Antibacterial peptide for preventing and treating rice blast and application thereof

Abstract

The application discloses an antibacterial peptide for preventing and treating rice blast and application thereof, and relates to an antibacterial peptide SAFM160 for preventing and treating rice blast, which is used for improving the resistance of rice to rice blast fungus, and can be used for preparing a medicine for resisting rice blast fungus and application in preventing and treating rice blast fungus infection. The application finds that the polypeptide SAFM160 can be used as a medicine for preventing and treating rice blast. The antibacterial peptide SAFM160 has obvious inhibiting effect on appressorium of rice blast fungus, and when the concentration of the SAFM160 is 0.2 ug / ul, the appressorium formation rate of spores of the rice blast fungus is 0.57%. Therefore, the antibacterial peptide SAFM160 can be used for improving the resistance of rice to rice blast fungus.

Description

Technical Field

[0001] This invention belongs to the field of rice blast disease control technology, specifically relating to an antimicrobial peptide for rice blast disease control and its application. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is an important staple crop. However, rice blast fungus (L.) Magnaporthe oryzae Rice blast, caused by [unspecified disease], is characterized by its rapid spread and high destructive potential. In severe cases, it can lead to plant death and complete crop failure, posing a significant threat to agricultural production. Therefore, active prevention and control of rice blast is of paramount importance.

[0003] The infection of rice by *Oryza sativa* is a highly specialized active pathogenic process, the basic principles of which can be summarized as follows: Conidia attach to the surface of the rice plant. Signals such as the hydrophobicity of the leaf surface and the presence of micronutrients induce spore germination and the development of a key infection structure—the appressorium—at the apex. The appressorium accumulates enormous turgor pressure through a "glycerol-mediated osmotic pressure engine" mechanism, driving the formation of delicate invasion nails beneath it. These nails pierce the hard cuticle and cell wall of the rice, completing the initial invasion. After invasion, the hyphae spread between and within the host cells, suppressing the rice's immune response by secreting effector proteins and depriving the plant of nutrients, ultimately leading to cell death and manifesting as typical spindle-shaped lesions with a grayish-white center and brown edges. Under suitable conditions, multiple cycles of "invasion-spore production-re-spreading" can occur within a single growing season, resulting in exponential disease growth and a high risk of large-scale epidemics. Depending on the infection site, it can manifest as leaf blast, node blast, neck blast, and grain blast, with neck blast being the most devastating to yield.

[0004] This infection mechanism, where the pathogen directly penetrates the host epidermis using mechanical force generated by appressorium, is the most characteristic pathogenic mechanism of rice blast fungus and the reason for its immense harm. Therefore, the principle of rice blast control is to prevent the germination and invasion of fungal spores, or to inhibit or kill already invading fungal hyphae, both externally and internally, thereby achieving preventative and curative effects. Currently, the most common, efficient, and rapid method for preventing rice blast is pesticide spraying. However, pesticide spraying has certain limitations; it not only pollutes the environment but also increases the fungus's resistance to pesticides. In agricultural production, antimicrobial peptides have significant potential and unique advantages in controlling rice blast. Many antimicrobial peptides have strong inhibitory or killing effects on multiple growth stages of rice blast fungus, including mycelial growth, spore germination, and appressorium formation. Antimicrobial peptides are essentially proteins or polypeptides and do not cause residues or environmental pollution like some chemical pesticides. They have low toxicity to non-target organisms (such as humans, livestock, and beneficial insects), meeting the requirements of agricultural product safety and ecological agriculture development.

[0005] A Chinese invention application with publication number CN118791582A discloses an antibacterial peptide OsSspr1b for rice blast prevention and application thereof, wherein it is disclosed that the antibacterial peptide OsSspr1b has obvious inhibitory effect on the appressorium of Magnaporthe oryzae, and when the concentration of OsSspr1b is 0.05 μg / μL, the appressorium formation rate of Magnaporthe oryzae spores is 80.67%.

