Application of magnaporthe oryzae gene MoCA5 in regulation and control of pathogenicity of magnaporthe oryzae

Abstract

The invention belongs to the field of plant genetic engineering, and relates to application of a magnaporthe oryzae gene MoCA5 in regulation of pathogenicity of magnaporthe oryzae. The nucleotide sequence of the magnaporthe oryzae gene MoCA5 is as shown in SEQ ID NO.1, and the coded amino acid sequence of the magnaporthe oryzae gene MoCA5 is as shown in SEQ ID NO.2. The MoCA5 gene is knocked out from magnaporthe oryzae to obtain a knockout mutant, and experiments show that compared with wild type WT, the growth and development capability of spores of the knockout mutant delta MoCA5 is obviously reduced. The MoCA5 has a carbonic anhydrase activity or has an affinity for carbonic anhydrase. Pathogenicity experiments show that the deficiency of MoCA5 significantly reduces the pathogenicity of spores and hyphae of pathogenic bacteria. The research of the invention is helpful to deeply clarify the pathogenic molecular mechanism of the pyricularia grisea-related carbonic anhydrase gene, and provides a target gene for the development of effective bactericides.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and relates to the application of a rice blast fungus gene MoCA5 in regulating the pathogenicity of the rice blast fungus, thereby providing a target for preventing and controlling the rice blast fungus. Background Art

[0002] Rice blast, caused by infection with Magnaporthe oryzae, is one of the most serious and destructive fungal diseases of rice. Inadequate or no control measures result in significant economic losses each year. Therefore, in-depth research on the pathogenic mechanisms of rice blast not only helps reveal the pathogenesis of fungal diseases but also facilitates the development of green control strategies, thus having important theoretical and practical significance.

[0003] Currently, research on the functional genes of the rice blast fungus Magnaporthe oryzae is helping to deepen our understanding of the infection mechanism of the fungus and provide new directions for its control. However, no studies have yet reported on the effect of the MoCA5 gene on the pathogenicity of the fungus. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of the Rice Blast Fungus MoCA5 gene in regulating the pathogenicity of the Rice Blast Fungus, thereby providing a target for preventing and controlling the Rice Blast Fungus.

[0005] The present invention discloses a novel, unknown protein gene, MoCA5, from the rice blast fungus and the function of the protein it encodes, MoCA5. The nucleotide sequence of the MoCA5 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. The MoCA5 protein contains a carbonic anhydrase domain. The present invention uses double enzyme digestion technology to construct a gene knockout vector, which is then introduced into AGL-1 Agrobacterium. Homologous recombination is used to knock out the MoCA5 gene from the rice blast fungus, resulting in a knockout mutant, ΔMoCA5. This mutant exhibits defects in spore growth and development. Pathogenicity assay results show that the knockout mutant, ΔMoCA5, significantly reduces its pathogenicity to rice. The above experiments demonstrate that the MoCA5 gene is a pathogenic gene of the rice blast fungus and is involved in both the pathogenicity and spore growth and development of the fungus.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides an application of the rice blast fungus gene MoCA5 in regulating the pathogenicity of the rice blast fungus. The nucleotide sequence of the rice blast fungus gene MoCA5 is shown in SEQ ID NO.1, and the amino acid sequence encoded by the rice blast fungus gene MoCA5 is shown in SEQ ID NO.2.

[0008] In the above technical solution, further, the application is to regulate the growth and development of rice blast fungus spores.

[0009] In the above technical solution, further, the application is to reduce the number of conidiophores and conidia of rice blast fungus; or inhibit the germination of rice blast fungus spores and reduce the formation of rice blast fungus attachment spores.

[0010] In the above technical solution, further, the application is to maintain the activity of carbonic anhydrase of rice blast fungus.

[0011] The present invention provides an application of a rice blast fungus gene MoCA5 in preventing and controlling rice blast disease caused by the blast fungus. The nucleotide sequence of the rice blast fungus gene MoCA5 is shown in SEQ ID NO.1.

[0012] In the above technical solution, further, the prevention and treatment is achieved by knocking out the MoCA5 gene.

[0013] The present invention provides an application of the rice blast fungus gene MoCA5 as a target for a drug for preventing and controlling plant diseases. The plant disease is rice blast caused by rice blast fungus. The nucleotide sequence of the rice blast fungus gene MoCA5 is shown in SEQ ID NO.1.

[0014] The present invention provides a method for treating rice blast caused by rice blast fungus, comprising blocking or inhibiting the expression of gene MoCA5 in rice blast fungus, wherein the nucleotide sequence of the gene MoCA5 is shown in SEQ ID NO.1.

[0015] The present invention provides use of an agent for blocking or inhibiting the expression of gene MoCA5 in preparing a medicament for preventing and treating rice blast, wherein the rice blast is caused by rice blast fungus. The nucleotide sequence of the gene MoCA5 is shown in SEQ ID NO.1.

