Antibacterial peptide and application thereof in resisting bacteria and / or enhancing disease resistance of plants

Abstract

The invention provides an antibacterial peptide and application thereof in resisting bacteria and / or enhancing the disease resistance of plants. Specifically, on one hand, the invention provides two antibacterial peptides which are novel functional antibacterial peptides ZJUAMP3 and ZJUAMP4, sequences of the novel functional antibacterial peptides ZJUAMP3 and ZJUAMP4 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the antibacterial peptides ZJUAMP3 and ZJUAMP4 both have broad-spectrum antibacterial activity, have good antibacterial activity on bacteria such as gram-negative bacteria, gram-positive bacteria and fungi, and are suitable for scenes needing sterilization or fungus and bacterium growth inhibition. Besides, the ZJUAMP4 can activate an MAPK signal channel in a plant body and induce the plant to generate systematic disease resistance, and is suitable for the fields of crop disease prevention and control and plant protection. The antibacterial peptide is simple in structure and easy to synthesize.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an antimicrobial peptide and its use in antimicrobial and / or enhancing the disease resistance of plants. BACKGROUND

[0002] Antimicrobial peptides (AMPs) are a class of natural polypeptides with broad-spectrum bactericidal activity, widely existing in plants, animals and microorganisms and other organisms. These polypeptides usually have a short amino acid sequence, a positive charge characteristic and a high hydrophobicity, and can effectively act on the cell membrane structure of bacteria, leading to an increase in membrane permeability, and ultimately achieving the purpose of killing or inhibiting pathogenic microorganisms. In recent years, with the increasing problem of bacterial drug resistance caused by the abuse of antibiotics, the use effect of traditional antibiotics has gradually decreased, and drug-resistant strains have rapidly increased. This global problem has posed a serious threat to human health, agricultural production and environmental safety. Therefore, finding antimicrobial substances with new mechanisms of action and not prone to drug resistance has become one of the important directions of current research.

[0003] At the same time, plant disease problems are also increasingly prominent. In the long-term game and arms race between plants and pathogenic bacteria, plant immunity has evolved into a two-layer system, namely the zig-zag model [1] The first is PAMPS-triggered immunity (pattern-triggered immunity, PTI). When infected by pathogenic bacteria, plants can quickly recognize the conserved related molecular patterns (pathogen-associated molecular patterns, PAMPs) of pathogenic bacteria by using pattern recognition receptors (pattern-recognition receptors, PRRs) located on the cell membrane surface, activate the cytoplasmic receptor-like kinases (receptor-like cytoplasmic kinases, RLCKs) through binding with co-receptors and phosphorylation, activate the mitogen-activated protein kinases (mitogen-activated protein kinases, MAPKs) cascade signal pathway, calcium dependent protein kinases (calcium dependent protein kinases, CDPKs) pathway and cause reactive oxygen species (reactive oxygenspecies, ROS) burst, etc. These reactions are collectively referred to as PTI immune response (pattern-triggered immunity); the second is effector-triggered immunity (effector-triggered immunity, ETI).

[0004] The excessive use of traditional pesticides leads to serious pesticide residues in agricultural products, which seriously threatens food safety and the sustainable development of the ecological environment. People are constantly seeking various strategies to enhance the defense ability of plants against pathogenic bacteria from the perspective of improving the immunity and basic disease resistance of plants. As a new green prevention and control method, antibacterial peptides not only can effectively inhibit pathogenic bacteria, but also can stimulate the immune response of plants (such as the Mitogen-Activated Protein Kinase (MAPK) signaling cascade pathway, i.e., the mitogen-activated protein kinase cascade pathway). The MAPK signaling cascade pathway is an important part of the PTI immune defense system of plants [2] , which can amplify external stimulation signals through signal transduction pathways to regulate the expression of downstream defense genes, thereby enhancing the ability of plants to resist pathogenic microorganisms. It has been reported in Arabidopsis, tobacco, rice and tomato [3][4] that the MAPK cascade reaction plays an important role in the immune response of plants against pathogenic bacteria, i.e., the activation of the MAPK pathway, such as the increased phosphorylation level of kinases MPK3, 4 and 6, can enhance the ability of plants to resist pathogenic bacteria [5] . Therefore, the development of new antibacterial peptides with bactericidal effect and the ability to induce the MAPK immune response of plants has great research value and application prospect.