[0006] However, there are few new agents for effectively preventing and controlling rice blast at present, and it is of practical significance to develop new antibacterial peptides for preventing and controlling rice blast. SUMMARY

[0007] Through research, the present application discloses the regulation mechanism of rice antibacterial peptide. Based on its functions of inhibiting the germination of Magnaporthe oryzae, inhibiting the formation of appressorium and inhibiting the infection of Magnaporthe oryzae on rice leaves, it is effectively applied to the prevention and control of rice blast, and efficient agents are developed to achieve effective prevention and control of rice blast.

[0008] The present application first provides an antibacterial peptide for rice blast prevention and control, which is antibacterial peptide SAFM160, and the amino acid sequence is shown in SEQ ID No. 4.

[0009] The present application further provides a gene encoding the antibacterial peptide for rice blast prevention and control.

[0010] Preferably, the gene has a nucleotide sequence shown in SEQ ID No. 3.

[0011] The present application further provides an expression vector or expression cassette comprising the gene.

[0012] The present application further provides the application of the antibacterial peptide for rice blast prevention and control in improving the resistance of rice to Magnaporthe oryzae.

[0013] The present application further provides the application of the antibacterial peptide for rice blast prevention and control in preparing a drug against Magnaporthe oryzae.

[0014] The present application further provides a drug against Magnaporthe oryzae, and the active ingredient is the antibacterial peptide for rice blast prevention and control.

[0015] Preferably, the concentration of the antibacterial peptide for rice blast prevention and control is not less than 0.08 μg / μL. More preferably, the concentration of the antibacterial peptide SAFM160 is not less than 0.16 μg / μL. More preferably, the concentration of the antibacterial peptide SAFM160 is not less than 0.2 μg / μL. More preferably, the concentration of the antibacterial peptide SAFM160 is 0.2-0.85 μg / μL.

[0016] The present application further provides the application of the drug in preventing and controlling the infection of Magnaporthe oryzae on rice.

[0017] The present invention also provides a method for preventing and controlling rice blast infection by spraying the drug onto rice leaves.

[0018] The beneficial effects of this invention are:

[0019] This invention has discovered that the peptide SAFM160 can be used as a pesticide to control rice blast. This antimicrobial peptide SAFM160 has a significant inhibitory effect on the appressorium of rice blast fungus; when the concentration of SAFM160 is 0.2 μg / μL, the appressorium formation rate of rice blast fungus spores is 0.57%. Therefore, the antimicrobial peptide SAFM160 of this invention can be used to improve the resistance of rice to rice blast fungus. Attached Figure Description

[0020] Figure 1 Image of purified SAFM160 antimicrobial peptide protein, in which, Figure 1 A in the diagram represents the purified protein copying, where lanes 1-8 are respectively PET-supernatant (empty plasmid control group supernatant), Marker (protein standard molecular weight), SAFM160-supernatant (supernatant), Elution 1 (elution 1), Elution 2 (elution 2), Elution 3 (elution 3), Elution 4 (elution 4), and Elution 5 (elution 5). Figure 1 B in the figure is a Western blot experiment of purified protein, in which lanes 1 to 7 are PET-supernatant (supernatant of empty plasmid control group), Marker (protein standard molecular weight), SAFM160-supernatant (supernatant), Elution 1 (elution 1), Elution 2 (elution 2), Elution 3 (elution 3), and Elution 4 (elution 4), respectively.

[0021] Figure 2 For antimicrobial peptide SAFM160 (rice) SAFM160 The image shows the results of the purified protein's inhibition of appressorium formation by *Strombus haemolyticus*. Figure 2 A in the diagram represents the formation of appressorium by the rice blast fungus. Figure 2 B in the figure represents the statistical results of adherent cell formation.

