Preparation method of fusion antibacterial peptide targeting phytophthora sojae

Abstract

The invention discloses a preparation method of a fusion antibacterial peptide targeting phytophthora sojae, and belongs to the technical field of biology. The invention aims to design a preparation method of a target phytophthora sojae fusion antibacterial peptide by taking phytophthora sojae chitin synthetase as a good bactericide target. The target phytophthora sojae fusion antibacterial peptide is a target phytophthora sojae fusion antibacterial peptide. A pichia pastoris recombinant expression vector of a plurality of antibacterial peptides is constructed, a pichia pastoris expression system is utilized to express the plurality of antibacterial peptides, the pichia pastoris recombinant expression vector is constructed, and the pichia pastoris expression system is utilized to perform high-density fermentation to successfully express the antibacterial peptides. The antibacterial peptide is helpful for enhancing the antibacterial ability of the antibacterial peptide, and has important significance for preventing and treating phytophthora sojae.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology. BACKGROUND

[0002] Phytophthora sojae is a soil-borne pathogenic microorganism of Oomycota, which can rapidly spread under suitable conditions, infect the roots, stems, leaves and seeds of soybean (Glycine max), and cause soybean root rot. Phytophthora sojae mainly spreads through oospores. At present, the main measures for preventing and controlling soybean root rot in agricultural production are breeding disease-resistant varieties and spraying fungicides. However, due to the strong resistance of Phytophthora sojae to chemical pesticides, the Phytophthora sojae remaining in the soil is difficult to be completely eliminated, resulting in the occurrence of soybean Phytophthora disease for many years. In the process of infection of Phytophthora sojae to soybean, it needs to break through the wax layer, cutin layer and cell wall of the epidermis of soybean, and also needs to break through the immune system of the plant, so as to successfully infect the soybean and cause soybean root rot.

[0003] Chitin is a component of the structure and function of fungal cell wall, and is also a pathogen-associated molecular pattern that triggers the innate immune response of host plants. However, chitin is rarely present in oomycetes. However, in the genus Phytophthora, chitin synthase (CHS) genes encoding chitin synthesis enzymes have been found to exist in their genomes. Studies have shown that chitin synthase is closely related to the growth and infection ability of Phytophthora sojae.

[0004] At present, the methods for preventing and controlling Phytophthora sojae mainly include chemical control, breeding of disease-resistant varieties, cultivation management measures for prevention and control, and biological control. In practical application, a single control method is often difficult to achieve a lasting and effective control effect. SUMMARY

[0005] The purpose of the present application is to use the chitin synthase of Phytophthora sojae as a good fungicide target, and to design a preparation method of a fusion antibacterial peptide targeting Phytophthora sojae.

[0006] The steps of the present application are as follows: (1) A plurality of antibacterial peptide Pichia pastoris recombinant expression vectors were constructed, and the corresponding high expression yeast transformants were screened; (2) A plurality of antibacterial peptides were expressed by using Pichia pastoris expression system, and it was determined by inhibition zone experiment that LFcinB is an antibacterial peptide having antibacterial ability to Phytophthora sojae; (3) Pichia pastoris recombinant expression vectors of LFcinB and LFcinB-peptide#1 were constructed, and the corresponding high expression yeast transformants were screened; (4) The Pichia pastoris expression system high-density fermentation successfully expressed LFcinB and LFcinB-peptide#1 antibacterial peptides; (5) The LFcinB-peptide#1 fusion antibacterial peptide has stronger bacteriostatic ability through mycelial growth inhibition experiment, hypocotyl inoculation experiment and qPCR experiment.

[0007] The LfcinB nucleotide sequence is SEQ ID NO:1: FKCRRWQWRMKKLGAPSITCVRRAF / TTTAAATGTCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCACCGAGCATTACCTGTGTTCGTCGCGCATTT; The LFcinB-peptide#1 nucleotide sequence is SEQ ID NO:2: FKCRRWQWRMKKLGAPSITCVRRAFKVSDTVVEPYNATLSVHQLVENADE / TTTAAATGTCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCACCGAGCATTACCTGTGTTCGTCGCGCATTTAAGGTGTCCGACACCGTCGTGGAACCTTACAACGCTACACTGAGCGTGCACCAGCTGGTGGAGAACGCCGATGAG.

[0008] The Pichia pastoris recombinant expression vectors of LFcinB and LFcinB-peptide#1 are constructed: (1) The pPIC9K-his plasmid is double enzyme cut by EcoRI and NotI, then the LfcinB and LFcinB-peptide#1 are cloned into the pPIC9K-his vector by using seamless cloning technology and transformed into the E.coli Trans T1 competent cells, then the plasmid is extracted, i.e.pPIC9K-LfcinB-his and pPIC9K-LfcinB-A-his; (2) The pPIC9K-LfcinB-his and pPIC9K-LfcinB-A-his recombinant plasmids are transformed into GS115.

[0009] The application helps to enhance the bacteriostatic ability of antibacterial peptides, and has important significance for preventing and treating soybean Phytophthora. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1are MD plate and different concentration G418 plate screening pPIC9K-LFcinB-his and pPIC9K-LFcinB-peptide#1-his high expression transformant colonies; Figure 2 are LFcinB-his and LFcinB-peptide#1-his SDS-PAGE; Figure 3 are LFcinB-his and LFcinB-peptide#1-his pretreated soybean hypocotyl lesion map; Figure 4 are LFcinB-his and LFcinB-peptide#1-his pretreated soybean hypocotyl lesion length; data are expressed as mean ± standard deviation (n = 3). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 (two-tailed t test); Figure 5 are LFcinB-his and LFcinB-peptide#1-his pretreated PsCHS1 and PsCHS2 expression analysis; data in the figure are expressed as mean ± standard deviation (n = 3). * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001 (two-tailed t test); Figure 6 are antibacterial peptide gene amplification product electrophoresis (M: Marker); Figure 7 is the pPIC9K-AMP-his recombinant vector map; Figure 8 are MD screening pPIC9K-AMP-his positive transformant colonies; Figure 9 are G418 screening antibacterial peptide high expression transformant colonies; Figure 10 are different antibacterial peptides on soybean Phytophthora infestans inhibition zone; (A) Mellttin (B) MsrA1 (C) Magainin (D) CecropinB (E) Plectasin (F) LfcinB; Figure 11 are LFcinB-peptide#1 gene amplification product electrophoresis (M: Marker); Figure 12 is the pPIC9K-LFcinB-peptide#1-his recombinant vector map; Figure 13MD plate and different concentration G418 plate screening pPIC9K-LFcinB-peptide#1-his high expression transformant colony; Figure 14 Pichia pastoris high-density fermentation system; Figure 15 LFcinB-his and LFcinB-peptide#1-his mycelial growth inhibition of Phytophthora sojae; Figure 16 LFcinB-his and LFcinB-peptide#1-his mycelial growth diameter and inhibition rate of Phytophthora sojae. DETAILED DESCRIPTION

