Antibacterial peptide and application thereof

Abstract

The present application belongs to the field of agricultural biotechnology, and through a large number of sequence analysis and tests, a short peptide with a function of antagonizing fungi in an extracellular protein Atp2 beta subunit protein of Rhodopseudomonas palustris is found. Tests show that the short peptide with the function of antagonizing fungi has low production cost, is non-toxic and environmentally friendly, can significantly inhibit the formation of appressorium of rice blast fungus and the pathogenicity on the host, and has potential broad-spectrum antagonistic function activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biotechnology, in particular, the present application relates to an antibacterial peptide and its application. BACKGROUND

[0002] The quality and disease control of rice have always been the focus of people's attention. Rice blast is one of the main diseases of rice, which can occur throughout the growth period of rice. This disease can occur in leaves, nodes, panicle necks, grains and other parts, and common ones are leaf blast and panicle neck blast, and stem blast. Severe occurrence of rice blast can cause rice seedling leaf to die, node to be short, white panicle and grain to be formed, and when it occurs seriously, it has a great impact on rice yield. The prevention and control of rice blast mainly adopts measures such as breeding of disease-resistant varieties, chemical control and strengthening of cultivation management. Among them, disease-resistant varieties are mainly vertical resistance varieties based on the "gene for gene" theory, and the genetic variation of rice blast fungus is very frequent. The long-term use of single disease-resistant varieties in large areas can easily change the composition and structure of physiological races, resulting in the loss of disease resistance. Chemical control has the advantages of economic efficiency, convenience, quick effect, etc., therefore, chemical control has always been an important part of the comprehensive management system of rice blast. However, due to the increase of drug resistance and the delay of discovery, the unreasonable use of pesticides, the over-standard of pesticide application dose and frequency, and the environmental pollution and quality safety of agricultural products, etc. Therefore, how to reduce the amount of pesticide and green comprehensive prevention and control of rice blast is still an important problem in current rice production.

[0003] Previous studies have found that the extracellular protein Atp2 β subunit of Rhodopseudomonas palustris has excellent prevention and control effect on rice blast. CN201610333926.1 discloses the application of Rhodopseudomonas palustris fermentation broth in preventing and controlling rice blast, wherein the Rhodopseudomonas palustris fermentation broth is obtained by culturing Rhodopseudomonas palustris LY-6. CN201610333888.X discloses the application of Rhodopseudomonas palustris biocontrol agent in preventing and controlling rice blast, wherein the Rhodopseudomonas palustris biocontrol agent is obtained by culturing Rhodopseudomonas palustris LY-6. CN201910613980.5 discloses the application of Rhodopseudomonas palustris Atps2 protein in preventing and controlling rice blast, wherein the Rhodopseudomonas palustris biocontrol protein is obtained by separating the extracellular fermentation broth of Rhodopseudomonas palustris.

[0004] Throughout the course of evolution, the ability of organisms to protect themselves from microbial or other species invasion has been a key factor for survival. From bacteria to humans, all species resist microbial invasion by relatively simple mechanisms, but exhibit functional complexity. Antimicrobial peptides (AMPs) are involved in the process of antibacterial immunity, AMPs are components of innate immunity, forming the first line of defense against invading pathogens in many organisms. AMPs are short (<100 amino acids) amphipathic molecules encoded by genes, with broad-spectrum antimicrobial activity, exhibiting multiple modes of action, including bacteriostatic, microbicidal, and cytolysic properties. Currently, there is no related report on peptides in the biological control of rice blast. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the present application provides an antimicrobial peptide and its application through extensive computational biology analysis and practical research testing. Specifically as follows:

[0006] In one aspect of the present application, an antimicrobial peptide is provided, which is derived from the Atp2 beta subunit of extracellular protein of Rhodopseudomonas palustris, and the amino acid sequence of the antimicrobial peptide is VIGAVV (SEQ ID NO. 1).