[0016] In the above technical solution, further, the aforementioned rice blast fungus is the wild type Y34 of rice blast fungus.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The rice blast fungus gene MoCA5 provided by the present invention contains the carbonic anhydrase Pro_CA domain. Experiments have shown that the knockout mutant ΔMoCA5 exhibits significantly reduced spore growth and development compared to its wild-type WT, as evidenced by fewer conidiophores, a lower conidia number, a reduced germination rate, a lower appressorium formation rate, and an increased appressorium deformity rate, indicating that MoCA5 is involved in the growth and development of rice blast fungus spores. In a carbonic anhydrase inhibitor phenotypic experiment, the hyphae of ΔMoCA5 were significantly inhibited, indicating that MoCA5 has carbonic anhydrase activity or affinity for carbonic anhydrase. Pathogenicity experiments have shown that the deletion of MoCA5 significantly reduces the pathogenicity of the pathogenic fungus spores and hyphae. The present invention confirms that MoCA5 is a protein with carbonic anhydrase activity that is involved in the growth and development of rice blast fungus spores and pathogenicity. The research presented in the present invention helps to further elucidate the molecular pathogenicity mechanism of the carbonic anhydrase gene in rice blast fungus and provides a target gene for the development of effective fungicides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of homologous recombination construction in rice blast fungus.

[0020] Figure 2 PCR amplification of the target gene sequence for the knockout mutant.

[0021] Figure 3 PCR amplification of the upstream outer -hph of the knockout mutant target gene.

[0022] Figure 4 qPCR detection of target gene knockout mutants.

[0023] Figure 5 Microscopic observation of conidiophores of the knockout mutant ΔMoCA5.

[0024] Figure 6 This is a statistical graph of the conidia number of the knockout mutant ΔMoCA5.

[0025] Figure 7 Microscopic observation of conidia germination and appressorium formation of the knockout mutant ΔMoCA5.

[0026] Figure 8 Statistical graphs of the conidia germination rate (A), appressorium formation rate (B), and appressorium deformity rate (C) of the knockout mutant ΔMoCA5.

[0027] Figure 9 Analysis of hyphal carbonic anhydrase activity in the knockout mutant ΔMoCA5.

[0028] Figure 10 Pathogenicity analysis of the knockout mutant ΔMoCA5; A. Colony growth comparison; B. Colony diameter statistics. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The test methods in the following examples that do not specify specific experimental conditions are generally carried out according to conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0030] Example 1 Construction of MoCA5 gene knockout vector

[0031] Experimental materials and test strains: Lijiang Xintuan Black Valley, rice blast fungus wild-type strain Y34 (laboratory preservation), Escherichia coli DH5α (laboratory preservation), Agrobacterium tumefaciens AGL-1 (laboratory preservation and preparation), and pXEH2.0 vector (laboratory preservation).

[0032] Experimental steps: First, PCR technology was used to amplify the left and right homology arms of the MoCA5 gene using Y34 genomic DNA as a template. Specific primers were designed and constructed on the pXEH2.0 vector (such as Figure 1 ), the constructed vector was transformed with Agrobacterium by homologous recombination replacement, and the transformants were verified by PCR of the target gene and PCR of the upstream outer-hph fragment. At the same time, the expression level of the gene was verified by qPCR technology of the wild type and knockout mutant.

[0033] Experimental results: The transformants 1, 7, 8, 16, 17, 27, 29, 43, 49 and the wild type WT obtained by genetic transformation were screened and verified by PCR. The transformants with no band of the target gene at 990 bp (such as Figure 2 ) and the transformants with a band at 2497 bp in the upstream outer-hph fragment (e.g. Figure 3 ) proved that 1, 7, 16, 17, 27, 29, and 43 were transformants that met the knockout conditions, and the expression levels of genes in three of the transformants were verified by qPCR technology (e.g. Figure 4 ) obtained 29 and 43 with no gene expression, which were knockout mutants. The knockout mutant 29 was used in the following examples.

[0034] Table 1 PCR reaction system

[0035]

[0036]

[0037] Table 2 PCR reaction procedure

[0038]

[0039] Table 3 qPCR reaction system

[0040]

[0041] Table 4 qPCR reaction procedure

[0042]

[0043] Table 5 Primer sequences

[0044]

[0045] Example 2 Spore Growth and Development Experiment of ΔMoCA5

[0046] (1) Observation of conidiophores and counting of conidia

[0047] The wild-type WT and ΔMoCA5 of rice blast fungus were activated in PDA medium. After 7 days at 28°C, sterile water was added and the mycelia were scraped off with a cotton swab. 200 μL of the bacterial solution was then applied to OMA medium. After 4 days at 28°C, sterile water was added to hang off the mycelia. The culture was covered with two layers of gauze and incubated in the dark for 3 days. The spores were then washed and filtered. The spore concentration of the strain was adjusted to 1×10 5 / mL, and then evenly apply 200μL to OMA culture medium. After culturing at 28℃ for 4 days, add sterile water to hang the mycelium, cut a piece of culture medium with a blade and place it on a slide, and then put it in a moisturizing box. After cutting, observe the changes in conidiophores and spore production of each strain under a microscope 12h, 24h, 48h, and 72h after cutting. At the same time, at 72h, the conidiophores were stained with lactic acid phenol cotton blue to observe the number of conidiophores. Each strain was repeated 3 times, and the experiment was repeated 3 times. The experimental results showed that the conidiophores of ΔMoCA5 were significantly less than those of WT (such as Figure 5 ). Under the above culture conditions, the spores were washed with 2 mL of sterile water and counted with a hemocytometer. Each strain was repeated 3 times, and the experiment was repeated 3 times. The experimental results showed that the number of conidia of ΔMoCA5 was significantly less than about 40% of the number of WT spores (such as Figure 6 ).