[0005] With the rapid development of bioinformatics, computational biology and machine learning technology, the prediction and design of new antibacterial peptides through artificial intelligence methods such as machine learning have become one of the current research hotspots. This method not only greatly improves the research and development efficiency of new antibacterial peptides, but also significantly reduces the research and development cost and cycle. However, although some computational methods have successfully predicted a variety of new antibacterial peptides, antibacterial peptides that show high bactericidal ability and can simultaneously activate the MAPK disease resistance pathway of plants are still relatively rare. SUMMARY

[0006] As mentioned earlier, although some studies have used computational prediction methods to screen antibacterial peptides, antibacterial peptides with broad-spectrum antibacterial activity are still relatively rare. After actual experimental screening and verification of a number of antibacterial peptides, the inventors unexpectedly found that the antibacterial peptides ZJUAMP3 and ZJUAMP4 of the present application have excellent antibacterial activity, not only can inhibit common pathogenic bacteria and fungi, but also have good inhibitory effect on the plant pathogenic fungus Fusarium graminearum, and can also significantly induce the MAPK disease resistance signaling pathway of plants. The present application not only provides a new method for the efficient development of antibacterial peptides, but also provides a new technical reserve for the green prevention and control of plant diseases, which has broad application prospects and important social and economic value.

[0007] In this regard, one aspect of the object of the present application is to provide an antibacterial peptide, which is a new functional antibacterial peptide ZJUAMP3 and ZJUAMP4, the sequences of which are respectively shown as SEQ ID NO: 1

[0008] (Gly-Trp-Tyr-Asn-Ala-Phe-Arg-Lys-Leu-Leu-Cys-Lys-Ile-Ala-Gly-Lys-Cys

[0009] (GWYNAFRKLLCKIAGKC)) and SEQ ID NO: 2

[0010] (Ile-Gly-Trp-Phe-Lys-Val-Phe-Lys-Lys-Val-Leu-Lys-Arg-Leu-Ala(IGWFKVFKKVLKRLA)). Among them, ZJUAMP3 includes 17 amino acid residues, the theoretical molecular weight is 1971.39 Da, the isoelectric point is 9.7, the net charge is +5, and it belongs to an alkaline antibacterial peptide, rich in lysine and other positively charged amino acids, easy to combine with bacterial cell membranes and destroy their integrity; ZJUAMP4 includes 15 amino acid residues, the theoretical molecular weight is 1833.31 Da, the isoelectric point is 11.33, the net charge is +5, and it belongs to an alkaline antibacterial peptide, rich in lysine and other positively charged amino acids, easy to combine with bacterial cell membranes and destroy their integrity. In addition, the antibacterial peptide of the present application has a simple structure and is easy to synthesize. Specifically, the antibacterial peptide of the present application can be obtained by conventional solid-phase polypeptide synthesis method or genetic engineering expression method, has good stability and synthesis feasibility, and is suitable for large-scale preparation and product development.

[0011] The antibacterial peptide described in the present application has broad-spectrum antibacterial activity and one of them can induce plant immune response. Specifically, the antibacterial peptides ZJUAMP3 and ZJUAMP4 of the present application exhibit significant bacteriostatic effect on a variety of pathogenic microorganisms in vitro, including the plant pathogenic fungus Fusarium graminearum, suitable for the field of biotechnology, and also suitable for the fields of crop disease prevention and control and plant protection; in addition, ZJUAMP4 can also activate the MAPK signal pathway in plants to induce plants to produce disease resistance. When the antibacterial peptide of the present application is applied to plants (for example, Arabidopsis or wheat), the MAPK (Mitogen-Activated Protein Kinase) signal pathway can be significantly activated, including the increase of the phosphorylation level of key kinases such as MAPK3 and MAPK6, thereby guiding the expression of downstream disease resistance-related genes and improving the disease resistance of plants. This function expands the application boundary of antibacterial peptides in the field of plant disease prevention and control, and provides a theoretical basis and technical reserve for the development of new green plant protection agents.