[0022] Figure 3 The figure shows the results of in vitro inoculation of rice leaves with the antimicrobial peptide SAFM160 (rice SAFM160 supernatant) to inhibit the infection of rice blast fungus. Figure 3 Figure A shows the results of in vitro inoculation of rice leaves with rice blast fungus. Figure 3 B in the figure represents the statistical results of the length of lesions on rice leaves.

[0023] Figure 4 Figure 1 is a graph showing the results of spraying and inoculating rice leaves with the rice blast fungus using the antibacterial peptide SAFM160 (rice SAFM160 albumin) to inhibit the infection of the rice leaves by the rice blast fungus, Figure 4 Figure 1 is a graph showing the results of spraying and inoculating rice leaves with the rice blast fungus using the antibacterial peptide SAFM160 (rice SAFM160 albumin) to inhibit the infection of the rice leaves by the rice blast fungus, Figure 4 Figure 1 is a graph showing the results of spraying and inoculating rice leaves with the rice blast fungus using the antibacterial peptide SAFM160 (rice SAFM160 albumin) to inhibit the infection of the rice leaves by the rice blast fungus, DETAILED DESCRIPTION

[0024] The amino acid sequence of the antibacterial peptide SAFM160 (the amino acid sequence is shown as SEQ ID No. 1):

[0025] MRTSSLVLFAAVAVFGAACTAAAGDESWKTIDANDRHVQDVALWAVAETDWASATGGLTLNTVDGAEKRFEAGVNYYRLTLEASSRVVAKYLRFQAVVYEEGDEHKLVSFVPIH.

[0026] The complete gene sequence of the antibacterial peptide SAFM160 (as shown in SEQ ID No. 2, which further includes the sequence encoding the signal peptide):

[0027] ATGAGGACCAGCAGCCTCGTCCTGTTCGCCGCCGTCGCCGTCTTCGGCGCTGCCTGCACGGCGGCGGCCGGCGACGAATCCTGGAAGACGATCGACGCGAACGACCGGCACGTCCAGGACGTCGCCCTGTGGGCGGTGGCGGAGACAGACTGGGCGTCGGCGACGGGCGGCCTCACGCTCAACACGGTGGACGGCGCCGAGAAGAGGTTTGAGGCCGGCGTGAACTACTACCGCCTCACCCTCGAGGCGTCGAGCCGCGTCGTCGCCAAGTACCTCAGGTTCCAGGCGGTGGTGTACGAGGAGGGCGACGAGCACAAGCTCGTCTCCTTCGTCCCCATCCACTGA.

[0028] The gene sequence of the antibacterial peptide SAFM160 after removing the sequence encoding the signal peptide (as shown in SEQ ID No. 3):

[0029] ATGGACGAATCCTGGAAGACGATCGACGCGAACGACCGGCACGTCCAGGACGTCGCCCTGTGGGCGGTGGCGGAGACAGACTGGGCGTCGGCGACGGGCGGCCTCACGCTCAACACGGTGGACGGCGCCGAGAAGAGGTTTGAGGCCGGCGTGAACTACTACCGCCTCACCCTCGAGGCGTCGAGCCGCGTCGTCGCCAAGTACCTCAGGTTCCAGGCGGTGGTGTACGAGGAGGGCGACGAGCACAAGCTCGTCTCCTTCGTCCCCATCCAC.

[0030] The amino acid sequence of the antibacterial peptide SAFM160 after removing the signal peptide coding sequence (the amino acid sequence is shown as SEQ ID No. 4):

[0031] MDESWKTIDANDRHVQDVALWAVAETDWASATGGLTLNTVDGAEKRFEAGVNYYRLTLEASSRVVAKYLRFQAVVYEEGDEHKLVSFVPIH.

[0032] Example 1

[0033] Obtaining of the antibacterial peptide SAFM160 (the amino acid sequence is shown as SEQ ID No. 4).

