Antimicrobial peptides, expression vectors and their applications

Abstract

The invention discloses an antimicrobial peptide, an expression vector and an application thereof. Aiming at the problem that the current antimicrobial peptide gene family of Monochamus alternatus is incomplete, the scheme of the invention proposes four antimicrobial peptides MaltCec, MaltDef, MaltAtt-1 and MaltAtt-2 of Monochamus alternatus and their corresponding antimicrobial peptide genes and corresponding exogenous expression vectors and products, and also discloses corresponding induced expression and purification methods. The scheme enriches the antimicrobial peptide gene family of Monochamus alternatus and realizes the exogenous expression of the antimicrobial peptide genes MaltCec, MaltDef, MaltAtt-1 and MaltAtt-2 of Monochamus alternatus; the scheme of the invention also successfully uses Pichia pastoris to express the antimicrobial peptides MaltCec, MaltDef, MaltAtt-1 and MaltAtt-2 of Monochamus alternatus, and proves that they have antibacterial activity. The antimicrobial peptides of Monochamus alternatus in the scheme have good antibacterial effect, are reliably and stably prepared and have positive application prospects.

Description

[0001] Divisional Application Statement

[0002] This application is a divisional application of the Chinese invention patent application with application number 202210468553.4 filed on April 29, 2022, and invention name “Antibacterial peptides from pine sawyer beetles, their encoding genes and applications”. Technical Field

[0003] The present invention relates to the field of biological gene engineering technology, in particular to antimicrobial peptides, expression vectors and applications thereof. Background Art

[0004] Antimicrobial peptides are a class of peptide active substances produced by the host's nonspecific immune defense system to fight against exogenous pathogens. They are effector molecules of natural immunity. In recent years, with the extensive use of traditional antibiotics, the emergence of drug-resistant bacteria has brought huge hidden dangers to human health. Antimicrobial peptides have the advantages of broad-spectrum antibacterial, fungal, viral, and parasitic activities, and are not easy to produce drug-resistant strains. At present, as a substitute for antibiotics or synergistic with antibiotics, they are expected to become new antibacterial drugs with broad application value and development prospects.

[0005] Recombinant protein expression technology can efficiently obtain target proteins in large quantities. Compared with other eukaryotic and prokaryotic expression systems, the Pichia pastoris expression system has the advantages of easy operation, multiple post-transcriptional modifications (such as polypeptide folding, acylation, glycosylation, methylation, etc.), stable genetic system and high-level protein expression.

[0006] In the prior art: Yin Na et al. expressed the antimicrobial peptide Cecropin D in Pichia pastoris (Yin Na, Chinese Journal of Biological Products, 2008); Wang Zhen et al. expressed the antimicrobial peptide Cecropin AD in Pichia pastoris (Wang Zhen et al., Chinese Journal of Animal Husbandry, 2010; Jin Fengliang et al. cloned and expressed the housefly defensin in Pichia pastoris (Jin Fengliang, Journal of Sun Yat-sen University, 2006); Wu Songquan et al. expressed the Culex pipiens defensin in Pichia pastoris (Wu Songquan, Journal of Immunology, 2013); however, the antimicrobial peptide gene family of Monochamus alternatus is incomplete and the corresponding research is relatively small. Therefore, it is a very practical research topic to conduct research on Monochamus alternatus and enrich its antimicrobial peptide gene family. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an antimicrobial peptide, an expression vector and its application which have good antibacterial effect, reliable and stable preparation and positive application prospects.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0009] An antimicrobial peptide from Monochamus alternatus, wherein the antimicrobial peptide is MaltCec, MaltDef, MaltAtt-1 or MaltAtt-2;

[0010] in,

[0011] The amino acid sequence of the antimicrobial peptide MaltCec from Monochamus alternatus is shown in SEQ ID NO.1.

[0012] The amino acid sequence of the antimicrobial peptide MaltDef from Monochamus alternatus is shown in SEQ ID NO.2.