[0012] Therefore, in another aspect, the present application also aims to provide use of the antibacterial peptide of the present application in antibacterial and activating plant MAPK signaling pathway, inducing plant immune response, enhancing plant disease resistance and / or preventing plant diseases.

[0013] In this case, the present application includes but is not limited to the following:

[0014] In one aspect, the present application provides an antibacterial peptide, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] In another aspect, the present application provides use of the antibacterial peptide of the present application in preparing an antibacterial drug or preparation.

[0016] In one aspect, the antibacterial drug or preparation of the present application is an antibacterial drug or preparation and an antifungal drug or preparation.

[0017] In one aspect, the bacteria of the present application is a gram-positive bacterium or a gram-negative bacterium.

[0018] In one aspect, the gram-positive bacterium of the present application is selected from Staphylococcus aureus. Preferably, the Staphylococcus aureus is Staphylococcus aureus CMCC26003.

[0019] In one aspect, the gram-positive bacterium of the present application is selected from Staphylococcus aureus CMCC26003.

[0020] In one aspect, the gram-negative bacterium of the present application is selected from Escherichia coli or Pseudomonas aeruginosa. Preferably, the Escherichia coli is Escherichia coli CMCC(B)44102, and the Pseudomonas aeruginosa is Pseudomonas aeruginosa ATCC27853.

[0021] In one aspect, the gram-negative bacterium of the present application is selected from Escherichia coli CMCC(B)44102 or Pseudomonas aeruginosa ATCC27853.

[0022] In one aspect, the fungus of the present application is selected from Candida albicans or Fusarium graminearum. Preferably, the Candida albicans is Candida albicans YW02, and the Fusarium graminearum is Fusarium graminearum PH-1.

[0023] In another aspect, the present application provides use of the antibacterial peptide of the present application in any one of the following:

[0024] (1) activating plant MAPK signaling pathway;

[0025] (2) inducing plant immune response;

[0026] (3) enhancing the disease resistance of plants;

[0027] (4) preventing and treating plant diseases,

[0028] The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 2.

[0029] In one aspect, the plant according to the present application is selected from Arabidopsis thaliana and wheat.

[0030] The present application has the following beneficial effects:

[0031] The antibacterial peptides ZJUAMP3 and ZJUAMP4 provided by the present application are verified and screened from a plurality of antibacterial peptides, and have broad-spectrum antibacterial activity, and have significant bacteriostatic effects on gram-positive bacteria, gram-negative bacteria and fungi, and are biologically safe, and can be used for the treatment of bacterial and fungal infections, the prevention and treatment of plant fungal diseases, and can also be applied to other scenarios requiring sterilization or inhibition of bacterial and fungal growth. In addition, the antibacterial peptide ZJUAMP4 can also activate the MAPK signaling pathway of plants, induce plant disease resistance, and improve the immune level of plants, and can be used for preventing and treating plant diseases, and has a wide application prospect. The antibacterial peptide sequence provided by the present application is short, the molecular weight is small, the chemical synthesis difficulty is low, and the scale production cost can be saved. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 For Western blot detection of the activation effect of the antibacterial peptide ZJUAMP3 on the MAPK signaling pathway of Arabidopsis thaliana. The antibody is a specific antibody for the phosphorylation level of MAPK signaling - Extracellular Regulated Protein Kinase Antibody (Extracellular Regulated Protein Kinases (a-pERK1 / 2) antibody, product number #4370S (Cell Signaling Technology)), flg22 1 μM (Pseudomonas syringae N-terminal conserved 22 polypeptides, Glpbio, USA) is a positive control, and the negative control CK is sterile water treatment.

[0033] Figure 2For Western blot detection of the activation effect of MAPK signaling pathway induced by antibacterial peptide ZJUAMP3 in wheat leaves. The antibody is a specific antibody for the phosphorylation level of MAPK signal - Extracellular Regulated Protein Kinases (a-pERK1 / 2) antibody (item number #4370S (Cell Signaling Technology)), flg22 1 μM (Pseudomonas syringae N-terminal conserved 22 polypeptides, Glpbio, USA) is a positive control, and the negative control CK is sterile water treatment.