[0034] According to the CDS sequence (shown as SEQ ID No. 3) of the gene SAFM160 , the primers SAFM160-F / R were designed. The primer sequences are as follows:

[0035] SAFM160-F: cagcaaatgggtcgcggatccATGGACGAATCCTGGAAGACG;

[0036] SAFM160-R: ctcgagtgcggccgcaagcttGTGGATGGGGACGAAGGAGA.

[0037] Construction of the vector: taking the genome of rice variety Zhonghua 11 as the template, the CDS sequence of the gene SAFM160 was amplified by using the primers SAFM160-F / R. The amplified fragment was connected to the PET-28aHIS vector which was digested by BamHI and Hind III. The connected plasmid was transformed into E. coli by using the heat shock method, and the positive clones were selected for detection.

[0038] Induction of SAFM160-HIS protein (C-terminal fusion of SAFM160 protein with HIS tag sequence) expression: After the SAFM160-HIS vector is sequenced correctly, the plasmid is transformed into E. coli strain BL21 by heat shock method. After selecting positive clones and culturing at 37°C until the OD value is 0.6, the positive clones are induced at 28°C for 10 h, and the IPTG concentration is 1 mM.

[0039] Purification of SAFM160-HIS protein:

[0040] (1) Centrifuge 10,000 rpm for 2 min per 150 mL of bacterial cells to remove the supernatant; add 15 mL of HIS binding / washing buffer (20 mM Na2HPO4, 0.5 M NaCl, 35 mM imidazole, pH adjusted to 7.4) to suspend the bacterial pellet, then add 300 μL of 10 mg / mL lysozyme and 150 μL of 0.1 M phenylmethylsulfonyl fluoride (PMSF); shake slowly at 4°C for 30 min for enzymatic hydrolysis;

[0041] (2) After the enzymatic hydrolysis process is completed, the bacterial cells are broken by ultrasonic, then centrifuged at 10,000 rpm for 10 min at 4°C;

[0042] (3) Take 200 μL of the supernatant as Input, transfer the remaining supernatant to a HIS protein resin flow column that has been equilibrated, adjust the flow rate to 7-9 s / drop, collect 200 μL of the effluent as Flowthroµgh; suspend the bacterial pellet with 15 mL of water, take 200 μL as Bacteriapellet; the whole process is carried out in a 4°C refrigerator to prevent protein denaturation;

[0043] (4) Add 5 mL of binding / washing buffer, control the flow rate at 4-6 s / drop, collect 200 μL of the effluent below the column as Wash1; repeat this step once, collect 200 μL of the effluent as Wash2;

[0044] (5) Elute the target protein with 1 mL of HIS elution buffer (20 mM Na2HPO4, 0.5 M NaCl, 500 mM imidazole, pH adjusted to 7.4), collect the eluate (containing the target protein) as Elution1; repeat this step 4 times, labeled as Elution2, Elution3, Elution4, and Elution5, respectively.

[0045] The purified protein (the sequence is shown as SEQ ID No. 4, i.e. the antibacterial peptide SAFM160 described in the present application) was detected by Coomassie brilliant blue staining and Western-blot, and then the concentration of the purified protein was determined. The Coomassie brilliant blue staining result is shown as A in Figure 1 , and the Western-blot result of detecting the protein expression is shown as B in Figure 1 .

[0046] Example 2

[0047] SAFM160 purified protein inhibits the formation of appressorium of Magnaporthe oryzae.