[0011] The application discloses a preparation method of a fusion antibacterial peptide targeting Phytophthora sojae. The antibacterial peptide LFcinB-peptide#1 is constructed by taking peptide#1 (KVSDTVVEPYNATLSVHQLVENADE) as a targeting peptide domain and LFcinB (FKCRRWQWRMKKLGAPSITCVRRAF) as an antibacterial peptide domain, so that the antibacterial peptide targets the cell membrane of Phytophthora sojae and enhances the antibacterial activity. The two antibacterial peptides are expressed by a Pichia pastoris expression system and the antibacterial activity is evaluated, and the polypeptide obtained by the yeast expression is closer to the natural state than the polypeptide obtained by prokaryotic expression, so that the normal exercise of the antibacterial peptide function is ensured. The fusion antibacterial peptide obtained in the application has obviously enhanced antibacterial capacity for Phytophthora sojae compared with single bovine lactoferricin peptide, and has important reference value for agricultural disease prevention and treatment.

[0012] Construction of a Pichia pastoris recombinant expression vector: 1. Construction of pPIC9K-LFcinB-his and pPIC9K-LFcinB-peptide#1-his plasmid vectors First, the pPIC9K-his plasmid is subjected to double enzyme digestion of EcoRI and NotI, then the LfcinB (FKCRRWQWRMKKLGAPSITCVRRAF / TTTAAATGTCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCACCGAGCATTACCTGTGTTCGTCGCGCATTT) and LFcinB-peptide#1 (FKCRRWQWRMKKLGAPSITCVRRAFKVSDTVVEPYNATLSVHQLVENADE / TTTAAATGTCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCACCGAGCATTACCTGTGTTCGTCGCGCATTTAAGGTGTCCGACACCGTCGTGGAACCTTACAACGCTACACTGAGCGTGCACCAGCTGGTGGAGAACGCCGATGAG) was cloned into pPIC9K-his vector and transformed into E. coli Trans T1 competent cells, then the plasmid was extracted for the following transformation of Pichia pastoris recombinant expression strain.

[0013] 2. Transformation of pPIC9K-LfcinB-his and pPIC9K-LfcinB-A-his recombinant plasmids into GS115 (1) The extracted pPIC9K-LfcinB-his and pPIC9K-LfcinB-A-his recombinant plasmids were linearized by Sac I enzyme digestion and then transformed into GS115 competent cells and plated on MD plates.

[0014] (2) After the positive transformants grew, they were collected and plated on plates containing different concentrations of G418 antibiotics. After the high expression transformants grew, single colonies were picked for expansion culture. The growth of transformant colonies is shown in Figure 1

[0015] Expression and identification of antibacterial peptides The above obtained yeast was placed in liquid medium and cultured at 30 °C, 250 rpm for 72 h. 100% methanol was added every 24 h to a final concentration of 1.0%. After expression, the fermentation supernatant was obtained by centrifugation (20 min, 5000 g, 4 °C) and stored on ice. The fermentation supernatant was purified using NI-NTA resin and analyzed by SDS-PAGE, and the results are shown in Figure 2 . The LfcinB-his sample had a main band near 4.1 kD, and the LfcinB-peptide#1-his had a main band in the 4.1-9.5 kD range, which was consistent with our predicted molecular weight. The above results showed that we successfully purified the LfcinB-his and LfcinB-peptide#1-his antibacterial peptides.

[0016] Detection of antibacterial activity ​1. Remove 10-day-old soybean plants from their pots, wash away all mud from the roots, and immerse the entire root system in a 1 mg / mL antimicrobial peptide solution (PBS solution as a control). After soaking for 2 hours, inoculate the hypocotyl with a 5 mm *Phytophthora soybeanis* mycelium cake. Incubate at 25°C in the dark for 36 hours, then measure the lesion length. Results are as follows: Figure 3 and 4 As shown, compared with the control group, both LFcinB-his and LFcinB-peptide#1-his significantly reduced the lesion length of soybean hypocotyls, and the lesion length of soybean hypocotyls treated with LFcinB-peptide#1-his was shorter than that treated with LFcinB-his. These results indicate that both LFcinB-his and LFcinB-peptide#1-his can enhance the disease resistance of soybeans, with LFcinB-peptide#1-his showing a more significant effect in controlling *Phytophthora indica*.

[0017] 2. RNA was extracted from soybean hypocotyl samples and reverse transcribed into cDNA for qPCR. ACTIN2 was used as the reference gene, and 2... −△△Ct Methods for calculating the expression level of the target gene; To compare the antifungal activity of LFcinB-his and LFcinB-peptide#1-his against *Phytophthora spp.*, the transcriptional levels of PsCHS1 and PsCHS2 in soybean hypocotyls inoculated with *Phytophthora spp.* for 36 h after treatment with LFcinB-his and LFcinB-peptide#1-his were detected by quantitative real-time PCR, thereby assessing the relative biomass of *Phytophthora spp.* in treated soybeans. Results are as follows: Figure 5 As shown, compared with the control group, both LFcinB-his and LFcinB-peptide#1-his treatments significantly reduced the transcriptional levels of PsCHS1 and PsCHS2, and the downregulation of PsCHS1 and PsCHS2 was more significant after LFcinB-peptide#1-his treatment. These results indicate that both LFcinB-his and LFcinB-peptide#1-his can significantly inhibit the infectivity of Phytophthora soybeanis, and LFcinB-peptide#1-his has a stronger antibacterial ability than LFcinB-his.