[0034] Figure 3 For Western blot detection of the activation effect of MAPK signaling pathway induced by antibacterial peptide ZJUAMP4 in Arabidopsis. The antibody is a specific antibody for the phosphorylation level of MAPK signal - Extracellular Regulated Protein Kinases (a-pERK1 / 2) antibody (item number #4370S (Cell Signaling Technology)), flg22 1 μM (Pseudomonas syringae N-terminal conserved 22 polypeptides, Glpbio, USA) is a positive control, and the negative control CK is sterile water treatment.

[0035] Figure 4 For Western blot detection of the activation effect of MAPK signaling pathway induced by antibacterial peptide ZJUAMP4 in wheat. The antibody is a specific antibody for the phosphorylation level of MAPK signal - Extracellular Regulated Protein Kinases (a-pERK1 / 2) antibody (item number #4370S (Cell Signaling Technology)), flg22 1 μM (Pseudomonas syringae N-terminal conserved 22 polypeptides, Glpbio, USA) is a positive control, and the negative control CK is sterile water treatment. DETAILED DESCRIPTION

[0036] In order to more clearly understand the present application, the antibacterial peptides ZJUAMP3 and ZJUAMP4 and their applications provided by the present application will be further described below in combination with specific examples. It should be understood that the following examples are only used to illustrate the present application and do not constitute a limitation on the scope of protection of the present application.

[0037] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0038] Example 1: Synthesis of antibacterial peptides ZJUAMP3 and ZJUAMP4

[0039] This example provides antibacterial peptides with amino acid sequences of GWYNAFRKLLCKIAGKC (SEQ ID NO: 1) (ZJUAMP3) and IGWFKVFKKVLKRLA (SEQ ID NO: 2) (ZJUAMP4), which can be prepared by solid-phase peptide synthesis technology (Solid Phase Peptide Synthesis, SPPS). In this experiment, the antibacterial peptides were synthesized by a biological company through solid-phase synthesis. The synthesized ZJUAMP3 and ZJUAMP4 are white or white-like powders, which are soluble in water or conventional buffer and suitable for subsequent biological activity evaluation.

[0040] Example 2: Evaluation of antibacterial activity of ZJUAMP3 and ZJUAMP4

[0041] According to the MIC determination operation guide of the American Clinical and Laboratory Standards Association (CLSI), the MIC values of antibacterial peptides ZJUAMP3 and ZJUAMP4 against different pathogenic microorganisms were determined by micro-broth dilution method. First, the antibacterial peptides were dissolved in sterile distilled water with an initial concentration of 5120 μg / mL and stored at 4°C. When used, the antibacterial peptides were diluted to 512 μg / mL with Mueller-Hinton broth (MH) (Solebo, LA6740), and then 2-fold serial dilutions were performed in 96-well plates. The bacterial culture solution was adjusted to 0.5 McFarland concentration, diluted 100-fold with fresh MH, and 100 μL was added to the 96-well plate containing the antibacterial peptides, so that the final concentration of bacteria was 5×10 5 CFU / mL. After incubation at 37°C for 18 hours, the MIC value was the minimum concentration of the antibacterial peptide at which no obvious bacterial growth was observed by naked eye.

[0042] When fungi were determined, RPMI1640 culture solution (Mingte Biological, BIO-000001) was used, and the final concentration of Candida albicans was 2.5×10 3 CFU / mL, which was incubated at 30°C; the final concentration of Fusarium graminearum spores was 5×10 3 CFU / mL, which was incubated at 25°C.

[0043] 1 Experimental materials:

[0044] Bacterial and fungal strains: Escherichia coli CMCC (B) 44102, Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus CMCC26003, Candida albicans YW02 or Fusarium graminearum PH-1;

[0045] Peptide solution: ZJUAMP3 and ZJUAMP4 were prepared in sterile water to 5120 μg / mL;

[0046] Culture medium: MH broth (bacteria); RPMI-1640 (fungi);

[0047] 96-well microplate, constant temperature shaking incubator, microplate reader.