[0048] The wild Magnaporthe oryzae strain B157 was activated on OA medium and cultured in dark at 25°C for 3 days and cultured under light for 4 days. Sterile ddH2O was added to the culture dish, and the mycelium was lightly scraped with an inoculation loop to elute the spores from the medium. The eluate was filtered through a magic filter cloth to obtain a spore suspension. The spore suspension was placed in a 2 mL centrifuge tube and centrifuged at 10000 rpm for 1 min. The supernatant was discarded (to avoid pouring out the bottom spores), sterile ddH2O was added, and the spore concentration was adjusted to not less than 3×10 4 / mL using a hemocytometer. A control group (H2O) and different concentrations of SAFM160-HIS purified protein were set up, and the concentrations thereof in the suspension were 0 (i.e. only buffer was used, buffer: 20 mM Na2HPO4, 0.5 M NaCl, pH adjusted to 7.4), 0.08, 0.1, 0.128, 0.16, 0.2 µg / µL, respectively. The spore solution was dropped on the surface of a hydrophobic glass slide, and the formation of Magnaporthe oryzae spores and appressorium was observed under a microscope after 24 h.

[0049] As shown in Figure 2 , the SAFM160-HIS protein has a significant inhibitory effect on the appressorium of Magnaporthe oryzae. The appressorium formation rate of Magnaporthe oryzae spores in H2O was 97.61%, and the appressorium formation rate of Magnaporthe oryzae spores was 0.57% when the concentration of SAFM160-HIS was 0.2 µg / µL, which was decreased by 97.04% compared with the control group, and there was a highly significant difference. This result indicates that the antibacterial peptide SAFM160 is expected to be used as a medicament for preventing and treating rice blast.

[0050] Example 3

[0051] SAFM160 supernatant protein inhibits the infection of Magnaporthe oryzae to rice leaves.

[0052] The wild Magnaporthe oryzae strain B157 was activated on OA medium, and the culture conditions were the same as those in the process of obtaining the spore suspension in Example 2. The spore concentration was adjusted to 1×106 The concentration of SAFM160-HIS protein supernatant in the suspension was set to 0.85 μg / μL, and the concentration of the rice blast fungus inoculum was set to 1.5 x 105spores / mL.

[0053] Water agar configuration: 3% water agar was prepared with water and sterilized at 110°C for 30 min. After the water agar was slightly cooled, kinetin was added to make the working concentration 2 μg / mL, 100 μg / mL carbenicillin, and 100 μg / mL streptomycin. After uniform mixing, the liquid was poured into 15 cm culture dishes.

[0054] In vitro inoculation: 10,000 rpm centrifugation for 2 min, and the supernatant was removed. The precipitated bacteria were suspended in 15 mL of HIS suspension buffer (20 mM Na2HPO4, 0.5 M NaCl, pH 7.4), and the bacteria were broken by ultrasonic crushing. Then, the bacteria were centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was collected. The PET-His empty control group, buffer solution (20 mM Na2HPO4, 0.5 M NaCl, pH 7.4), and SAFM160-HIS supernatant protein with a concentration of 0.85 μg / μL in the suspension were set. Group

[0055] The 4-leaf rice rice blast susceptible material CO39 was taken, and about 5 cm was taken from the lower part of the second leaf. Two wounds were made on the main vein with a needle without penetrating. The drawn leaves were gently wiped with alcohol for 3 times, and then washed with ddH2O for 3 times. The washed leaves were placed on the water agar medium with the front side facing up, and the water agar block was used to press the two ends of the leaves to prevent the leaves from curling. 10 μL of spore mixture was dropped on the wound with a gun, and after 30 s, the inoculated CO39 leaves were placed in a 25°C constant temperature incubator, and after 48 h of dark culture, light was restored. After 7 d of culture under light and dark alternating conditions, the disease incidence was investigated.

[0056] The inoculation situation is shown in Figure 3 The results showed that the addition of 0.85 μg / μL of SAFM160-HIS supernatant protein could reduce the lesion area compared with the control group, indicating that the antibacterial peptide SAFM160 could inhibit the infection of rice blast fungus on rice leaves.

[0057] Example 4

[0058] SAFM160 supernatant protein inhibits the infection of rice blast fungus on rice leaves.