[0013] The amino acid sequence of the antimicrobial peptide MaltAtt-1 from Monochamus alternatus is shown in SEQ ID NO.3.

[0014] The amino acid sequence of the antimicrobial peptide MaltAtt-2 from Monochamus alternatus is shown in SEQ ID NO.4.

[0015] Based on the above, the present invention also provides a gene encoding the above-mentioned Monochamus alternatus antimicrobial peptide, wherein:

[0016] The nucleotide sequence encoding the antimicrobial peptide MaltCec of Monochamus alternatus is shown in SEQ ID NO.5.

[0017] The nucleotide sequence encoding the antimicrobial peptide MaltDef of Monochamus alternatus is shown in SEQ ID NO.6.

[0018] The nucleotide sequence encoding the antimicrobial peptide MaltAtt-1 of Monochamus alternatus is shown in SEQ ID NO.7.

[0019] The nucleotide sequence encoding the antimicrobial peptide MaltAtt-2 from Monochamus alternatus is shown in SEQ ID NO.8.

[0020] Based on the above, the present invention also provides an expression vector containing the gene described above.

[0021] As a possible implementation manner, further, the expression vector is Pichia pastoris.

[0022] Based on the above, the present invention also provides a method for preparing the above-mentioned Monochamus alternatus antimicrobial peptide, wherein the Monochamus alternatus antimicrobial peptide is obtained by microbial expression, or prepared by artificial synthesis.

[0023] As a possible implementation mode, further, the method for obtaining the antimicrobial peptide of Monochamus alternatus by microbial expression includes: screening the gene sequence corresponding to the antimicrobial peptide of Monochamus alternatus, and adding a SnaBI restriction site and a start codon at its 5' end, and adding a stop codon and a NotⅠ restriction site at its 3' end to complete the synthesis of the gene sequence, and then double-digesting it with the pPIC9K plasmid by SnaBI / NotⅠ, connecting the synthesized target gene to the Pichia pastoris exocrine expression vector pPIC9K to obtain a recombinant expression vector, and then inducing the expression of the recombinant expression vector to obtain the antimicrobial peptide of Monochamus alternatus.

[0024] Based on the above, the present invention also provides the use of the above-mentioned Monochamus alternatus antimicrobial peptide in the preparation of a drug or reagent for inhibiting pine wood nematode disease.

[0025] Based on the above, the present invention also provides a pesticide, which includes the above-mentioned Monochamus alternatus antimicrobial peptide.

[0026] Based on the above, the present invention also provides an antibacterial drug, which includes the above-mentioned Monochamus alternatus antibacterial peptide.

[0027] By adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art: the scheme of the present invention aims at the problem that the antimicrobial peptide gene family of Monochamus alternatus is incomplete at present, and proposes four antimicrobial peptides MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 of Monochamus alternatus and their corresponding antimicrobial peptide genes and corresponding exogenous expression vectors and products, and also discloses corresponding induced expression and purification methods. The scheme enriches the antimicrobial peptide gene family of Monochamus alternatus, and realizes the exogenous expression of the antimicrobial peptide genes MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 of Monochamus alternatus; the scheme of the present invention also successfully uses Pichia pastoris to express the antimicrobial peptides MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 of Monochamus alternatus, and proves that they have antibacterial activity. The antimicrobial peptides of Monochamus alternatus in the scheme have good antibacterial effect, are reliable and stable in preparation, and have positive application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0029] Figure 1This is the technical roadmap for the efficient recombinant expression of MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 antimicrobial peptides in Pichia pastoris according to the present invention;

[0030] Figure 2 It is a schematic diagram for characterizing the induced expression results of MaltCec, MaltDef, MaltAtt-1 and MaltAtt-2 of the present invention;

[0031] Figure 3 This is a characterization diagram of the antibacterial activity identification of the recombinant antimicrobial peptides MaltCec, MaltDef, MaltAtt-1 and MaltAtt-2 of the present invention;

[0032] Figure 4 0.3 mg·mL in the embodiment of the present invention -1 Characterization curve of nematicidal activity of antimicrobial peptide protein from Monochamus alternatus;

[0033] Figure 5 0.6 mg·mL in the embodiment of the present invention -1 Characterization curve of the nematicidal activity of the antimicrobial peptide protein of Monochamus alternatus.