[0048] 2. Experimental procedure:

[0049] After activation, each strain was inoculated into liquid medium and cultured to the logarithmic growth phase; the bacterial solution was diluted to a final concentration of about 10 6 CFU / mL, C. albicans was diluted to a concentration of 5 x 10 3 CFU / mL, F. graminearum spores were diluted to a concentration of 10 4 CFU / mL; different concentrations of ZJUAMP3 or ZJUAMP4 peptide solution were mixed with the bacterial solution into a 96-well plate; incubated for 16-24 hours under appropriate conditions; the absorbance (OD 600 ) was measured by microplate reader and the growth was determined.

[0050] 3. Experimental results:

[0051] ZJUAMP3 and ZJUAMP4 showed good antibacterial activity against the tested strains, and the MICs were as follows:

[0052]

[0053] Note: All quality controls were within the allowed range, all growth control wells were turbid, and blank control wells remained clear, verifying that the experimental system was operating normally.

[0054] The above results show that ZJUAMP3 and ZJUAMP4 have broad-spectrum antibacterial ability.

[0055] Example 3 Determination of the effect of antibacterial peptides ZJUAMP3 and ZJUAMP4 on the MAPK signaling pathway of Arabidopsis thaliana and wheat

[0056] Elicitors include proteins, polypeptides, lipids, glycoproteins and oligosaccharides, which can activate plant pattern recognition receptors (PRRs) to induce typical pattern-triggered immunity (PTI) in plants, one of the early responses of which is the mitogen-activated protein kinase (MAPK) cascade. In Arabidopsis, the MAPK cascade has been extensively studied and is involved in a variety of biological processes, including plant immune response, stress response and growth and development, etc. For example, among the 20 MAPK members in Arabidopsis, MPK3, MPK4 and MPK6 can be activated in response to the recognition of different pathogen-associated molecular patterns (PAMPs), and the MEKK1-MKK4 / 5-MPK3 / 6 cascade pathway has been reported to be involved in pathogen activation and disease resistance in Arabidopsis. In wheat, the MAPK cascade is also a core immune signaling pathway for the plant to resist pathogen invasion. For example, Puccinia striiformis f. sp. tritici targets the wheat MAPK-SGT1 cascade pathway by secreting effector proteins to directly inhibit MAPK signaling, thereby weakening the host immune response. In particular, according to the following documents [1]-[5], a person skilled in the art can fully understand that the activation of the MAPK pathway represents the enhancement of the plant's defense and disease resistance ability:

[0057] [1] Ngou, B.P.M., Ding, P. & Jones, J.D.G. Thirty years of resistance: Zig-zag through the plant immune system. The Plant Cell 34, 1447-1478 (2022);

[0058] [2] Cristina, M., Petersen, M. & Mundy, J. Mitogen-Activated Protein Kinase Signaling in Plants. Annu. Rev. Plant Biol. 61, 621-649 (2010);

[0059] [3] Pozo, O. del, Pedley, K.F. & Martin, G.B. MAPKKKalpha is a positive regulator of cell death associated with both plant immunity and disease. EMBO J 23, 3072-3082 (2004);

[0060] [4] Frye, C. A., Tang, D. & Innes, R. W. Negative regulation of defense responses in plants by a conserved MAPKK kinase. Proc Natl Acad Sci U S A 98, 373-378 (2001); and

[0061] [5] Meng, X. & Zhang, S. MAPK Cascades in Plant Disease Resistance Signaling. Annu. Rev. Phytopathol. 51, 245-266 (2013).

[0062] To explore the plant MAPK cascade response induced by ZJUAMP3 and ZJUAMP4 in Arabidopsis and wheat, three seven-day-old Arabidopsis or three seven-day-old wheat leaves were placed in one well of a 24-well plate containing sterile water overnight. The next day, the sterile water was removed, and the Arabidopsis leaves or wheat leaves were divided into three groups, and 500 μL of bacterial flagellin N-terminal conserved 22 polypeptide (flg22) diluent with a final concentration of 1 μM (positive control group), 10 μM ZJUAMP3, ZJUAMP4 and sterile water (negative control group) were added to each group, respectively, and the treatment time of each group was 15 min, 30 min and 45 min, respectively. The samples treated in each group were quickly frozen in liquid nitrogen, and the protein was extracted. The protein samples were subjected to SDS-PAGE (10% gel, voltage 110V, electrophoresis time about 3h), and then western blot was used to detect the MAPK phosphorylation activation level of Arabidopsis or wheat after treatment with antibacterial peptides.

