Antibacterial peptide KT-12 and application thereof

Abstract

The invention belongs to the technical field of biology, particularly relates to an antibacterial peptide KT-12 and application thereof, and provides a novel antibacterial peptide KT-12, the amino acid sequence of the novel antibacterial peptide KT-12 is as shown in SEQ ID No.1, and the purity of the synthetic peptide is gt; 90%; through screening, the peptide KT-12 has antibacterial activity to the helminthosporium bombycis, and within 12-72 hours of inoculation, the bacteriostasis rate to the helminthosporium bombycis is 47.9%-72.3%, which indicates that the peptide KT-12 can effectively inhibit the growth of the helminthosporium bombycis and shows significant antibacterial activity; after the peptide KT-12 is added, spores of the helminthosporium bombycis do not germinate, and the peptide KT-12 on the surface has antibacterial activity on the helminthosporium bombycis. The application is beneficial to expanding the application scene of the antibacterial peptide in plant disease control, and provides theoretical support and novel candidate molecules for research and development of biopesticides.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an antimicrobial peptide and application thereof. Background Art

[0002] Corn (Zea mays L.) is one of the world's three major staple crops, playing a crucial role in global food security, animal feed, and industrial raw material supply. With the expansion of cultivated areas and the promotion of high-density cultivation techniques, corn faces threats from a variety of pathogens during its growth. Among them, northern corn leaf blight, due to its wide range of occurrence and severe damage, has become a major disease affecting corn yield and quality. Caused by the fungus Helminthosporium macrosporum, this disease forms long, spindle-shaped lesions on leaves, leading to leaf dryness and premature plant aging. In severe cases, yield reductions can exceed 30%, or even total crop failure.

[0003] Currently, the main methods for controlling corn leaf blight include breeding resistant varieties, crop rotation, and spraying chemical pesticides. However, the pathogen's high genetic diversity allows it to quickly overcome resistance. Long-term reliance on chemical fungicides can easily lead to the development of resistance, resulting in environmental pollution and food safety risks. Therefore, developing new, efficient, green, and safe anti-disease measures has become a research hotspot in plant protection.

[0004] Antimicrobial peptides are a class of natural immune factors widely found in animals, plants, and microorganisms. They are typically composed of 2-100 amino acids and are highly cationic and amphiphilic. They can exert their bactericidal effects by disrupting microbial cell membranes or interfering with their metabolic processes. Compared to traditional pesticides, antimicrobial peptides offer significant advantages, including broad-spectrum antimicrobial activity, high efficacy, low toxicity, and resistance to drug resistance. Due to the global overuse of antibiotics, an increasing number of superbugs are emerging, which are increasingly resistant to traditional antibiotics. This has led to an urgent search for alternatives to traditional antibiotics. This has made antimicrobial peptides a promising antimicrobial agent, showing great potential for application in plant disease prevention and control. Patent 202110981649.6 discloses a peptide, KC-19, with antimicrobial activity. Peptide KC-19 primarily inhibits fungi by aggregating hyphae. KC-19 has shown inhibitory effects against the pathogens Helicoverpa zeae, Curvularia lunata, and Fusarium graminearum.

[0005] In recent years, studies have reported that antimicrobial peptides from various sources exhibit significant inhibitory activity against plant fungal pathogens, but research on antimicrobial peptides targeting the pathogen of corn leaf blight is still relatively limited. Summary of the Invention

[0006] To solve the above problems, the present invention provides an antimicrobial peptide KT-12 and its application.

[0007] The technical solution of the present invention is achieved as follows:

[0008] On the one hand, the present invention applies to protect an antimicrobial peptide KT-12, the amino acid sequence of which is shown in SEQ ID No. 1.

[0009] In a second aspect, the present invention applies to protect the use of the antimicrobial peptide KT-12 in antifungal treatment.

[0010] Preferably, the antifungal effect is achieved by the antimicrobial peptide KT-12 binding to the fungal cell membrane and causing damage to the cell membrane structure.

[0011] Preferably, the fungus is Helicoverpa macromaculata.