[0034] Figure 6 1.0 mg·mL in the embodiment of the present invention -1 Characterization curve of the nematicidal activity of the antimicrobial peptide protein of Monochamus alternatus. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] Combination Figure 1 As shown, this embodiment provides a method for exogenous expression of antimicrobial peptide genes MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 of Monochamus alternatus and induced expression and purification of the products, which comprises the following steps:

[0038] (1) Antimicrobial peptide gene synthesis and expression vector construction

[0039] The antimicrobial peptide MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 gene sequences were screened out from the transcriptome data of the larvae of Monochamus alternatus. A SnaBⅠ restriction site and a start codon were added to the 5' end of each antimicrobial peptide gene sequence, and a stop codon and a NotⅠ restriction site were added to the 3' end. Finally, the target genes were named MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2, and the sequences were synthesized by Wuhan Jinkairui Co., Ltd.

[0040] Among them, the nucleotide sequence encoding the antimicrobial peptide MaltCec of Monochamus alternatus is shown as SEQ ID NO.5, the nucleotide sequence encoding the antimicrobial peptide MaltDef of Monochamus alternatus is shown as SEQ ID NO.6, the nucleotide sequence encoding the antimicrobial peptide MaltAtt-1 of Monochamus alternatus is shown as SEQ ID NO.7, and the nucleotide sequence encoding the antimicrobial peptide MaltAtt-2 of Monochamus alternatus is shown as SEQ ID NO.8.

[0041] The synthesized gene sequence and pPIC9K plasmid were double-digested with EcoRⅠ / NotⅠ, and the synthesized target gene was connected to the Pichia pastoris exocytosis expression vector pPIC9K to obtain recombinant expression vectors pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, and pPIC9K-MaltAtt-2.

[0042] (2) Verification of recombinant expression vectors pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, and pPIC9K-MaltAtt-2

[0043] The puncture bacteria containing the recombinant expression vectors pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, and pPIC9K-MaltAtt-2 were obtained, and they were picked into LB liquid culture medium respectively. After culturing for 12 hours, the plasmids were extracted using the Novozymes kit, and the extracted plasmids were identified by 1% agarose gel electrophoresis.

[0044] (21) Sequencing verification

[0045] The double enzyme digestion results can only preliminarily verify the successful construction and correct size of the recombinant vector, and cannot determine whether deletions or mutations will occur in the base sequence. Therefore, Fuzhou BoShang Biotechnology Co., Ltd. was commissioned to sequence the recombinant plasmid.

[0046] (3) Transform the recombinant expression vectors pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, and pPIC9K-MaltAtt-2 into Pichia pastoris

[0047] (31) Linearization and dephosphorylation of recombinant expression vector

[0048] After the circular plasmid is linearized, both ends of the vector can undergo homologous recombination with the homologous sequences of yeast, which greatly improves the efficiency of its integration into the yeast genome. Therefore, in order to increase the probability of the target gene being inserted into the yeast genome, the restriction endonuclease Sal I is required to linearize and dephosphorylate the recombinant expression plasmids pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, and pPIC9K-MaltAtt-2 before electroporation. At the same time, the pPIC9K empty vector is digested as a control for subsequent expression. The Sal I digestion system is shown in Table 1

[0049]

[0050] The above solution was mixed by instant centrifugation, and digested at 37℃ for several hours or overnight. During this period, the digestion effect was detected by 0.8% agarose gel electrophoresis, and the plasmid without digestion was used as a control. After complete digestion, 1ul of dephosphorylase CAIP (1U / ul) was added and incubated at 37℃ for 1-2h for dephosphorylation. Then, phenol: chloroform: isoamyl alcohol (25:24:1) was used for extraction, and the linear plasmid was precipitated and concentrated with 3mol / LNaAc (PH5.2) solution and anhydrous ethanol, and stored at -20℃ for later use.