[0018] The present invention will now be described in detail: 1. Experimental Materials and Methods 1.1 Bacterial strains, cells, and vectors The tested plant material, soybean Williams82, was preserved in our laboratory. The wild-type strain of Phytophthora soybeanis P6497 was kindly provided by Professor Liu Xili. E. coli Trans T1 competent cells were purchased from Zoman Biotech Co., Ltd., which were used for the construction of expression vectors. Pichia pastoris GS115 competent cells were purchased from Pint Biotech Co., Ltd., and the Pichia pastoris secretion expression vector pPIC9K was preserved in the laboratory.

[0019] 1.2 Main reagents and instruments 1.2.1 Main reagents 2x Phanta Max Master Mix was purchased from Novozyme, restriction endonucleases were purchased from NEB, seamless cloning kit was purchased from Zoman, BCA kit was purchased from Thermo, YNB was purchased from Lambolind, small molecular weight protein marker was purchased from Shanghai Sangon, 4-20% Bis-Tris precast gel was purchased from Duyu Biotech, methanol was purchased from Macklin, V8 juice medium (2x solution) was purchased from Pint, RNA extraction kit was purchased from Zoman, reverse transcription kit was purchased from Lambolind, and qPCR kit was purchased from Lambolind. 1.2.2 Main instruments PCR instrument (Bio-rad), constant temperature incubator, shaker, nucleic acid electrophoresis tank, protein electrophoresis tank, clean bench, small desktop high-speed centrifuge (Eppendorf), plant light incubator, constant temperature incubator, shaker, fermenter, protein purification instrument, clean bench, etc.

[0020] 1.3 Methods 1.3.1 Construction of Pichia pastoris recombinant expression strain vector (1) Amplification of target genes ① According to the amino acid sequence of the antibacterial peptide, the different antibacterial peptide base sequences were optimized according to the preference of Pichia pastoris codon, and the forward / reverse primers were designed. The double-stranded DNA fragments were obtained by primer annealing, and the annealing reaction system is shown in Table 1.

[0021] Table 1 Primer annealing system Component Volume Forward primer F 1 μL Reverse primer R 1 μL ddH2O 8 μL

[0022] ② The amplification of the target gene was carried out with the double-stranded DNA fragments obtained by annealing as the template, and the primers were designed according to the In-Fushion principle (the primer information is shown in Table 2). The primer synthesis and subsequent gene sequencing were completed by Shanghai Sangon Biotech Co., Ltd.

[0023] Table 2 Primer information Name Sequence pPIC9K-Mellttin-his-F 5' gcttacgtagaattcggcatcggcgccgtgctgaa 3' pPIC9K-Mellttin-his-R 5' gcgtggtggtggtggtggtgctgctgtcttttacgcttaa 3' pPIC9K-MsrAI-his-F 5' gcttacgtagaattcatggctttagaacacatgaa 3' pPIC9K-MsrAI-his-R 5' cgtggtggtggtggtggtgtttggtcagcttcagcgccg 3' pPIC9K-Magainin-his-F 5' gcttacgtagaattcggaatcggtaaattcctgca 3' pPIC9K-Magainin-his-R 5' cgtggtggtggtggtggtggctattcataatttcaccaa 3' pPIC9K-CecropinB-his-F 5' gcttacgtagaattcaagtggaaggttttcaagaa 3' pPIC9K-CecropinB-his-R 5' cgcgtggtggtggtggtggtgtaaagccttagcttcaccta 3' pPIC9K-Plectasin-his-F 5' gcttacgtagaattcggttttggttgtaatggtcc 3' pPIC9K-Plectasin-his-R 5'cgcgtggtggtggtggtggtgataacatttacaaacaaaac 3' pPIC9K-LFcinB-his-F 5' gcttacgtagaattctttaaatgtcgtcgttggca 3' pPIC9K-LFcinB-his-R 5' attcgcggccgcgtggtggtggtggtggtgaaatgcgcgacgaacac 3' pPIC9K-LFcinB-peptide#1-his-F 5' gcttacgtagaattctttaaatgtcgtcgttggca 3' pPIC9K-LFcinB-peptide#1-his-R 5' attcgcggccgcgtggtggtggtggtggtgaaatgcgcgacgaacac 3' 5' cgcggccgcttagtgatggtgatggtgatgctcatcggcgttctccacc 3'

[0024] After receiving the primers, the target gene was amplified using Novozyme high-fidelity DNA polymerase, and the amplification system and program are shown in Tables 3 and 4.

[0025] Table 3 PCR reaction system Components Volume (50 μL) 2x Phanta Max Master Mix 25 μL Template 1 μL Primer F 2 μL Primer R 2 μL ddH2O 20 μL

[0026] Table 4 PCR program Reaction Temperature Reaction Time Cycle Number 95℃ 30 s-5 min 1 95℃ 15 s 29 58℃ 15 s 29 72℃ 30-60 s / kb 29 72℃ 5 min 1

[0027] (2) Obtaining linearized vector Take pPIC9K-his vector as an example, the vector is cut by EcoRI and NotI, and the cutting system is prepared on ice, as shown in Table 5.