[0063] 1 Effect of antibacterial peptides ZJUAMP3 and ZJUAMP4 on the activity of Arabidopsis MAPK pathway.

[0064] 1.1 Experimental materials:

[0065] Test treatment: several seven-day-old Arabidopsis seedlings on 1 / 2MS plates, antibacterial peptides ZJUAMP3 and ZJUAMP4 (final concentration 10 μM, diluted with water), flg22 (QRL STG SRI NSA KDD AAG LQI A) (final concentration 1 μM, diluted with water), 24-well plate, liquid nitrogen, grinder.

[0066] SDS-PAGE: 2x SDS-loading buffer, 1x Running buffer, 10% protein gel, ice box.

[0067] Western blot: semi-dry transfer apparatus (1.5A, 12min), methanol, 1x transfer, 0.22μm polyvinylidene fluoride (PVDF) membrane, 5% skim milk, TBST wash buffer, primary antibody (Extracellular Regulated protein Kinases (a-pERK1 / 2) antibody, Cat# 4370S, dilution ratio 1:2,000, Cell Signaling), secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, Cat# 7074S, dilution ratio 1:5000, Cell Signaling), Super sensitive exposure solution.

[0068] 1.2 Experimental procedure:

[0069] Three 7-day-old Arabidopsis thaliana were selected and soaked in 24-well plates containing distilled water overnight. The next day, the sterile water was removed and the leaves were treated with 500μL of the final concentration of 10μM of the antimicrobial peptide ZJUAMP3 or ZJUAMP4, 1μM of flg22 (positive control) and water (negative control), each treatment containing 3 replicates, and each replicate was treated for 15min, 30min and 45min, respectively. After treatment, the samples were placed in liquid nitrogen, ground with a pre-cooled grinder, and then placed in 100μL of SDS buffer (62.5mM Tris-HCl pH 6.8, 1% [w / v] SDS, 0.025% [w / v] bromophenol blue, 10% [v / v] glycerol, 4% [v / v] β-mercaptoethanol, 1x protease inhibitor, 2mM NaF, 2mM Na3VO4), followed by boiling at 95℃ for 10 minutes. The homogenate after boiling was centrifuged at 14,000g for 10 minutes at 4℃, and the supernatant was collected for subsequent analysis. After 10% SDS-PAGE gel electrophoresis, the supernatant was transferred to a 0.22μm polyvinylidene fluoride (PVDF) membrane, and immunoblotting pMAPKs detection was performed using a phosphorylated extracellular regulated protein kinase antibody (Extracellular Regulated protein Kinases (a-pERK1 / 2) antibody, Cat# 4370S, dilution ratio 1:2,000, Cell Signaling), and a rabbit anti-antibody (Anti-rabbit IgG, HRP-linked Antibody, Cat# 7074S, dilution ratio 1:5000, Cell Signaling) was used as the secondary antibody.

[0070] 1.3 Experimental results:

[0071] The results are shown inFigure 1 As shown in the figure, the positive control group and 10 μΜ ZJUAMP4 can activate the Arabidopsis MAPK signaling pathway (using Rubisco enzyme as internal reference), while ZJUAMP3 cannot activate the Arabidopsis MAPK signaling pathway.

[0072] 2. Effect of antibacterial peptides ZJUAMP3 and ZJUAMP4 on the activity of wheat MAPK pathway.

[0073] 2.1 Experimental materials:

[0074] Test treatment: several 7-day-old wheat, antibacterial peptides ZJUAMP3 and ZJUAMP4 (final concentration 10 μΜ, surfactant dilution), flg22 (final concentration 1 μΜ, surfactant dilution), 0.1% Tween20-ddH2O solution (surfactant), 24-well plate, liquid nitrogen, grinding instrument.