[0059] ​Rice variety CO39 seeds were soaked at 37°C for 1 day, and then germinated at 37°C for 2 days. Most of the seeds germinated to about 0.5 cm. The germinated seeds with uniform growth were selected and sowed in pots at 5 seeds per pot. The soil was kept appropriately dry after sowing. When the rice grew to about 2 leaves, 3 rice seedlings with uniform growth were reserved. The 4-leaf stage to tillering stage rice seedlings were used for inoculation.

[0060] Wild rice Magnaporthe oryzae strain B157 was activated on OA medium, and the culture conditions were the same as those in the process of obtaining the spore suspension in Example 2. The spore concentration was adjusted to 1×10 6 The concentration of the SAFM160-HIS protein supernatant in the suspension was set to 0.85 μg / μL for Magnaporthe oryzae inoculation.

[0061] Rice spray inoculation: 10,000 rpm centrifugation of 150 mL of bacterial bodies for 2 min, and removal of the supernatant; 15 mL of HIS suspension buffer (20 mM Na2HPO4, 0.5 M NaCl, pH adjusted to 7.4) was added to the precipitated bacterial mass to suspend the bacteria. The bacterial bodies were broken by ultrasonic crushing, and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was taken. The control group (water), the PET-His empty load group (PET group), the buffer solution group, and the SAFM160-HIS supernatant protein with a concentration of 0.85 μg / μL in the suspension were set.

[0062] Four-leaf stage to tillering stage rice CO39 was prepared, and transparent PVC film was made into a roll to seal the rice to be inoculated. Inoculation was performed in the evening, and a small spray bottle was used to spray the spore solution with added Geltin (agarose) on the rice leaves. After inoculation, the upper part was covered with a wet preservative film for moisturizing. The temperature was 22°C, and the culture was performed in the dark for 1 day, followed by alternating light for 16 h and dark for 8 h. Observation was performed every day, and when obvious lesions appeared on the leaves, the leaves were photographed or observed and photographed when the difference in disease was the largest. The diseased leaves were pasted on A4 with double-sided tape.

[0063] The inoculation conditions are shown in Table 1. Figure 4 The results show that the addition of 0.85 μg / μL of SAFM160-HIS supernatant protein can reduce the lesion area compared with the control group, indicating that the antimicrobial peptide SAFM160 can inhibit the infection of Magnaporthe oryzae on rice leaves.

Claims

1. Use of an antibacterial peptide for the prevention and treatment of rice blast in improving the resistance of rice to Magnaporthe grisea, characterized in that, The antibacterial peptide for preventing and treating rice blast is antibacterial peptide SAFM160, and the amino acid sequence is shown as SEQ ID No.

4.

2. Use of the antibacterial peptide for preventing rice blast in the preparation of a medicine for resisting Pyricularia oryzae, characterized in that, The antibacterial peptide for preventing and treating rice blast is antibacterial peptide SAFM160, and the amino acid sequence is shown as SEQ ID No.

4.

3. The use of a medicament for anti-Magnaporthe grisea for preventing and treating Magnaporthe grisea infection in rice, characterized in that, The active pharmaceutical ingredient for resisting Magnaporthe grisea is an antibacterial peptide for preventing and treating rice blast, and the antibacterial peptide for preventing and treating rice blast is antibacterial peptide SAFM160, and the amino acid sequence is shown as SEQ ID No.

4.

4. Use according to claim 3, characterized in that, The concentration of the antibacterial peptide for preventing and treating rice blast is not less than 0.08 μg / μL.

5. A method of controlling Magnaporthe grisea infection in rice, characterized by, The active pharmaceutical ingredient for resisting Magnaporthe grisea is an antibacterial peptide for preventing and treating rice blast, and the antibacterial peptide for preventing and treating rice blast is antibacterial peptide SAFM160, and the amino acid sequence is shown as SEQ ID No.

4.

6. The method for preventing and controlling rice blast fungus infection according to claim 5, characterized in that, The concentration of the antibacterial peptide for preventing and treating rice blast is not less than 0.08 μg / μL.

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