[0028] Table 5 Enzymatic reaction system Enzymatic Reaction Components Volume (50 μL) 10x rCutsmart Buffer 5 μL pCI-neo Plasmid 3 μg EcoRI 1 μL XmaI 1 μL ddH2O To 50 μL

[0029] (3) DNA agarose gel electrophoresis ①Prepare 1% agarose gel. Weigh 0.35 g of agarose and dissolve it in 35 mL of 1×TAE. Heat it in a microwave oven until it is clear and transparent. Add nucleic acid dye to make the final concentration 1×. Mix well and pour into the gel module. Insert the comb. Wait for solidification at room temperature; ②Add 6×Loading Buffer to the PCR product and the cutting product to make the final concentration 1×; ③Remove the comb and place the agarose gel in the electrophoresis tank. Place the DNA samples in the gel wells in turn. Electrophorese at 160 V for 20 min; ④After electrophoresis, place the gel block under a blue light gel cutter and observe the size of the target band. Cut the correct band and place it in a 1.5 mL centrifuge tube.

[0030] (4) DNA purification and recovery ①Add 400-600 μL PC solution to the centrifuge tube and heat to dissolve the gel block; ②Balance 400 μL of the equilibration solution BL to the adsorption column; ③After the gel block is completely dissolved, add the obtained liquid to the adsorption column. Centrifuge at 12000 rpm for 50 s and discard the filtrate; ④Add 600 μL of the rinsing solution to the adsorption column. Stand for 50 s, centrifuge at 12000 rpm for 20 s, and discard the waste liquid; ⑤Repeat step ④ once; ⑥Place the adsorption column back into the collection tube and centrifuge at 12000 rpm for 3 min; ⑦Then place the adsorption column into a new centrifuge tube and place it in a 50℃ metal bath for 6 min; (8) Add 35 μL of ddH2O preheated at 65°C to the adsorption column, stand at room temperature for 1 minute and 30 seconds, centrifuge at 12000 rpm for 1 minute and 30 seconds, collect the PCR product and store at -20°C.

[0031] (5) Construction of recombinant vector The target gene was cloned into the target vector using the Golden Gate Seamless Cloning Kit, and the reaction system is shown in Table 6.

[0032] Table 6 Seamless cloning reaction system Components Volume (5 μL) DNA Assembly Mix 1.5 μL Linearized Vector 1.75 μL PCR Fragment 1.75 μL The above system is placed at 50°C for 1-2 hours.

[0033] (6) E. coli transformation ① Place the ligation product on ice for standby, take Trans T1 competent cells from -80°C, and slowly thaw on ice; ② Prepare a sterile 1.5 mL centrifuge tube, add 50 μL of competent cells and 5 μL of ligation product, mix gently with fingers, and incubate on ice for 25 minutes; ③ Heat shock at 42°C metal bath for 45 seconds, then insert into ice for 3 minutes; ④ Add 450 μL of LB liquid medium without antibiotics to the centrifuge tube, and incubate at 37°C for about 45 minutes; ⑤ Centrifuge at 5000 rpm for 4 minutes, remove part of the supernatant with a pipette, resuspend the bacterial pellet with 100 μL of supernatant, evenly spread on LB plates containing ampicillin, dry in a clean bench, and then place in a 37°C incubator for 12-16 hours.

[0034] (7) Bacterial liquid PCR identification of positive clones ① Use a sterile gun to pick a single colony in a clean bench, inoculate into LB liquid medium with the same resistance as the target vector, and incubate at 37°C for 12-16 hours; ② Perform PCR reaction with the shaking bacterial liquid as template, and the amplification system is shown in Table 7; Table 7 Bacterial liquid PCR amplification system Reaction Components Volume (μL) 2 x Taq Master Mix 10 μL Primer F 0.5 μL Primer R 0.5 μL Template 1 μL ddH2O 8 μL After vortexing the above liquid, place it in a PCR instrument for reaction, and the reaction program is shown in Table 8 Table 8 Bacterial liquid PCR amplification program Temperature (°C) Time Cycle Number 95 3 min 1 95 15 sec 30 60 15 sec 30 72 1 kb / min 30 72 5 min 1 16 - 1 After the end of the PCR reaction, agarose gel electrophoresis was used to detect whether the target gene was successfully cloned into the target vector. The PCR positive bacterial solution was sent for sequencing. The correct bacterial solution was added with an equal volume of 40% glycerol and stored at -20°C for standby.

[0035] (8) Extraction of plasmid The plasmid was extracted by using the high-purity small-scale plasmid extraction kit from Tiangeng. The extracted plasmid can be used for cell transfection, enzyme digestion, transformation, etc. The specific steps are as follows: ① Take the bacteria solution stored at -20°C, add 100 μL of bacterial solution to 100 mL of LB liquid medium containing ampicillin, and incubate at 37°C and 220 rpm overnight. Then collect the bacterial precipitate by high-speed centrifugation; ② Add 3 mL P1 to resuspend the precipitate; ③ Add 3 mL P2 and mix gently; ④ Add 4.2 mL P3 and quickly invert until a white flocculent precipitate appears. Centrifuge at high speed to retain the supernatant; ⑤ Add 500 μL BL to the equilibration adsorption column, then add the supernatant from step ④, centrifuge at 12000 rpm for 25 s, and discard the waste liquid; ⑥ Add 650 μL PW and centrifuge at 12000 rpm for 40 s; ⑦ Repeat step ⑥; ⑧ Place the adsorption column back into the collection tube and centrifuge at 12000 rpm for 1 min, then dry; ⑨ Place the adsorption column into a new centrifuge tube, add elution solution, and centrifuge at 12000 rpm for 1 min. Collect the plasmid solution and store it at -20°C.

[0036] 1.3.2 Transformation and screening of Pichia pastoris recombinant expression strain (1) Linearization of recombinant plasmid After enzyme digestion of different antimicrobial peptide plasmids with endonuclease Sac1, about 2 volumes of PC solution were added to the digested liquid for liquid recovery. The experimental steps were the same as in section

[0031] (2) Transformation ① Take a sterile 1.5 mL EP tube and add 5 μg of pre-cooled linear plasmid, 5 μL of yeast transformation promoter, 100 μL of ice-melted GS115 competent cells, and 500 μL of Pichia pastoris transformation solution. Mix gently for 6-8 times; ② 30°C water bath for 25 min; ③ 42°C water bath for 20 min; (4) 12,000 rpm supernatant was discarded, resuspended with 100 μL of 0.9% NaCl solution and then coated on MD plates, and incubated at 30°C for 3-7 days; (3) High copy number transformant screening of antibacterial peptide The single colony on the MD plate was scraped into 1 mL of 0.9% NaCl solution, 50 μL of the resuspension was taken and coated on G418 plates with different concentrations, and incubated at 30°C in the dark for about 5 days to screen high copy transformants.

