Application of the rice OsSAFM6 gene or its encoded protein in enhancing rice resistance to rice blast fungus

Abstract

This invention discloses the application of the rice OsSAFM6 gene or its encoded protein in enhancing rice resistance to rice blast fungus, relating to the field of rice blast control technology. Research by this invention has found that the protein encoded by the rice OsSAFM6 gene can be used as a pesticide to control rice blast. The OsSAFM6 protein has a significant inhibitory effect on the appressorium of rice blast fungus. When the SAFM6-GST concentration is 0.05 μg / μL, the appressorium formation rate of rice blast fungus spores is 26.67%; at a concentration of 0.1 μg / μL, the appressorium formation rate is 5.33%. Therefore, the protein encoded by the rice OsSAFM6 gene can be used to enhance rice resistance to rice blast fungus.

Description

Technical Field

[0001] This invention relates to the field of rice blast disease control technology, specifically to the application of the rice OsSAFM6 gene or its encoded protein in enhancing rice resistance to rice blast fungus. Background Technology

[0002] Rice has a long history of cultivation and is an important economic crop in many countries. Due to its high nutritional value and strong adaptability to different growing environments, it is a vital source of food and nutritional security for developing countries. Among rice's important food crops, the most typical fungal disease is rice blast, caused by the rice blast fungus (Magnaporthe oryzae). This fungus can infect the above-ground tissues of rice at all growth stages, causing yield reduction. Furthermore, the blast fungus varies depending on different resistant rice varieties, growing conditions, growth stages, and the location of infection within the rice plant.

[0003] Rice blast can occur at different stages and in different parts of rice growth and development, mainly including leaf blast, neck blast, and grain blast. The rice blast fungus primarily spreads in the field through asexual reproduction, completing its infection cycle. Three-celled conidia of the rice blast fungus, scattered on weeds or plant debris in the field the previous year, are spread to the surface of rice leaves by air and rainwater. When the spores land on the surface of hydrophobic leaves, they secrete a sticky substance to adhere to the leaf surface and form germ tubes. These germ tubes accumulate, bend, and swell to form appressoriums, which accumulate large amounts of melanin. The appressoriums differentiate into penetrating spikes, which penetrate the cuticle and cell walls of the leaf epidermis. The resulting infectious hyphae continuously infect adjacent cells, and the infected cells continuously produce infectious hyphae and conidia, repeatedly infecting adjacent cells. In humid environments, gray or grayish-brown spindle-shaped lesions appear on rice leaves; this is the process by which the rice blast fungus transforms from a biotrophic form to a necrotrophic form. Newly produced conidia at the lesions can be spread by wind and rain to new host plants, initiating a new infection cycle. Spraying relevant pesticides is currently the fastest and most effective method for controlling rice blast, and it is also the most widely used method, generally applied at the late booting and heading stages. Tricyclazole and isoprothiolane are commonly used pesticides for controlling rice blast, but prolonged continuous use of chemical pesticides can lead to pesticide resistance in the rice blast fungus and pollute the surrounding air environment. In current agricultural production, antimicrobial peptides play an important role. Antimicrobial peptides are a class of naturally occurring small molecule peptide chains with strong antimicrobial activity, capable of combating various pathogenic microorganisms. Applying antimicrobial peptides to agricultural production is a green and efficient strategy that can effectively ensure crop yield and quality, and is of great significance for achieving more sustainable agricultural development.

[0004] However, little is known about novel agents for the effective control of rice blast. This invention elucidates the regulatory mechanism of rice antimicrobial peptides. Based on their functions of inhibiting rice blast fungus germination, inhibiting appressorium formation, and inhibiting rice blast fungus infection of rice leaves, these peptides are effectively applied to the control of rice blast, and highly efficient agents are developed to achieve effective control of rice blast. Summary of the Invention

[0005] This invention has found that the OsSAFM6 protein encoded by the rice OsSAFM6 gene has a significant inhibitory effect on the formation of appressoriums of rice blast fungus, and can be used as a pesticide to control rice blast.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides the application of the rice OsSAFM6 gene or its encoded protein in enhancing rice resistance to rice blast fungus.

[0008] This invention also provides the application of the rice OsSAFM6 gene or the protein it encodes in the preparation of drugs against rice blast fungus.

[0009] This invention also provides the application of the rice OsSAFM6 gene or its encoded protein in rice breeding, by screening rice plants that highly express the rice OsSAFM6 gene or the protein encoded by the rice OsSAFM6 gene to obtain rice lines resistant to rice blast fungus.

[0010] The CDS nucleotide sequence of the rice OsSAFM6 gene is shown in SEQ ID No. 2, and the amino acid sequence of the protein encoded by the rice OsSAFM6 gene is shown in SEQ ID No. 1.

[0011] The present invention also provides an antifungal drug for rice blast fungus, the active ingredient of which comprises a protein encoded by the rice OsSAFM6 gene with the amino acid sequence shown in SEQ ID No. 1. The concentration of the protein encoded by the rice OsSAFM6 gene is 0.05–0.1 g / μL.