[0012] In a third aspect, the present invention applies to protect the use of the antimicrobial peptide KT-12 in inhibiting the hyphae growth and / or spore germination of pathogenic bacteria.

[0013] Preferably, the pathogen is Helicoverpa macrophyte.

[0014] In a fourth aspect, the present invention applies to protect the use of the above-mentioned antimicrobial peptide KT-12 in preventing and controlling corn leaf blight.

[0015] In a fifth aspect, the present invention applies to protect the use of the above-mentioned antimicrobial peptide KT-12 in the preparation of products for preventing and treating corn leaf blight.

[0016] In a sixth aspect, the present invention applies to protect a product for preventing and treating corn leaf blight, wherein the product comprises the above-mentioned antimicrobial peptide KT-12.

[0017] In a seventh aspect, the present invention applies to protect a method for preventing and controlling corn leaf blight, which comprises spraying the above-mentioned product for preventing and controlling corn leaf blight on corn leaves.

[0018] Preferably, the antimicrobial peptide KT-12 in the above product is used at a concentration of 5-100 μM.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides a novel antimicrobial peptide KT-12, whose amino acid sequence is shown in SEQ ID No. 1. The structure and molecular weight of the synthetic peptide are in line with expectations, and the purity of the synthetic peptide is >90%. After screening, peptide KT-12 has antimicrobial activity against Helminthosporium macrosporum. Within 12-72 hours of inoculation, the antibacterial rate against Helminthosporium macrosporum is 47.9%-72.3%, indicating that peptide KT-12 can effectively inhibit the growth of Helminthosporium macrosporum and exhibit significant antimicrobial activity. After adding peptide KT-12, the spore germination of Helminthosporium macrosporum is inhibited, which again indicates that peptide KT-12 has antimicrobial activity against Helminthosporium macrosporum. This application helps to expand the application scenarios of antimicrobial peptides in plant disease prevention and control, and provides theoretical support and new candidate molecules for the research and development of biopesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Mass spectra of 12 synthetic peptides.

[0023] Figure 2 HPLC chromatograms of 12 synthetic peptides.

[0024] Figure 3 The present invention is to screen small peptides against Helicoverpa macrophylla.

[0025] Figure 4 This is the effect of antimicrobial peptide KT-12 on the mycelial growth of Helicoverpa macrophylla at different times.

[0026] Figure 5 Statistical analysis of the effect of antimicrobial peptide KT-12 on the colony diameter of Helicoverpa macrophylla at different times.

[0027] Figure 6 This is an analysis of the inhibition rate of antimicrobial peptide KT-12 on the growth of Helicoverpa macrophylla at different times.

[0028] Figure 7 This is the effect of antimicrobial peptide KT-12 on the spore germination of Helicoverpa macrophyte. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0031] KT-12 is a novel antimicrobial peptide recently identified through sequence screening and in vitro activity validation. Its sequence is rich in lysine residues and is generally cationic, theoretically capable of binding to fungal cell membranes and causing structural disruption. Preliminary experiments have demonstrated that KT-12 significantly inhibits spore germination and hyphal growth of Helicoverpa macrosporum. These findings will help expand the application of antimicrobial peptides in plant disease control and provide theoretical support and novel candidate molecules for biopesticide development.

[0032] Example 1: Synthesis and mass spectrometry identification of novel corn peptides

[0033] 1. Peptide synthesis

[0034] The synthesis was carried out using the Fmoc (9-fluorenylmethoxycarbonyl) peptide solid-phase synthesis method, with the order of synthesis from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). First, the Fmoc-protected amino acid was attached to the resin. The Fmoc protecting group in the resin was removed using a 20% piperidine / DMF solution to expose the active NH2 group. The second Fmoc-protected amino acid was attached using the HBTU method until the entire peptide chain was connected. The peptide chain was eluted from the resin with DMF solvent to obtain a crude product. The specific experimental method is as follows:

[0035] (1) Resin synthesis

[0036] a.Fmoc-AA-Wang resin

[0037] It is obtained by the reaction of the carboxyl group on Fmoc-AA-OH with the -OH group on Wang resin, removing one molecule of H2O. The reaction is as follows:

[0038] The specific synthesis steps are as follows:

[0039] Add weighed Wang resin to a round-bottom flask and an appropriate amount of dry DMF to swell the mixture. Stir with a magnetic stirrer. Add 1.5 eq of Fmoc-AA-OH and then 6 eq of pyridine. Once the Fmoc-AA-OH has dissolved, slowly add 3 eq of 2,6-dichlorobenzoyl chloride dropwise using a dropper. Cover tightly with a rubber stopper and shake for at least 3 hours. Remove the solution by suction using a fritted funnel. Wash the resin with DMF (3 times), MeOH (1 time), DCM (3 times), and MeOH (2 times). Transfer the mixture to a collection tube and dry in a vacuum desiccator overnight.

[0040] b. Synthesis of Fmoc-AA-2-Cl Trt resin

[0041] Fmoc-AA-2-Cl Trt Resin is obtained by reacting the carboxyl group on Fmoc-AA-OH with the -Cl on 2-Cl Trt resin to remove one HCl molecule. It is mainly used for the synthesis of three amino acids with Pro, Cys, and His at the C-terminus. The reaction is as follows:

[0042] The specific synthesis steps are as follows:

[0043] To a round-bottom flask, add weighed 2-Cl Trt resin and 2 eq of Fmoc-AA-OH. Add an appropriate amount of dry DCM to swell the resin and dissolve the amino acid, followed by 6 eq of DIPEA. Secure the flask with a stopper and stir at room temperature for 2-3 hours. Add HPLC-grade MeOH (10 mL per 1 g of resin) and stir for 30 minutes. Remove the solution by suction filtration using a fritted funnel, then wash the resin with DMF (3 times), MeOH (1 time), DCM (3 times), and MeOH (2 times). Transfer the mixture to a collection tube and dry in a vacuum desiccator overnight.

[0044] c. Synthesis of Rink Amide MBHA resin

[0045] It is obtained by the reaction of linker amide group and MBHA resin. The specific steps are as follows:

[0046] Add MBHA resin to a reactor, add 2 eq HB to activate Rink Amide, and add 6 times the amount of NMM. React for 3-4 h. Cap the ends and transfer the resin to a fritted funnel. Wash with DMF three times, MeOH once, DCM three times, and MeOH three times. Drain the resin under vacuum.

[0047] (2) Weighing and swelling of resin

[0048] Calculate the required amount of resin based on the number of moles of peptide to be synthesized and the degree of substitution of the resin (resin amount = number of moles of peptide to be synthesized / degree of substitution of resin). Weigh the resin and place it into the reactor labeled with the corresponding number. Place the reactor with resin on a stand and add 3-5 times the volume of the resin bed in DMF. Allow to swell for 30 minutes.

[0049] (3) Deprotection and washing

[0050] Add about 3 times the volume of the resin bed 20% piperidine / DMF solution, let nitrogen react for 5 minutes, then remove it, add about 3 times the volume of the resin bed 20% piperidine / DMF solution, let nitrogen react for 15 minutes.

[0051] (4) Ninhydrin detection after deprotection

[0052] Place a small amount of resin in a small test tube, add ninhydrin, and react at 110-120°C for 3 minutes. Observe the color development. Different amino acids will display different colors. Most amino acid resins will appear dark blue or blue, while Pro, Asn, Asp, Gln, Glu, His, and Cys will often appear red or reddish-brown. However, due to variations in peptide sequence during synthesis, the colors displayed by amino acids may not always match the above description and may sometimes be affected by the presence of preceding amino acids, resulting in different colors. However, if the color displayed by the resin in the ninhydrin test after deprotection is clearly different from that during ligation, the deprotection reaction has been completed.

[0053] (5) Ligation reaction

[0054] Solvent:

[0055] DMF (N,N-dimethylformamide), DCM (dichloromethane)

[0056] Raw material ratio:

[0057] AA:HBTU:NMM=3:2.85:6 (molar ratio)

[0058] The amino acid was added to the reactor, and the minimum amount of DMF that could dissolve the amino acid was added, followed by HBTU and NMM, and the reaction was continued for 60 min.