[0051] (32) Preparation of Pichia pastoris competent cells

[0052] A. Activation of Pichia pastoris strain KM71

[0053] Take 50ul of strain KM71 frozen at -80℃ and spread it on YPD solid plate, and culture it at 30℃ for 36h. Pick the same single colony and streak it on MD and MDH solid plates respectively. Screen the amino acid-deficient strain that cannot grow on MD plate but can grow on MDH plate. Streak it on fresh YPD plate again and culture it at 30℃ for 2 days until single colonies appear.

[0054] B. Preparation of KM71 competent cells

[0055] a. Pick a single colony from the activated YPD plate, inoculate it into 5 ml YPD liquid medium, and culture overnight at 28°C and 250 rpm.

[0056] b. Centrifuge at 4℃, 1500×g for 5 min to collect the bacteria. Add 100 ml of ice-cold sterile water to resuspend the bacterial cells and repeat this step once.

[0057] c. Centrifuge at 4°C, 1500×g for 5 min to collect the bacteria. Add 50 ml of ice-cold 1 mol / L sorbitol to resuspend the bacterial cells.

[0058] d. Centrifuge at 4℃ and 1500×g for 5 min to collect the bacteria. Add 1 ml of ice-cold 1 mol / L sorbitol to resuspend the bacterial cells and place on ice for later use (the competent cells can be divided into 80 ul aliquots and frozen in centrifuge tubes, but the transformation efficiency will be greatly reduced).

[0059] (33) Electroporation transformation of Pichia pastoris competent cells and screening of high-copy transformants

[0060] A. Electroporation transformation of Pichia pastoris

[0061] Bio-Ead Gene Pulse electroporator, voltage 1500V, capacitor 25uF, resistor 200Ω

[0062] a. Mix 80ul of Pichia competent cells and about 10ug of linearized recombinant expression plasmids pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, and pPIC9K-MaltAtt-2 in a 1.5ml centrifuge tube and precool in an ice bath. At the same time, mix 80ul of Pichia competent cells and about 10ug of linearized pPIC9K empty vector in another centrifuge tube and precool in ice as a control. At the same time, place a 0.2cm electroporation cuvette on ice for 10 minutes.

[0063] b. Transfer the pre-cooled plasmid and competent cell mixture into an ice-cold 0.2 cm electrode cup, place it in an ice bath for 5 minutes, wipe off the moisture on the surface of the electroporation cup, quickly place it on the electroporator and shock it once, immediately add 1 ml of pre-cooled 1 mol / L sorbitol to the cup, mix well, transfer it into a 1.5 ml centrifuge tube, and place it on ice.

[0064] c. Take 0.4 ml of the electroporated product and spread it on an RPB plate, transform the strain KM71 with the empty plasmid pPIC9K as a negative control, and culture at 30°C until transformants appear.

[0065] B. Screening of high copy number transformants

[0066] After pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, pPIC9K-MaltAtt-2 and pPIC9K transformants grow on MD plates, resuspend the bacteria with sterile water, determine the concentration so that OD600 = 1, and spread the bacterial solution on YPD plates with different concentrations (0-4 mg / mL) of geneticin (G-418). The bacterial solution containing the pPIC9K empty plasmid only needs to be spread on the YPD plate without resistance and placed in a 30°C incubator for culture. After the colonies grow on the YPD plate, pick some colonies and streak them on the YPD plate of the corresponding concentration. Use a pair of primers (a-Factor / 5'AOX (PPIC9K-F) and 3'AOX (PPIC9K-R) for colony PCR verification. The positive colonies grown on the G-418 plate with high concentration are the transformants with high insertion copies.