[0012] OsSAFM6 purified protein (SAFM6-GST protein) has a significant inhibitory effect on the appressorium of rice blast fungus. When the concentration of SAFM6-GST is 0.05 μg / μL, the appressorium formation rate of rice blast fungus spores is 26.67%, and when the concentration is 0.1 μg / μL, the appressorium formation rate of rice blast fungus spores is 5.33%.

[0013] The present invention also provides the application of the aforementioned anti-rice blast fungus drug in the prevention and control of rice blast fungus infection.

[0014] This invention also provides a method for constructing transgenic rice resistant to rice blast fungus, wherein the rice OsSAFM6 gene is transferred into rice plants to obtain transgenic rice with high expression of the rice OsSAFM6 gene, and the CDS nucleotide sequence of the rice OsSAFM6 gene is shown in SEQ ID No. 2.

[0015] Specifically, the CDS region nucleotide sequence of the rice OsSAFM6 gene was cloned into a vector, first transformed into Agrobacterium, and then transformed into rice cells through callus transformation to obtain transgenic rice that highly expresses the rice OsSAFM6 gene. The vector used was PGEX-4T-1.

[0016] The beneficial effects of this invention are:

[0017] This invention has discovered that the protein encoded by the rice gene OsSAFM6 can be used as a pesticide to control rice blast. The OsSAFM6 protein has a significant inhibitory effect on the appressorium of rice blast fungus. When the concentration of OsSAFM6 is 0.05 μg / μL, the appressorium formation rate of rice blast fungus spores is 26.67%, and when the concentration is 0.1 μg / μL, the appressorium formation rate is 5.33%. Therefore, the protein encoded by the rice gene OsSAFM6 can be used to improve the resistance of rice to rice blast fungus. Attached Figure Description

[0018] Figure 1 The graph shows the inhibition of rice blast fungus appressorium formation by purified rice OsSAFM6 protein. In the graph, A is the rice blast fungus appressorium formation graph, and B is the statistical result graph of appressorium formation.

[0019] Figure 2 The graph shows the inhibition of rice blast fungus mycelial growth by purified rice OsSAFM6 protein. In the graph, A is the mycelial growth of rice blast fungus, and B is the colony inhibition rate of rice blast fungus.

[0020] Figure 3 Figure showing the results of using purified OsSAFM6 protein to inhibit rice blast fungus infection in rice leaves after inoculation. Detailed Implementation

[0021] Example 1

[0022] Obtaining purified OsSAFM6 protein.

[0023] Primers OsSAFM6GST-F / R were designed based on the CDS sequence of the OsSAFM6 gene (as shown in SEQ ID No. 2). The primer sequences are as follows:

[0024] OsSAFM6GST-F: ggttccgcgtggatccATGAAGACCGCC;

[0025] OsSAFM6GST-R:gtcgacccgggaattcGTTCTCGCACGA.

[0026] Vector Construction: Using the Nipponbare genome as a template, the CDS sequence of the OsSAFM6 gene was amplified using primers OsSAFM6GST-F / R. The amplified fragment was ligated into the PGEX-4T-1 vector, which had been digested with BamHI and EcoRI, using seamless cloning technology. The ligated plasmid was then transformed into *E. coli* using a heat shock method, and positive clones were selected for detection.

[0027] Induction of SAFM6-GST protein expression: After the SAFM6-GST vector was correctly sequenced, the plasmid was transformed into E. coli strain BL21 by heat shock. Positive clones were selected and cultured at 37℃ until the OD value reached 0.6. Then, the cells were induced at 16℃ for 16 h with an IPTG concentration of 1 mM. Subsequently, protein expression was detected by Coomassie brilliant blue staining and Western blot assay to confirm successful protein induction.

[0028] OsSAFM6-GST protein purification:

[0029] (1) Centrifuge 150 mL of bacterial cells at 10,000 rpm for 2 min and discard the supernatant; add 15 mL of GST binding / washing buffer to the precipitated bacterial clump to suspend the bacterial cells, and then add 300 μL of 10 mg / mL lysozyme, 30 μL of 0.5 M MgCl2, and 150 μL of 0.1 M benzyl sulfonyl fluoride (PMSF). Incubate at 4 °C with gentle shaking for 30 min.

[0030] (2) After the enzymatic hydrolysis process is completed, the bacterial cells are broken by ultrasonication and then centrifuged at 4℃ for 10 min at 10000 rpm.

[0031] (3) Take 200 μL of supernatant and label it as Input. Transfer the remaining supernatant to a pre-equilibrated glutathione transferase (GST) protein resin flow column. Adjust the flow rate to 7-9 s / drop and collect 200 μL of effluent and label it as Flowthrough. Add 15 mL of water to suspend the bacterial cell residue and take 200 μL and label it as Bacteria pellet. The entire process is carried out in a 4℃ refrigerator to prevent protein denaturation.