[0059] Note:

[0060] ① Turn on nitrogen before adding DMF to dissolve to prevent amino acids from leaking.

[0061] ② Be sure to wait until the amino acids are completely dissolved before adding HBTU.

[0062] ③During the reaction, the amount of DMF solvent added should be sufficient to completely immerse the resin.

[0063] ④The nitrogen stirring airflow should be appropriate.

[0064] (6) NT detection after connection

[0065] Use a pipette to transfer a small amount of resin to a test tube. Wash the resin once with DMF and once with MeOH. Follow the same steps as for the deprotection test. After the connection is complete, the resin is generally colorless and transparent, but may occasionally appear pale yellow. If the color is significantly different from the color displayed during deprotection, the connection is complete.

[0066] (7) End-capping

[0067] Ratio: 5%-10% acetic anhydride / DMF+NMM (acetic anhydride:NMM=1:1)

[0068] Add 3 times the volume of the resin layer of end-capping solution, stir under nitrogen for 30 minutes, then remove the solution, rinse the reactor port with DCM first, and then wash with DMF 5 times to clean the end-capping solution.

[0069] (8) Washing after connection

[0070] The washing method after ligation is the same as that after deprotection, except that three washes are required after ligation.

[0071] Repeat the previous steps until a peptide chain is completed

[0072] (9) Resin transfer

[0073] Transfer the deprotected and washed resin to a fritted funnel with DMF. Wash once with methanol, three times with DCM, and twice more with methanol. For the final wash, use methanol to concentrate the resin in the center of the funnel. Drain the resin and transfer it to a small collection tube. Label the tube and place it in a vacuum desiccator to dry before cutting.

[0074] (10) Cutting

[0075] a. Preparation of cutting fluid

[0076] Solution A: TFA: thioanisole: phenol: EDT: water = 87.5: 5: 2.5: 2.5: 2.5

[0077] Solution B: TFA:TIS:water = 95:3:2

[0078] b. Selection and dosage of cutting fluid

[0079] Generally, peptide chains containing Cys, Met and unprotected Trp side chains use solution A, and other peptides use solution B. Generally, 10-15 mL of cutting solution is added to 1 g of resin.

[0080] c. Cutting time

[0081] This refers to the time from the addition of the cutting solution to the resin to the precipitation of the filtrate with ether. For short peptides (such as pentapeptides and hexapeptides), the cutting time is 2 hours, for peptides with more than 10 peptides, the cutting time is 2.5 hours, and for peptides with more than 30 peptides, the time can be appropriately extended.

[0082] Add an appropriate amount of cutting fluid, place on a shaker for reaction, filter under reduced pressure, collect the filtrate, and rinse with ether in a centrifuge tube. Centrifuge the pellet in a centrifuge (at a speed of approximately 4000 rad / min). Discard the supernatant, crush the pellet with a glass rod, rinse with ether, and centrifuge again. Repeat this washing process three times.

[0083] The crude product was placed in a vacuum desiccator and dried, and then purified by HPLC to obtain a pure product with a purity of >90%, which was then identified by mass spectrometry.

[0084] 2. Preparation of peptide solution

[0085] In order to screen small peptides with antimicrobial activity, 12 candidate peptides were synthesized in this study (Table 1). Mass spectrometry analysis showed that their structures and molecular weights were consistent with expectations ( Figure 1 ). HPLC analysis showed that the purity of the synthesized peptide was >90% ( Figure 2 The successfully synthesized peptide was dissolved in sterile water to prepare a 5 mM stock solution and stored at -20°C.

[0086] Table 1 Characteristics of the synthesized novel peptides

[0087]

[0088] Example 2: Screening of small peptides against Helicoverpa spp.