[0037] 1.3.3 Shake flask induced expression of antibacterial peptide (1) A single colony was picked into a 100 mL Erlenmeyer flask containing 25 mL of YPD medium, and incubated at 30°C, 250 rpm overnight; (2) The above bacterial solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of BMGY medium at a ratio of 5%, and incubated at 30°C, 250 rpm overnight; (3) The bacterial solution was transferred to a 500 mL shake flask containing 100 mL of BMMY medium at a ratio of 5%, and incubated at 30°C, 250 rpm for 72 h, 100% methanol was added every 24 h to a final concentration of 1.0%.

[0038] 1.3.4 Antibacterial peptide inhibition zone experiment on soybean Phytophthora Several 5 mm fungus cakes were taken from the soybean Phytophthora plate covered with mycelium and inoculated on a new V8 medium plate, then four Oxford cups were placed on the plate at equal distance from the fungus cake, 200 μL of the sample to be tested and PBS (PBS as negative control) were added into the Oxford cups, and the plate was placed in a 25°C incubator for 5 days to observe the antibacterial effect.

[0039] 1.3.5 High-density fermentation culture of LFcinB and LFcinB-peptide#1 (1) Primary seed liquid culture: 25 mL of YPD medium was prepared in a 250 mL shake flask, the mouth of the flask was wrapped with a sealing film, and sterilized at 115°C for 20 min, and then cooled to room temperature; the antibacterial peptide strain preserved in a glycerol tube was inoculated into the YPD medium in a clean bench, and then incubated at 30°C in a shaker overnight at a speed of 250 rpm; (2) Secondary seed liquid culture: 250 mL of BMGY medium was prepared and divided into 5 500 mL shake flasks, the mouths of the flasks were wrapped with a sealing film, and sterilized at 121°C for 20 min, and then cooled to room temperature; 10x YNB and the primary seed liquid were inoculated into the BMGY medium in a clean bench, and the inoculation amount was 5%, and then incubated at 30°C in a shaker overnight at a speed of 250 rpm; (3) High-density fermentation in 5 L fermenter: 2.5 L BMMY medium was prepared and sterilized at 121°C for 30 min in a 5 L fermenter. 10xYNB and secondary seed liquid were inoculated into the 5 L fermenter, and the tank temperature was controlled at 30°C. The pH was adjusted and controlled at 6.0 with ammonia water, the aeration rate was 1.0-1.2 vvm, the tank pressure was 0.08-0.12 MPa, the rotation speed was 900 rpm, and the dissolved oxygen was controlled at >30%. When the dissolved oxygen quickly rose, methanol was added to induce the expression of the antibacterial peptide after 1 h of starvation. The tank temperature was controlled at 30°C, the pH was 6.0, the rotation speed was 900 rpm, the aeration rate was 1.0-1.2 vvm, the tank pressure was 0.04-0.05 MPa, and the methanol flow rate was controlled by the dissolved oxygen feedback control to control the dissolved oxygen at >30%. The fermentation was completed after 96 h of induction, and the supernatant was collected.

[0040] 1.3.6 Isolation and purification of LFcinB and LFcinB-peptide#1 LFcinB and LFcinB-peptide#1 were purified using Ni-NTA Agarose beads based on the principle of affinity chromatography. The side chain of histidine (His) contains an imidazole group, which can chelate with nickel ions in Ni-NTA Agarose beads. Therefore, the target protein with a 6xHis tag can be combined with Ni-NTA Agarose beads and hung on the column. In addition, imidazole can competitively bind to nickel ions with the 6xHis tag, and low-concentration imidazole buffer can be used to elute non-specifically bound impurities with nickel, while high-concentration imidazole buffer can be used to elute the target protein. (1) The process of protein purification using a protein purification instrument is system cleaning, column loading, equilibration, sample loading, impurity washing, and elution. The specific steps are as follows: ① System cleaning: Place the A and B pump heads of the purification system in PBS buffer, set the flow rate to 10 ml / min, and remove 20% ethanol from the system pipeline; ② Column loading: Reduce the flow rate to 0.5 mL / min, first connect the bottom of the nickel column to the base, then inject the liquid flowing out of the pipeline into the column, and connect the pipeline to the top of the column through drop-to-drop; ③ Equilibration: Adjust the flow rate to 5 mL / min, and equilibrate the column with PBS for at least 5 column volumes to the baseline; ④ Sample loading: Place the A1 pump head in a beaker containing the sample, and set the flow rate to 5 mL / min. After sample loading is complete, wash the column with PBS for about 5 column volumes; ⑤ Impurity washing: Set the flow rate to 5 mL / min, and wash the column with 40 mM imidazole buffer for about 5 column volumes to remove impurities; ⑥ Elution: The flow rate was set to 5 mL / min, and the target protein was eluted with 250 mM imidazole buffer. The system automatically collected the sample; ⑦ After the protein purification was completed, the system was cleaned with PBS, ultrapure water, and 20% alcohol, respectively. The column was removed, and the instrument was turned off. (2) Ultrafiltration desalination of LFcinB and LFcinB-peptide#1 The LFcinB and LFcinB-peptide#1 protein solutions obtained by nickel column affinity chromatography purification were subjected to ultrafiltration treatment using Macrosep® Advance Centrifugal Device ultrafiltration centrifuge tubes (Pall Corporation, USA) with a molecular weight cut-off of 1 kDa and 3 kDa, respectively, to remove imidazole to avoid the influence of imidazole on subsequent experiments. ① 10 mL of PBS solution was added to rinse the ultrafiltration tube, and centrifugation was performed at 6000 g and 4°C for 15 min. The effluent was discarded, and this step was repeated once. ② The protein solution was added in portions, and centrifugation was performed at 6000 g and 4°C for 15 min until the sample was concentrated to 5 mL. ③ 10 mL of PBS was added for liquid exchange, and centrifugation was performed at 6000 g and 4°C for 15 min. After repeating this step 3 times, the protein solution was collected.