[0048] (2) Spore germination and appressorium observation experiments of ΔMoCA5 spore strain

[0049] The spores of wild-type WT and ΔMoCA5 of rice blast fungus cultured on OMA medium were collected and adjusted to 1×105 / mL, dripped on the biconcave piece, and observed the germination of spores and the formation of appressorium of different strains under the microscope at 1h, 2h, 3h, 4h, and 6h (such as Figure 7 The experiment showed that the germination rate of ΔMoCA5 decreased by about 20% at 2h and 3h compared with WT, the appressorium production rate also decreased by about 20%, and the appressorium deformity rate increased by about 25% (e.g. Figure 8 ).

[0050] Studies have shown that the conidiophores, conidia, germination and appressorium formation of ΔMoCA5 spores were significantly reduced compared with WT, indicating that the MoCA5 gene is involved in the growth and development of rice blast fungus spores.

[0051] Table 6 PDA culture medium

[0052]

[0053] Table 7 OMA culture medium

[0054]

[0055] Example 3 Phenotypic Observation Experiment of Mycelial Carbonic Anhydrase Activity of ΔMoCA5

[0056] Melt the PDA solid culture medium and add carbonic anhydrase inhibitor when the temperature is about 50℃, so that the final concentration of carbonic anhydrase inhibitor in the culture medium is 50nM. Pour into the plate, take the strain of the same diameter and inoculate it to the center of the plate, invert it and culture it in a 28℃ incubator for 7 days, measure the colony diameter, and take pictures. The inoculation and culture process should be protected from light. Repeat 3 times for each strain and the experiment is repeated 3 times. The experiment shows that the hyphal growth of ΔMoCA5 is significantly inhibited by carbonic anhydrase inhibitor (such as Figure 9 ), proving that MoCA5 protein has carbonic anhydrase activity or affinity for carbonic anhydrase.

[0057] Example 4 Pathogenicity experiment of ΔMoCA5

[0058] Spores of wild-type WT and ΔMoCA5 of rice blast fungus cultured on OMA medium were collected and spore suspensions were adjusted with 0.25% gelatin to 1×10 5 1 mL of the bacteria was sprayed onto two-week-old rice leaves, sealed and placed in a 28°C light incubator. Photos were taken and compared after 7 days. The experiment was repeated three times.

[0059] Mycelia of wild-type WT and ΔMoCA5 rice blast fungi cultured on PDA medium were attached to two-week-old rice leaves, sealed to retain moisture, and placed in a 28°C light-incubator. Photos were taken and compared after 7 days. The experiment was repeated three times.

[0060] The experiment showed that the pathogenicity of spores and hyphae of ΔMoCA5 was significantly lower than that of WT, indicating that the MoCA5 gene is involved in the pathogenicity of rice blast fungus.

[0061] The above description is only a preferred specific implementation method of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Application of the rice blast fungus gene MoCA5 in regulating the pathogenicity of rice blast fungus. The nucleotide sequence of the rice blast fungus gene MoCA5 is shown in SEQ ID NO.1, and the amino acid sequence encoded by the rice blast fungus gene MoCA5 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The application is to regulate the growth and development of rice blast fungus spores.

3. The use according to claim 2, characterized in that The application is to reduce the number of conidiophores and conidia of rice blast fungus; or to inhibit the germination of rice blast fungus spores and reduce the formation of rice blast fungus attachment spores.

4. The use according to claim 1, characterized in that The application is to maintain the activity of carbonic anhydrase of rice blast fungus.

5. Use of the rice blast fungus gene MoCA5 in preventing and controlling rice blast disease caused by rice blast fungus, wherein the nucleotide sequence of the rice blast fungus gene MoCA5 is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that The prevention and treatment is achieved by knocking out the MoCA5 gene.

7. Use of the rice blast fungus gene MoCA5 as a target for a drug for controlling plant diseases, wherein the plant disease is rice blast caused by rice blast fungus, and the nucleotide sequence of the rice blast fungus gene MoCA5 is shown in SEQ ID NO.

1.

8. A method for treating rice blast caused by rice blast fungus, characterized in that: The method comprises blocking or inhibiting the expression of the gene MoCA5 in rice blast fungus, wherein the nucleotide sequence of the gene MoCA5 is shown in SEQ ID NO.

1.

9. Use of an agent for blocking or inhibiting the expression of gene MoCA5 in the preparation of a medicament for preventing and controlling rice blast, wherein the rice blast is caused by Magnaporthe oryzae, and the nucleotide sequence of the gene MoCA5 is shown in SEQ ID NO. 1.

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