[0007] In the present application, the antimicrobial peptide is from the 7-80 amino acid position (7-80aa) of the N-terminal representative region of the Atp2 beta subunit protein sequence of extracellular protein of Rhodopseudomonas palustris, which is named ATP5.

[0008] In one aspect of the present application, the application discloses the application of the aforementioned antimicrobial peptide in the resistance to rice blast.

[0009] In one aspect of the present application, the application discloses an antimicrobial agent, which contains an antimicrobial peptide, and the amino acid sequence of the antimicrobial peptide is VIGAVV (SEQ ID NO. 1).

[0010] In one embodiment, the antimicrobial agent is a biological pesticide dosage form, such as a liquid preparation, a wettable powder, a freeze-dried powder. Preferably, the dosage form is a freeze-dried powder, which is beneficial to maintain the biological activity of the peptide. The freeze-dried powder also contains a polypeptide stabilizer and a buffer. The polypeptide can be prepared by conventional chemical synthesis method in the art, or obtained by microbial expression and purification through prokaryotic or eukaryotic expression method. As for the liquid preparation, the freeze-dried powder can be mixed with deionized water or physiological saline or buffer solution to prepare. In the process of use, it is sprayed on the surface of crops to play the role of antibacterial / bacteriostatic. Beneficial effects

[0011] (1) The synthesis process of the functional short peptide is simple, low in cost, and the liquid preparation required for antibacterial effect can be prepared by simple, fast and low-cost operation method;

[0012] (2) The freeze-dried powder or liquid preparation of the functional short peptide has the characteristics of environmental friendliness, non-toxicity to humans and animals, no phytotoxicity to crops, good antibacterial effect, simple and convenient application, heat resistance and stability, etc.

[0013] (3) The freeze-dried powder of the functional short peptide can inhibit the formation of appressorium of Magnaporthe oryzae, inhibit the pathogenicity of Magnaporthe oryzae on rice, and has potential broad-spectrum antifungal activity. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 : A, chain structure of the functional short peptide of the application; B, homology alignment of the functional short peptide.

[0015] Figure 2 : A is the inhibition of the functional short peptide in Example 2 of the application on the formation of appressorium of Magnaporthe oryzae, ** represents significant difference (P < 0.01); B is the temperature tolerance and pH tolerance test of the functional short peptide; C-D are comparative diagrams of the inhibition of the functional short peptide in Example 3 of the application on the pathogenicity of Magnaporthe oryzae on rice, ** represents significant difference (P < 0.01). DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the application clearer, the application will be further described in detail below in combination with examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial channels, and the reagents used in the application are analytical grade reagents. The specific examples described herein are only used to explain the application and not to limit the application.

[0017] Example 1. Synthesis and screening of antifungal functional short peptides

[0018] The ATP2 β subunit protein sequence (NCBI Sequence ID: CP041387.1, 1431 bp) is analyzed by bioinformatics analysis method, and the N-terminal representative region (7-80 aa) of the Atp2 β subunit protein sequence is selected and named as ATP5. Further, the sequence is subjected to highlight analysis to obtain 2 Polar sequences, which are named as ATP5.1 and ATP5.2, and 4 Hydrophobic sequences, which are named as ATP5.3, ATP5.4, ATP5.5 and ATP5.6. Meanwhile, 6 antagonistic fungal functional short peptides are chemically synthesized, and the initial screening of the antibacterial ability is performed. The ATP5.3 short peptide shows high antifungal activity. Further, sequence alignment of the functional short peptides of different bacteria shows that the sequence of ATP5.3 is highly conserved. Figure 1 B)

[0019] Example 2. Functional short peptide ATP5.3 inhibits the formation of appressorium of Magnaporthe oryzae