[0041] 1.3.7 Mycelial growth inhibition experiment (1) After the purified LFcinB and LFcinB-peptide#1 antimicrobial peptide solutions were concentrated to 10 mg / mL, they were mixed uniformly with sterilized V8 solid medium cooled to about 50°C to prepare solid plates with concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. (2) After taking the agar cake from the edge of the activated P. sojae colony with a 5 mm puncher, it was inoculated into the center of the V8 medium plate containing different concentrations of antimicrobial peptides. After 7 days of culture at 25°C in the dark, the minimum concentration of the medium without P. sojae growth was the minimum inhibitory concentration. The diameter of the colony was measured using the cross method, and the inhibition rate was calculated. Each treatment had 3 replicates. Inhibition rate = [(colony diameter of control group - cake diameter) - (colony diameter of experimental group - cake diameter)] / (colony diameter of control group - cake diameter) x 100%.

[0042] 1.3.8 Hypocotyl inoculation method Ten-day-old soybean plants were taken out of the pots, washed to remove all the soil on the roots, and the whole plant roots were immersed in an antibacterial peptide solution of 1 mg / mL, with a PBS solution as a control. After 2 h of immersion, a 5 mm piece of P. sojae fungus cake was inoculated on the hypocotyl, and the lesion length was measured after 36 h of culture at 25°C in the dark. The sample was then frozen in liquid nitrogen and stored at -80°C for later use. Each treatment was repeated 5 times.

[0043] 1.3.9 Real-time fluorescent quantitative PCR (1) Extraction of soybean RNA ① The soybean sample was ground in a mortar with liquid nitrogen until it became a powder.

[0044] ② About 100 mg of the soybean sample was weighed, and total RNA was extracted using the high-purity RNA extraction kit from Qiagen. The detailed steps are as follows: Ⅰ. The sample was ground into a powder under liquid nitrogen. Ⅱ. The powdered sample was transferred to a 2 mL centrifuge tube treated with liquid nitrogen, 1 mL TransZol Up reagent and 0.2 mL RNA extraction reagent were added, and it was vortexed vigorously at room temperature for 6-8 min. Ⅲ. Centrifuge at 10000 g, 4°C for 12 min. Take 450 μL of the upper aqueous phase into a new 2 mL centrifuge tube, and mix with 450 μL of anhydrous ethanol. Ⅳ. The mixed solution was added to the centrifugal column in two portions, and centrifuged at 12000 g, room temperature for 40 s, and the filtrate was discarded. Ⅴ. Add 550 μL of CB9, centrifuge at 12000 g, room temperature for 40 s, and discard the filtrate. Ⅵ. Repeat step V. Ⅶ. Add 550 μL of WB9, centrifuge at 12000 g, room temperature for 40 s, and discard the filtrate. Ⅷ. Repeat step VII twice. Ⅸ. Centrifuge at 12000 g, room temperature for 3 min, and stand in a clean fume hood for 1-2 min. Ⅹ. Place the centrifugal column in an RNase-free collection tube, add 70 μL of RNase-free water to elute the RNA, and store at -80°C. Ⅺ. The RNA sample was separated by 1% agarose gel to detect its integrity, and the RNA sample was quantified by ultramicro spectrophotometer. (2) Reverse transcription (RT-PCR) 1 μg of total RNA from P. sojae was taken and reverse transcribed using the reverse transcription kit from Lamba Bio. The reverse transcription PCR reaction system is shown in Table 9: Table 9 Reverse transcription reaction system Components Amount RNA Template 1 μg All-in-one First-Strand Synthesis Master Mix 4 μL dsDNase 1 μL Nuclease-Free Water To 20 μL After the above system was prepared on ice, it was gently mixed and beaten, and then centrifuged, centrifuged, incubated at 37°C for 2 min to remove genomic DNA contamination, incubated at 55°C for 15 min, and then incubated at 85°C for 5 min to terminate the reaction. The obtained cDNA was stored at -20°C; (3) Real-time fluorescent quantitative PCR qPCR reaction system was prepared according to the GoTaq qPCR Master Mix instruction manual, and qPCR experiment was carried out. ACTIN2 was used as an internal reference gene, and the expression of the target gene was calculated by the 2 −△△Ct method. The qPCR primers are shown in Table 10, the reaction system is shown in Table 11, and the reaction program is shown in Table 12: Table 10 qPCR primers Name Sequence ACTIN2-F 5' actgcaccttccagaccatc 3' ACTIN2-R 5' ccaccaccttgatcttcatg 3' PsCHS1-F 5' gctgtaccatttgctgttcg 3' PsCHS1-R 5' gttatccaccgtcagttgtcc 3' PsCHS2-F 5' aagctgaactcccatctgtg 3' PsCHS2-R 5' gagtttgaagatggaggtaggc 3' Table 11 qPCR reaction system Components Volume (μL) SYBR Mix 10 μL Primer F 0.75 μL Primer R 0.75 μL Template 1 μL ddH2O 7.5 μL Table 12 qPCR reaction program Temperature (°C) Time Cycle Number 95 3 min 1 95 20 sec 35 60 45 sec 35 (4) Statistical analysis All statistical data were analyzed by GraphPad Prism (8.0.2), and the differences were statistically analyzed by two-tailed t test (two independent samples obeying normal distribution) or one-way ANOVA (three or more samples obeying normal distribution), *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. All data are expressed as mean ± standard deviation.