[0067] (4) Protein expression, purification and activity detection

[0068] (41) Protein expression

[0069] pPIC9K-MaltCec, pPIC9K-MaltDef, pPIC9K-MaltAtt-1, pPIC9K-MaltAtt-2 and pPIC9K transformants were picked up and inoculated into BMGY medium, cultured overnight at 30°C and 250-300 r / min, and the bacterial liquid was collected when OD600 was 4-6. The cells were centrifuged at 4°C and 3000 g / min for 5 min, and the cells were collected and resuspended in BMMY medium; 100% methanol was added every 24 h to make the final concentration of methanol 0.5%; after continuous induction for 96 h, the cells were centrifuged at 49°C and 6500 r / min for 10 min, and the supernatant was collected; the collected fermentation broth supernatant was freeze-dried and the concentrated supernatant was precipitated with ammonium sulfate.

[0070] The precipitated proteins were detected by Tricine-SDS-PAGE and Western-Blot to verify whether they contained the target proteins MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2. The results were as follows: Figure 2 shown.

[0071] (42) Inhibition zone test

[0072] In order to verify whether the antimicrobial peptides MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 have antibacterial activity, this example conducts an inhibition zone experiment for verification. Escherichia coli (because it is a model bacterium) and Serratia marcescens are selected as experimental strains. The specific method is: Escherichia coli and Serratia marcescens are cultured overnight, and the next day, the inoculation amount is transferred to OD600=0.2 at 1%, 100 puL is mixed with LB solid culture medium and poured into a plate, and after the culture medium solidifies, it is punched with a hole puncher, and 100 μL of MaltCec fermentation broth supernatant (MaltDef, MaltAtt-1, MaltAtt-2) is added to each well, and water (or pPIC9K fermentation broth supernatant) is used as a negative control, and 8 μuM Ampicillin is used as a positive control. The culture is cultured at 37°C and the growth is observed. The results are as follows: Figure 3 shown.

[0073] (43) Protein purification

[0074] According to the method of Nanjing GenScript HIS tag purification kit, the crude proteins of antimicrobial peptides MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 were purified, and the purification effect of the protein was detected by Tricine-SDS-PAGE.

[0075] Example 2

[0076] Determination of the nematicidal activity of antimicrobial peptides from Monochamus alternatus against pine wood nematodes

[0077] In order to verify whether the bacterial peptides MaltCec, MaltDef, MaltAtt-1 and MaltAtt-2 have an effect on the life activities of pine wood nematodes, the present embodiment adopts the insect immersion method to determine the nematicidal activity of the tracheal antimicrobial peptides of Monochamus alternatus against pine wood nematodes.

[0078] (1) Treatment of test nematodes

[0079] a. Inoculate Polytrichomonas discoides on PDA medium;

[0080] b. Inoculate pine wood nematodes on the colonies of Polytrichomonas discus and culture them in a constant temperature box at 28℃ until the colonies are completely eaten;

[0081] c. Use the Berman funnel method to separate the nematode solution, transfer it to a 15 ml centrifuge tube after 5-8 hours, and observe the strong activity of the nematodes under a stereomicroscope;

[0082] d. Place the collected fresh test nematodes into a 1.5 mL centrifuge tube and centrifuge at 4000 rpm for 5 min at room temperature;

[0083] e. Remove the supernatant with a pipette, add 0.5 mL of sterile water, centrifuge at 4000 rpm for 5 min at room temperature, and repeat twice;

[0084] f. Remove the supernatant with a pipette, add 0.5 mL of DEPC-H2O, centrifuge at 4000 rpm for 5 min at room temperature, and repeat twice;

[0085] g. Rapidly centrifuge to remove water and set aside for subsequent tests.

[0086] (2) Treatment of pine wood nematodes with chemicals

[0087] a. Dilute each concentration of antimicrobial peptide protein MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 to 0.3 mg / L with sterile water. -1 , 0.6mg·L -1 , 1mg·L -1 ;

[0088] b. Place the diluted protein and nematode suspension (about 100 nematodes) in a 24-well culture plate at a ratio of 1:9 and mix well with a pipette. Each concentration is one treatment, and each treatment is repeated 6 times. Sterile water is used as a blank control.