[0075] SDS-PAGE: 2x SDS-loading buffer, 1x Running buffer, 10% protein gel, ice box.

[0076] Western blot: semi-dry transfer instrument (1.5A, 12 min), methanol, 1x transfer, 0.22 μm polyvinylidene fluoride (PVDF) membrane, 5% skim milk, TBST membrane washing solution, primary antibody (Extracellular Regulated protein Kinases (a-pERK1 / 2) antibody, product number #4370S, dilution ratio 1:2,000, Cell Signaling Company), secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, product number #7074S, dilution ratio 1:5,000, Cell Signaling Company), ultra-sensitive exposure solution.

[0077] 2.2 Experimental steps:

[0078] Wheat seedlings were grown on pure vermiculite for one week before the second leaf was fully emerged. The leaves were cut into 1 cm long segments with a razor blade and discarded the tip and base sections. Three leaves were placed in a 24-well plate with surfactant (0.1% Tween 20-ddH2O solution) to ensure full leaf contact with the solution and left to soak overnight. To reduce error, the leaf segments in each well were preferably from different leaves. The next day, the solution was removed and the leaves were treated with 500 μL of 10 μM of the antimicrobial peptides ZJUAMP3 or ZJUAMP4, 1 μM of flg22 (positive control) and water (negative control) for 15 min, 30 min and 45 min, respectively. The samples were then frozen in liquid nitrogen and ground using a pre-cooled grinder before being placed in 100 μL of SDS buffer (62.5 mM Tris-HCl pH 6.8, 1% [w / v] SDS, 0.025% [w / v] bromphenol blue, 10% [v / v] glycerol, 4% [v / v] β-mercaptoethanol, 1 x protease inhibitor, 2 mM NaF, 2 mM Na3VO4) and then boiled at 95 °C for 10 min. The homogenate was then centrifuged at 14,000 g for 10 min at 4 °C and the supernatant was collected for subsequent analysis. The supernatant was subjected to 10% SDS-PAGE gel electrophoresis and then transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The membrane was then subjected to immunoblotting using an antibody against phosphorylated extracellular regulated protein kinases (a-pERK1 / 2) (Cat. No. 4370S, dilution ratio 1:2,000, Cell Signaling Technology) and an anti-rabbit IgG, HRP-linked antibody (Cat. No. 7074S, dilution ratio 1:5,000, Cell Signaling Technology) as the secondary antibody.

[0079] 2.3 Experimental results:

[0080] The results are shown in Figures Figure 1 , Figure 2 . The positive control and 10 μM ZJUAMP3 were unable to activate the MAPK signaling pathway in Arabidopsis and wheat leaves (using Rubisco enzyme as the internal control).

[0081] As shown in Figures Figure 3 , Figure 4 , ZJUAMP4 was able to activate the MAPK signaling pathway in Arabidopsis and wheat.

[0082] The above experiments show that 10 μM ZJUAMP4 can activate the MAPK signaling pathway of Arabidopsis and wheat leaves to enhance the disease resistance of the plants.

[0083] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibacterial peptide, characterized in that, The amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. Use of the antibacterial peptide according to claim 1 in the preparation of an antibacterial medicament or preparation.

3. Use according to claim 2, characterized in that, The antibacterial medicament or preparation is an antibacterial medicament or preparation or an antifungal medicament or preparation.

4. Use according to claim 3, characterized in that, The bacteria are gram-positive bacteria or gram-negative bacteria.

5. Use according to claim 4, characterized in that, The bacteria are gram-positive bacteria selected from Staphylococcus aureus.

6. Use according to claim 4, characterized in that, The bacteria are gram-negative bacteria selected from Escherichia coli or Pseudomonas aeruginosa.

7. Use according to claim 3, characterized in that, The fungi are selected from Candida albicans or Fusarium graminearum.

8. Use of the antibacterial peptide according to claim 1 in any one of the following: (1) activating the MAPK signaling pathway of plants; (2) inducing the immune response of plants; (3) enhancing the disease resistance of plants; (4) preventing and treating plant diseases, wherein The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO:

2.

9. Use according to claim 8, characterized in that, The plants are selected from Arabidopsis thaliana and wheat.

Images