[0032] (4) Add 5 mL of GST binding / washing buffer, control the flow rate at 4-6 s / drop, collect 200 μL of effluent at the bottom of the column and label it Wash1; repeat this step once and collect 200 μL of effluent and label it Wash2;

[0033] (5) Add an appropriate amount of glutathione to 1 mL of GST elution buffer to elute the target protein, and collect the eluent (containing the target protein) and label it as Elution 1; repeat this step 3 times and label them as Elution 2, Elution 3 and Elution 4 respectively.

[0034] The purified protein (sequence shown in SEQ ID No. 1) was detected by Coomassie brilliant blue staining and Western blot assay, and the concentration of the purified protein was then determined.

[0035] Example 2

[0036] OsSAFM6 purified protein inhibits the formation of appressoriums of rice blast fungus.

[0037] Wild-type rice blast fungus strain RB22 was activated on OA medium and cultured in the dark at 25°C for 3 days followed by light culture for 4 days. Sterile ddH2O was added to the culture dish, and the mycelia were gently scraped off with an inoculation loop to elute the rice blast fungus spores from the medium. The eluent was filtered through a filter cloth to obtain a spore suspension. The spore suspension was placed in a 2 mL centrifuge tube and centrifuged at 10,000 rpm for 1 min. The supernatant was discarded (avoiding the discarding of spores at the bottom). Sterile ddH2O was added, and the spore concentration was adjusted to no less than 1-2 × 10⁻⁶ using a hemocytometer. 5 The concentrations of SAFM6-GST purified protein in the suspension were set at 0, 0.05, and 0.1 μg / μL. The spore suspension was dropped onto a hydrophobic glass slide and incubated at 25°C for 12 h. The germination of *Oryza sativa* spores was then observed under a microscope. After 24 h, the formation of *Oryza sativa* spores and appressoriums was observed under a microscope.

[0038] like Figure 1 As shown, the SAFM6-GST protein significantly inhibited the appressorium formation of rice blast fungus. When the SAFM6-GST concentration was 0.05 μg / μL, the appressorium formation rate of rice blast fungus spores was 26.67%, and when the concentration was increased to 0.1 μg / μL, the appressorium formation rate was 5.33%. These results indicate that the OsSAFM6 protein holds promise as a potential agent for the control of rice blast.

[0039] Example 3

[0040] OsSAFM6 purified protein inhibits the mycelial growth of rice blast fungus.

[0041] SAFM6-GST purified protein was mixed into CM-C medium at concentrations of 0 and 0.05 μg / μL. 1 mm diameter mycelial blocks were excised from activated wild-type rice blast fungus strain RB22 and placed on CM-C medium. After incubation at 25°C for 7 days, the colony diameter was measured and recorded.

[0042] like Figure 2 As shown, SAFM6-GST protein has a certain inhibitory effect on the mycelial growth of rice blast fungus.

[0043] Example 4

[0044] OsSAFM6 purified protein inhibits rice blast fungus infection of rice leaves.

[0045] Wild rice blast fungus strain RB22 was activated on OA medium, and the culture conditions and spore suspension acquisition process were the same as in Example 2. The spore concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 6 Add 1 / 10 volume of 0.1% Gelatin (final Gelatin concentration 0.01%, v / v) to the spore solution, and set the concentrations of SAFM6-GST purified protein in the suspension to 0 and 0.05 μg / μL, respectively, for inoculation with rice blast fungus.

[0046] Live inoculation: Take 4-leaf stage rice blast-susceptible material CO39, and spray 1 mL of spore mixture evenly onto the leaf surface using a spray gun. Seal with PVC film and plastic wrap to maintain humidity. After 48 hours of dark incubation, restore light and incubate at 22℃ for 7 days before investigating disease incidence.

[0047] Vaccination status as follows ( Figure 3 The results showed that, compared with CK (SAFM6-GST purified protein concentration 0 μg / μL) and GST (GST purified protein concentration 0.05 μg / μL), the addition of 0.05 μg / μL SAFM6-GST purified protein reduced the lesion area, indicating that OsSAFM6 can inhibit rice blast fungus infection in rice leaves.

Claims

1. Rice OsSAFM6 Application of gene-encoded proteins in improving rice resistance to rice blast fungus. OsSAFM6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

1.

2. Rice OsSAFM6 Application of gene-encoded proteins in the preparation of drugs against rice blast fungus, rice OsSAFM6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

1.

3. The application of an anti-rice blast fungus drug in the prevention and control of rice blast fungus infection, wherein the active ingredient of the anti-rice blast fungus drug comprises rice amino acid sequences as shown in SEQ ID No.

1. OsSAFM6 Proteins encoded by genes.

4. The application as described in claim 3, characterized in that, When applying, the anti-rice blast fungus drug is sprayed onto the leaves of the plant.

5. The application as described in claim 4, characterized in that, The rice OsSAFM6 The concentration of the gene-encoded protein is 0.05~0.1 μg / μL.

Images