[0045] 2、Experimental results 2.1 Construction of Pichia pastoris recombinant expression vector for expressing antibacterial peptide 2.1.1 Amplification of antibacterial peptide gene The forward / reverse primers designed according to the base sequences of antibacterial peptides Mellttin, MsrA1, Magainin, CecropinB, Plectasin and LFcinB were annealed to obtain double-stranded DNA fragments, which were used as templates to amplify the genes by PCR technology. The electrophoresis results (Figure Figure 6 ) of the amplification products showed that the bands were relatively single and consistent with the expected size, indicating that the gene amplification was successful.

[0046] 2.1.2 Construction of pPIC9K-AMP-his recombinant vector To conduct subsequent antimicrobial experiments on the antimicrobial peptides Mellittin, MsrA1, Magainin, CecropinB, Plectasin, and LFcinB, the pPIC9K-AMP-his vector was constructed. First, the pPIC9K-his plasmid was double-digested with EcoRI and NotI. Then, using seamless cloning technology, the genes for Mellittin, MsrA1, Magainin, CecropinB, Plectasin, and LFcinB were cloned into the pPIC9K-his vector and transformed into *E. coli* Trans T1 competent cells. Subsequently, the plasmid was extracted for transformation into recombinant expression strains of *Pichia pastoris*. The recombinant vector map is shown below. Figure 7 As shown.

[0047] 2.2 Transformation and screening of recombinant Pichia pastoris strains for expressing antimicrobial peptides 2.2.1 Transformation of pPIC9K-AMP-his recombinant plasmid The GS115 strain is a histidine-deficient yeast strain, lacking the gene required for histidine synthesis, and therefore cannot grow on MD agar (a minimal medium containing histidine). The pPIC9K vector contains a gene that compensates for this histidine synthesis deficiency, enabling the transformed GS115 strain to synthesize histidine and grow on MD agar. Therefore, using MD agar as a selective medium allows for the screening of successfully transformed strains carrying the compensatory gene, thus achieving transformant selection.

[0048] To screen for transformants that successfully transformed the pPIC9K-AMP-his recombinant plasmid into the GS115 strain, the extracted pPIC9K-AMP-his recombinant plasmid was linearized by SacI digestion, transformed into GS115 competent cells, and plated on MD plates. Positive transformant colonies were visible after approximately 5 days. The growth of the transformant colonies is shown in the figure below. Figure 8 As shown.

[0049] 2.2.2 Screening of pPIC9K-AMP-his high-expression transformants The pPIC9K vector contains the G418 resistance gene, which encodes an enzyme that enables transformants to survive on plates containing G418 antibiotic. By using different concentrations of G418 antibiotic, transformants expressing high levels of both the resistance gene and the target protein can be screened. Under low G418 conditions, all successfully transformed strains can grow, but as the G418 concentration increases, only transformants with high expression of the resistance gene survive, thus allowing for the selection of high-expression transformants.

[0050] To screen for high-expression transformants of pPIC9K-AMP-his, colonies from MD plates were collected and spread onto plates containing different concentrations of G418 antibiotic. Transformant colonies were visible after approximately 5 days. The growth of the transformant colonies is shown in the figure below. Figure 9 As shown.

[0051] 2.3 Antimicrobial peptide antimicrobial activity test against Phytophthora soybeanis To test the antimicrobial activity of Mellittin, MsrA1, Magainin, CecropinB, Plectasin, and LFcinB against *Phytophthora sacchariformis*, the above antimicrobial peptides were expressed using a Pichia pastoris expression system, and yeast fermentation supernatant containing these antimicrobial peptides was obtained. Then, inhibition zone experiments demonstrated that under the same expression conditions, LFcinB had antimicrobial activity against *Phytophthora sacchariformis*, while the other antimicrobial peptides showed almost no antimicrobial activity against the same fungus. Figure 10 This may be because the content of antimicrobial peptides in the supernatant is too low. Considering the subsequent industrialization of antimicrobial peptides, LFcinB was selected as the antimicrobial peptide domain of the soybean Phytophthora infestans-specific antimicrobial peptide.

[0052] 2.4 Construction of Pichia pastoris recombinant expression vector for expressing antimicrobial peptides 2.4.1 Amplification of the LFcinB-peptide#1 gene After annealing the double-stranded DNA fragment obtained by forward / reverse primers designed based on the LFcinB-peptide#1 base sequence, the gene was amplified by PCR using the fragment as a template. The electrophoresis results of the amplification products were then analyzed. Figure 11 The bands were relatively uniform and matched the expected size (186 bp), indicating that the gene amplification was successful.

[0053] 2.4.2 Construction of the pPIC9K-LFcinB-peptide#1-his recombinant vector To facilitate the subsequent expression and purification of LFcinB-peptide#1, the recombinant vector pPIC9K-LFcinB-peptide#1-his was constructed. First, the pPIC9K-his plasmid was double-digested with EcoRI and NotI. Then, LFcinB-peptide#1 was cloned into the pPIC9K-his vector using seamless cloning technology and transformed into *E. coli* Trans T1 competent cells. Subsequently, the plasmid was extracted for transformation into Pichia pastoris recombinant expression strains. The recombinant vector map is shown below. Figure 12 As shown.

[0054] 2.5 Transformation and screening of the pPIC9K-LFcinB-peptide#1-his recombinant plasmid To screen for high-expression transformants of pPIC9K-LFcinB-peptide#1-his, the extracted pPIC9K-LFcinB-peptide#1-his recombinant plasmid was linearized by SacI digestion, transformed into GS115 competent cells, and plated on MD plates. Colonies grown on MD plates were then plated on plates containing different concentrations of G418 antibiotic. Transformant colonies were visible after approximately 5 days. The growth of transformant colonies is shown in the figure below. Figure 13 As shown.