[0089] Phenotypic identification:

[0090] After autoclaving, PDA culture medium was mixed with the peptide solution to a final concentration of 100 μM. Sterile water was used instead of the peptide solution as a control. The mixture was then poured into a 30 mm sterile Petri dish. After solidification, a 5 mm cake was cut from the edge of a 4-day-old fungal colony and inoculated with the mycelium side down in the center of the culture medium. Incubate at 28°C in the dark, observing colony growth every 12 hours and photographing the colonies.

[0091] 1. Screening of small peptides against Helminthosporium macrosporum

[0092] After culturing Helminthosporium macrosporum on peptide-containing medium (experimental group) and blank medium (control group) for 24 hours, it was observed that the colony size of the KT-12-treated group in the experimental group was significantly lower than that of the control group, while there was no significant difference between the other 11 peptide-treated groups and the control group ( Figure 3). This indicates that KT-12 has antibacterial activity against Helicoverpa macrophyte.

[0093] 2. Peptide KT-12 inhibits bacterial growth

[0094] Peptide KT-12 has a significant inhibitory effect on the growth of Helicoverpa macrosporum. By comparing the growth of colonies in peptide-containing medium and control medium (without peptide), it was found that although both groups of colonies expanded over time, the expansion rate of the colony in the peptide-containing group was significantly lower than that in the control group ( Figure 4 ).

[0095] Statistical analysis of colony diameters showed that starting from 12 hours after inoculation, the colony diameters of the peptide-containing group at each time point were significantly smaller than those of the control group (p<0.01). At 36 hours, the colony diameter of the control group was twice that of the peptide-containing group ( Figure 5 In addition, growth inhibition analysis showed that the inhibitory effect of peptide KT-12 reached a peak at 36h (72.3%), and the overall inhibition rate ranged from 47.9% to 72.3% ( Figure 6 These data confirmed that peptide KT-12 could effectively inhibit the growth of Helicoverpa macrophylla and exhibit significant antibacterial activity.

[0096] Example 3: Effect of peptide KT-12 on spore germination of Helicoverpa macrophyte

[0097] Spore germination experiment:

[0098] Prepare a spore suspension with sterile 5% sucrose solution from the culture medium. Pipette 36 μL of the spore suspension and 4 μL of antimicrobial peptide, mix thoroughly, and transfer to a clean glass slide. Place the mixture in a Petri dish lined with moistened filter paper and incubate in a hanging drop format at 28°C. Repeat three times, using a control without antimicrobial peptide. After 2 hours, refrigerate and observe spore germination under a microscope.

[0099] Peptide KT-12 inhibits spore germination:

[0100] To further investigate the antibacterial activity of peptide KT-12, we used spore germination experiments to verify the results. We found that spores did not germinate when peptide KT-12 was added, but spores without peptide germinated normally ( Figure 7 These results further confirmed that peptide KT-12 has antibacterial activity against Helicoverpa macrophytes.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An antimicrobial peptide KT-12, characterized in that: The amino acid sequence of the antimicrobial peptide KT-12 is shown in SEQ ID No.

1.

2. Use of the antimicrobial peptide KT-12 according to claim 1 in antifungal treatment.

3. The use according to claim 2, characterized in that: The fungus is Helicoverpa macrocarpon.

4. Use of the antimicrobial peptide KT-12 according to claim 1 in inhibiting mycelial growth and / or spore germination of pathogenic bacteria.

5. The use according to claim 4, characterized in that: The pathogen is Helicoverpa macrophyte.

6. Use of the antimicrobial peptide KT-12 according to claim 1 in preventing and treating corn leaf blight.

7. Use of the antimicrobial peptide KT-12 according to claim 1 in the preparation of products for preventing and treating corn leaf blight.

8. A product for preventing and treating corn leaf blight, characterized by: The product comprises the antimicrobial peptide KT-12 according to claim 1.

9. A method for preventing and controlling corn leaf blight, characterized by: The product for preventing and controlling corn leaf blight according to claim 8 is sprayed on corn leaves.

10. The method for preventing and controlling corn leaf blight according to claim 9, wherein: The antimicrobial peptide KT-12 in the product is used at a concentration of 5-100 μM.

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