Fusarium graminearum. Figure 5 In the diagram, A represents the phenotypic pattern of wheat after inoculation with different bacterial strains; Figure 5 B in the figure represents the statistical results of disease index of wheat after inoculation with different strains; Figure 5 C in the figure represents a semi-thin section of the lemma and spikelet axis of wheat after inoculation with different strains.

[0025] Figure 6 for Fg05119 The effect of gene deletion on the synthesis of DON toxin in Fusarium graminearum. Figure 6 A in the figure represents the DON toxin content in wheat grains; Figure 6 B in the figure represents the DON toxin production of different strains after culturing in TBI liquid toxin-producing medium; Figure 6 C in the image represents a fluorescence microscope image of the toxin-producing bodies from different strains; Figure 6 In the diagram, D represents the immunoblot images of Tri1 protein markers from different strains. Detailed Implementation

[0026] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0027] Fusarium graminearum ( F. graminearum The wild-type strain is PH-1. Fg05119 The gene deletion mutant was named ΔFg05119, and the complemented strain was named ΔFg05119-C.

[0028] The culture medium and its formulation used in the examples are as follows: Complete culture medium (CM): 10 g sucrose, 6 g yeast extract, 6 g acid-hydrolyzed casein, 20 g agar powder, double-distilled water to a final volume of 1 L, autoclaved at 121°C for 20 min.

[0029] 5×YEG medium: 5 g yeast extract, 10 g glucose, double distilled water to a final volume of 1 L, autoclave at 121℃ for 20 min.

[0030] TBI toxin-producing medium: Add an appropriate amount of double-distilled water to a beaker, then add 30 g of sucrose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of potassium chloride, 10 mg of ferrous sulfate heptahydrate, 871 mg of L-arginine, and 200 μL of trace element stock solution. Finally, add 0.3 g of plant gel and bring the volume to 1 L with double-distilled water. Mix thoroughly and adjust the pH of the medium to 5.6. Autoclave at 121℃ for 20 min.

[0031] Trace element stock solution: 5 g citric acid, 5 g zinc sulfate heptahydrate, 0.25 g copper sulfate pentahydrate, 50 mg manganese sulfate monohydrate, 50 mg boric acid, 50 mg sodium molybdate dihydrate, add ddH2O to make up to 100 mL.

[0032] Carrot culture medium: Wash and peel 200 g of carrots, then cut them into 1 cm pieces. 3 Add 400 mL of sterile water to the small pieces of agar and autoclave for 10 min; then blend in a juicer, bring the volume to 1 L with distilled water, add 15-20 g of agar, dispense, and autoclave at 121℃ for 35 min.

[0033] Example 1 This embodiment provides Fg05119 Construction of gene knockout mutants and complement strains.

[0034] The primer sequences used in this embodiment are shown in Table 1. The Fg05119 gene deletion mutant was constructed using a homologous recombination-mediated gene substitution method. Figure 1 First, using Fusarium graminearum PH-1 genomic DNA as a template, the following were amplified separately. Fg05119 Upstream homologous arm fragment A (1F, 2R) and downstream homologous arm fragment B (3F, 4R); to carry the hygromycin B resistance gene ( HPH Using plasmid pCB1003 as a template, selection marker fragments H1 (HYG / F, HY / R) and H2 (YG / F, HYG / R) were amplified. Then, fusion PCR (SOE) was performed. PCR involves splicing homologous arm fragments with selection marker fragments to obtain the results used for... Fg05119 Recombinant fragment A, which was knocked out and replaced by gene knockout H1 and H2 B. Using a PEG-mediated protoplast transformation method, the aforementioned... Fg05119 The gene knockout fragment was introduced into protoplasts of the PH-1 strain, and transformants were obtained by selection on a hygromycin B-containing selective medium. PCR detection of the transformants was performed using four pairs of primers: primers 5F / 6R (L1 lane). Fg05119 Gene knockout status; primers 7F / H855R (lane L2) and H856F / 8R (lane L3) were used to verify whether correct homologous recombination occurred in the upstream and downstream homologous arms, respectively; primers H850 / H852 (lane L4) were used to detect the hygromycin resistance gene. HPH The electrophoresis results of PCR detection are as follows: Figure 2 As shown, by Figure 2 It can be seen that the detection results of transformants ΔFg05119-2 and ΔFg05119-19 are completely correct.

[0035] Further construct containing Fg05119 The complement vector containing its own promoter, coding region, and termination sequence (nucleotide sequence as shown in SEQ ID NO:1) was amplified using HBF and HBR primers. After sequencing verification, it was introduced into the mutant strain ΔFg05119 to obtain the complement strain ΔFg05119-C.

[0036] Table 1 Primer sequences

[0037] Example 2 This embodiment provides Fg05119 The effects of gene deletion on colony growth and sexual reproduction of Fusarium graminearum.

[0038] 1. Colony growth experiment Wild-type strain PH-1, mutant strain ΔFg05119, and complement strain ΔFg05119-C were inoculated into the center of CM and 5×YEG agar plates, respectively, and incubated at 25℃ for 3 days. Colony growth phenotypes were observed, and colony diameters were measured. At least three biological replicates were set up for each strain. Results are shown below. Figure 3 As shown.

[0039] Depend on Figure 3 It can be seen that the colony morphology of the mutant strain ΔFg05119 is basically the same as that of the wild-type strain PH-1 and the complement strain ΔFg05119-C, but the colony growth rate is significantly faster.

[0040] 2. Sexual reproduction experiment of the strain To investigate Fg05119 The effect of genes on the sexual reproduction of Fusarium graminearum was investigated. Sexual reproduction of each strain was induced using carrot agar medium, and the cultures were observed after 7 days of incubation under black light. The results are as follows: Figure 4 As shown.