[0089] c. Place the culture plate in a constant temperature incubator at 26±2℃, observe and count the number of pine wood nematodes alive and dead under a microscope at 3h, 6h, 12h and 24h, and use the Abbott formula to correct the mortality rate of the treatment group. The mortality rate formula is as follows:

[0090] Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control group mortality rate) × 100%

[0091] Identification of live worms: All worms that move are live worms; suspended animation: worms that are in an "S" shape, curled or spiral shape need to be pricked with an insect needle to verify whether they are suspended animation nematodes: Identification of dead worms: worms that are not moving and are in a "J" or "C" shape, or worms that are rigid and have incomplete body walls are judged to be dead worms.

[0092] Pine wood nematode 0.3 mg·mL -1 , 0.6mg·mL -1 , 1mg·mL -1It was found that the antimicrobial peptides of Monochamus alternatus had a good toxic effect on pine wood nematodes under the treatment of 4 antimicrobial peptides. Compared with the treatment of fluopyram (90.0%) and avermectin (63.7%) at the same concentration for 24 hours, the nematicidal activity was significant. By comparison, it was found that the antimicrobial peptides of Monochamus alternatus reached the LC50 after 3 hours of treatment with pine wood nematodes; the mortality rate reached LC90 after 24 hours of treatment with pine wood nematodes, and the antimicrobial peptide MaltAtt-1 still had good nematicidal activity at a low concentration of 0.3 mg·mL-1, reaching (98.7%) at 6 hours. The second was MaltDef (83.4%), MaltAtt-2 (82.9%) and MaltCec (80.9%).

[0093] This protocol provides four antimicrobial peptides MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 from Monochamus alternatus, as well as their coding genes and exogenous expression, product induction expression, and purification methods, which enrich the antimicrobial peptide gene family of Monochamus alternatus and achieve exogenous expression of antimicrobial peptide genes MaltCec, MaltDef, MaltAtt-1, and MaltAtt-2 from Monochamus alternatus. It was found that the recombinant antimicrobial peptides after sequence modification have good nematode killing activity against pine wood nematodes. In view of this, antimicrobial peptides have shown great application potential as a green pesticide in inhibiting pine wood nematode disease.

[0094] The above descriptions are only some embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An antimicrobial peptide, characterized in that: The antimicrobial peptide is the antimicrobial peptide MaltDef of Monochamus alternatus; in, The amino acid sequence of the antimicrobial peptide MaltDef from Monochamus alternatus is shown in SEQ ID NO.

2.

2. The gene encoding the antimicrobial peptide according to claim 1, characterized in that: The nucleotide sequence encoding the antimicrobial peptide MaltDef from Monochamus alternatus is shown in SEQ ID NO.

6.

3. An expression vector containing the gene according to claim 2.

4. A method for preparing the antimicrobial peptide according to claim 1, characterized in that: The antimicrobial peptide is obtained by microbial expression or prepared by artificial synthesis.

5. The method according to claim 4, characterized in that: The method for obtaining the antimicrobial peptide by microbial expression comprises: screening the gene sequence corresponding to the antimicrobial peptide MaltDef of Monochamus alternatus, adding a SnaB Ⅰ restriction site and a start codon at its 5' end, and adding a stop codon and a Not Ⅰ restriction site at its 3' end to complete the synthesis of the gene sequence, and then respectively performing SnaB Ⅰ / Not Ⅰ double restriction digestion on the gene sequence and the pPIC9K plasmid, connecting the synthesized target gene to the Pichia pastoris exocrine expression vector pPIC9K to obtain a recombinant expression vector, and then inducing expression of the recombinant expression vector to obtain the antimicrobial peptide.

6. Use of the antimicrobial peptide according to claim 1 in the preparation of a drug or reagent for inhibiting pine wood nematode disease.

7. A pesticide, characterized in that: It comprises the antimicrobial peptide according to claim 1.

8. An antibacterial drug, characterized in that: It comprises the antimicrobial peptide according to claim 1.

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