[0020] The conidia of Magnaporthe oryzae Guy11 are collected, washed with ddH2O twice, and then 30 μL of the freeze-dried powder of the 6 functional short peptides prepared in Example 1 is added dropwise on the hydrophobic membrane. The formation of appressorium is observed under a microscope after 8 and 24 h, respectively. It is found that the formation of appressorium of Magnaporthe oryzae conidia treated with the functional short peptide ATP5.3 (amino acid sequence: VIGAVV) is significantly inhibited (see Figure 2 Fig. A) and the inhibition rate of appressorium formation is positively correlated with the concentration of the functional short peptide (see Figure 2 Fig. B). Statistical analysis shows that the appressorium formation rate of the 200 μg / mL treatment group for 8 h is about 15%, which is significantly lower than that of the 200 μg / mL Atp2 treatment group (63%) and the water control group (94.67%), i.e., the functional short peptide ATP5.3 inhibits the formation of appressorium of Magnaporthe oryzae.

[0021] Example 3. Functional short peptide ATP5.3 has heat-resistant stability

[0022] The pH stability of the functional short peptide is analyzed under the conditions of pH range of 4 to 9, pH interval of 0.5, and temperature range of 30 to 100 °C with an interval of 10 °C. After the short peptide is treated at different temperatures and pHs for 30 min, the appressorium formation inhibition experiment is performed to detect the stability of the antibacterial peptide. The results are shown in Figure 2As shown in Figure B, it is shown that the antibacterial peptide ATP5.3 has a broad spectrum of heat stability, and when the pH is less than 5 or greater than 7.5, the antibacterial activity is reduced to about 30%.

[0023] Example 4. Functional short peptide ATP5.3 inhibits the pathogenicity of Magnaporthe oryzae on rice

[0024] Select healthy rice seedlings (variety Co-39, which is sensitive to Magnaporthe oryzae) that have grown for about 2 weeks, collect spores from a sporulation plate, and add them to a 200 μg / mL functional short peptide ATP5.3 liquid preparation containing 2% gelatin to prepare a suspension with a final concentration of 5.0 x 10 4 The spore suspension was then uniformly sprayed onto the rice leaves using a throat sprayer. The spore suspension in water was used as a control. The experimental group was divided into three treatment groups: functional short peptide ATP5.3 liquid preparation was sprayed one day before spore inoculation (-1), on the day of spore inoculation (0), and one day after spore inoculation (+1). After inoculation, the rice was moved to a container with 28°C, darkness, and high humidity (RH > 95%) for 24 h, and then placed in a container with alternating light and darkness (12 h each) and high humidity for continuous cultivation. The results were observed and photographed after 7 days. The results showed that the functional short peptide ATP5.3 liquid preparation significantly inhibited the pathogenicity of Magnaporthe oryzae on rice leaves (see Figure C in the middle). Figure 2 The number of lesions per unit area was found to be lower in the functional short peptide ATP5.3 liquid preparation treatment group than in the control group (P < 0.01, see Figure D in the middle), indicating that the functional short peptide ATP5.3 liquid preparation significantly inhibited the pathogenicity of Magnaporthe oryzae on rice. Figure 2

[0025] The above content is a further detailed description of the present application in combination with a specific implementation, which cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can be made, which should be considered as belonging to the protection scope determined by the claims submitted by the present application.​

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide is derived from the Atp2β subunit of the extracellular protein of Rhodopseudomonas palustris, and the amino acid sequence of the antimicrobial peptide is VIGAVV.

2. The application of the antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide is used to combat rice blast.

3. An antibacterial reagent, characterized in that, The antimicrobial reagent includes an antimicrobial peptide derived from the Atp2β subunit of the extracellular protein of Rhodopseudomonas palustris, and the amino acid sequence of the antimicrobial peptide is VIGAVV.

4. The antibacterial reagent according to claim 3, characterized in that, The antibacterial agent is a biological pesticide formulation.

5. The antibacterial reagent according to claim 4, characterized in that, The dosage form is a liquid preparation, a wettable powder, or a lyophilized powder.

6. The application of the antibacterial agent according to claim 3 in the treatment of rice blast.

7. The application according to claim 6, characterized in that, The antibacterial reagent is formulated as a liquid preparation and sprayed onto the crop surface to exert its effect.

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