[0041] Depend on Figure 4 It was found that the mutant strain ΔFg05119, the wild-type strain PH-1, and the complement strain ΔFg05119-C all formed a large number of mature black ascospores; however, compared with the control strain, the number of ascospores emitted by the mutant strain ΔFg05119 was significantly reduced. These results indicate... Fg05119 Gene deletion does not affect the formation and development of ascocarps in *Fusarium graminearum*, but it significantly inhibits the eruption of ascospores, indicating that... Fg05119 Genes play an important role in the eruption of ascospores of Fusarium graminearum.

[0042] Example 3 This embodiment provides Fg05119 The effects of gene deletion on pathogenicity of Fusarium graminearum and DON toxin synthesis.

[0043] 1. Fg05119 The effect of gene deletion on the pathogenicity of Fusarium graminearum To test the pathogenicity of each strain, wild-type strain PH-1, mutant strain ΔFg05119, and complement strain ΔFg05119-C were inoculated into wheat ears at the flowering stage. Leminal and spikelet rachis tissues were collected from the inoculation sites 3 and 5 days after inoculation, and semi-thin sections were prepared for microscopic observation. Symptoms were observed and the number of diseased spikelets was counted 14 days after inoculation. The results are as follows: Figure 5 As shown.

[0044] Depend on Figure 5 It was found that the wild-type PH-1 could induce typical Fusarium head blight lesions in wheat, which spread along the spikelet axis; the mutant strain ΔFg05119 only formed slight lesions at the inoculated spikelet, and the disease could not spread to adjacent spikelets, resulting in a significantly reduced disease index in the wheat spike; the pathogenicity of the replacement strain ΔFg05119-C was restored to the level of the wild-type strain. Semi-thin section results showed that the mutant strain ΔFg05119 exhibited significantly reduced mycelial infection in the lemma, and no mycelial infection was observed in the spikelet axis; the replacement strain showed abundant mycelial infection in both the lemma and spikelet axis. These results indicate that... Fg05119 Gene deletion significantly weakens the ability of Fusarium graminearum to infect and spread wheat, indicating that this gene plays an important regulatory role in its pathogenicity.

[0045] 2. Fg05119 Effects of gene deletion on DON toxin synthesis in Fusarium graminearum After 14 days of moisturizing culture, seeds from the inoculation site were collected, and the DON toxin content was determined by high-performance liquid chromatography (HPLC). The results are as follows. Figure 6 As shown in A in the figure. Further analysis was conducted to determine the DON toxin production of each strain in TBI liquid toxin-producing medium. Wild-type strain PH-1, mutant strain ΔFg05119, and complement strain ΔFg05119-C were cultured at 25°C in the dark with shaking for 7 days. The supernatant was collected by centrifugation and analyzed by HPLC. The results are shown in Figure A. Figure 6 As shown in B. The toxin-producing body is the main site of DON toxin synthesis in Fusarium graminearum. Tri1 protein can be used as a marker, and the fluorescence microscopy results of the marker are shown in Figure 1. Figure 6 As shown in C, further Western blot analysis was performed on this marker, and the results are as follows. Figure 6 As shown in D in the diagram.

[0046] Depend on Figure 6It was found that the DON toxin content in wheat grains of the mutant strain ΔFg05119 was more than 4 times lower than that of the wild-type strain PH-1. Its toxin production culture results showed that the DON toxin yield of the mutant strain ΔFg05119 was more than 2 times lower than that of the wild type, and the replenished strain recovered to near-wild-type levels. Fluorescence microscopy observations showed that the wild-type strain formed numerous ring-shaped or crescent-shaped toxin-producing bodies, while the mutant strain ΔFg05119 only formed short linear toxin-producing bodies, indicating abnormal formation. Western blot results further showed that the Tri1 protein expression level in the mutant strain ΔFg05119 was significantly reduced. These results indicate that... Fg05119 The gene is involved in the regulation of DON toxin biosynthesis in Fusarium graminearum, and its deletion can lead to abnormal formation of toxin-producing bodies and significantly reduce DON toxin production.

[0047] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Fg05119 The application of genes in the control of wheat scab is characterized by, The Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The pathogen causing wheat scab is Fusarium graminearum. Fusarium graminearum ; The Fg05119 Genes positively regulate the pathogenicity of Fusarium graminearum.

2. The application according to claim 1, characterized in that, Knockout or silencing Fg05119 Genes that reduce the pathogenicity of Fusarium graminearum.

3. The application according to claim 1, characterized in that, Knockout or silencing Fg05119 Genes that reduce the amount of ascospores emitted by Fusarium graminearum.

4. The application according to claim 2, characterized in that, The reduction of Fusarium graminearum pathogenicity includes weakening its ability to spread within host tissues.

5. The application according to claim 2, characterized in that, The reduction of pathogenicity of Fusarium graminearum also includes at least one of reducing the synthesis of DON toxin and reducing the number of toxin-producing bodies.

6. A method for reducing the pathogenicity of Fusarium graminearum, characterized in that, Including knockout or silencing of Fusarium graminearum Fg05119 Gene; The Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

7. The application of this method as a screening target in screening active ingredients that can inhibit the pathogenicity and / or DON toxin synthesis of Fusarium graminearum, characterized in that, The screening target is Fg05119 Gene, Fg05119 Gene transcription products, Fg05119 One of the proteins encoded by a gene.

8. The application according to claim 7, characterized in that, The Fg05119 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The Fg05119 The nucleotide sequence of the gene transcription product is shown in SEQ ID NO:2; The Fg05119 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

3.

9. The application according to claim 7, characterized in that, The active ingredients include double-stranded RNA, siRNA, antisense oligonucleotides, protein function inhibitors, and small molecule compounds.

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