[0055] 2.6 High-density fermentation and purification of LFcinB-his and LFcinB-peptide#1-his High-expression transformants of pPIC9K-LFcinB-his and pPIC9K-LFcinB-peptide#1-his obtained after two screenings were selected and subjected to high-density fermentation according to method 5.1.3.5. Figure 14 After fermentation for 96 h, the mixture was transferred to a separate tank, and the fermentation supernatant was obtained by high-speed centrifugation. The LFcinB-his and LFcinB-peptide#1-his antimicrobial peptides were purified by Ni column affinity chromatography according to method 5.1.3.6. To determine the purity and molecular weight of the recombinant antimicrobial peptides, the LFcinB-his and LFcinB-peptide#1-his antimicrobial peptide samples were separated by 4-20% Bis-Tris pre-gel and stained with Coomassie Brilliant Blue. The results are as follows. Figure 2 As shown, the LFcinB-his sample exhibits a main band around 4.1 kDa, while the LFcinB-peptide#1-his sample shows a main band in the 4.1-9.5 kDa range, consistent with the predicted molecular weight. These results indicate successful purification of the LFcinB-his and LFcinB-peptide#1-his antimicrobial peptides. Further experiments will verify their antimicrobial activity.

[0056] 2.7 Validation of the antibacterial activity of LFcinB-his and LFcinB-peptide#1-his 5.2.7.1 LFcinB-peptide#1-his exhibited stronger antifungal activity against the mycelial growth of Phytophthora soybeanis. To compare the antifungal activity of LFcinB-his and LFcinB-peptide#1-his against *Phytophthora sojae*, this study conducted a mycelial growth inhibition experiment. The results are as follows: Figure 15 and 16As shown in Fig. 2A and Fig. 2B, both LFcinB-his and LFcinB-peptide#1-his could significantly inhibit the growth of P. sojae. When the concentration of LFcinB-his was 2 mg / mL, the growth inhibition rate of P. sojae mycelium was 100%, while the concentration of LFcinB-peptide#1-his was 1 mg / mL, the growth inhibition rate of P. sojae was 100%, which determined the minimum inhibitory concentration of LFcinB-his was 2 mg / mL, while the minimum inhibitory concentration of LFcinB-peptide#1-his was 1 mg / mL, which proved that the inhibitory ability of LFcinB-peptide#1-his on P. sojae was stronger than that of LFcinB-his.

[0057] 2.7.2 LFcinB-his and LFcinB-peptide#1-his enhanced the disease resistance of soybean hypocotyls To compare the effects of LFcinB-his and LFcinB-peptide#1-his on the disease resistance of soybean hypocotyls, the soybean was soaked with PBS, LFcinB-his and LFcinB-peptide#1-his respectively before inoculating P. sojae on the soybean hypocotyls. The results are shown in Fig. 2C and Fig. 2D. Figure 3 and 4 As shown in Fig. 2C and Fig. 2D, compared with the control group, both LFcinB-his and LFcinB-peptide#1-his could significantly reduce the lesion length of soybean hypocotyls, and the lesion length of soybean hypocotyls treated with LFcinB-peptide#1-his was shorter than that of LFcinB-his. The above results showed that both LFcinB-his and LFcinB-peptide#1-his could enhance the disease resistance of soybean, and the effect of LFcinB-peptide#1-his on preventing P. sojae was more obvious.

[0058] 2.7.3 qPCR analysis of the expression level of PSCHS1 and PSCHS2 after LFcinB-his and LFcinB-peptide#1-his pretreatment To compare the inhibitory activity of LFcinB-his and LFcinB-peptide#1-his on P. sojae, this study detected the transcription level of PsCHS1 and PsCHS2 in soybean hypocotyls inoculated with P. sojae for 36 h after treatment with LFcinB-his and LFcinB-peptide#1-his by fluorescence quantitative PCR, so as to evaluate the relative biomass of P. sojae in soybean after treatment. The results are shown in Fig. 2E and Fig. 2F. Figure 5As shown, compared with the control group, LFcinB-his and LFcinB-peptide#1-his treatments can significantly reduce the transcription level of PsCHS1 and PsCHS2, and the transcription level of PsCHS1 and PsCHS2 after LFcinB-peptide#1-his treatment is more significantly down-regulated, and the above results show that LFcinB-his and LFcinB-peptide#1-his can significantly inhibit the invasion ability of Phytophthora sojae, and LFcinB-peptide#1-his has stronger bacteriostatic ability than LFcinB-his.

[0059] The present application constructs a plurality of anti-bacterial peptide Pichia pastoris recombinant expression vectors, and obtains transformants capable of efficiently expressing anti-bacterial peptides by using MD culture medium screening and high-concentration G418 screening. Further, the anti-bacterial peptides are expressed by using the Pichia pastoris expression system, and the bacteriostatic circle experiment determines that LFcinB has bacteriostatic effect on Phytophthora sojae. Subsequently, LFcinB is used as the specific anti-bacterial peptide domain of the anti-bacterial peptide, and peptide#1 is used as the targeting domain of the specific anti-bacterial peptide, and the Pichia pastoris recombinant expression vector pPIC9K-LFcinB-peptide#1-his is constructed, and transformants capable of efficiently expressing pPIC9K-LFcinB-his and pPIC9K-LFcinB-peptide#1-his anti-bacterial peptides are obtained by twice screening.

[0060] The Pichia pastoris expression system is a eukaryotic expression system, which has the function of post-translational modification similar to mammalian cells, and has more obvious advantages in expressing anti-bacterial peptides. The prokaryotic expression system (such as E. coli) does not have this mechanism, and the anti-bacterial peptides expressed by the prokaryotic expression system are different in structure from the natural anti-bacterial peptides, so there is often a problem that the expressed anti-bacterial peptides have no antibacterial ability. Compared with other eukaryotic expression systems, the Pichia pastoris expression system also has the advantages of low fermentation nutritional requirement, low cost, and simpler purification steps. Based on the above, the Pichia pastoris expression system is selected to express two kinds of anti-bacterial peptides LFcinB-his and LFcinB-peptide#1-his by high-density fermentation, and the obtained fermentation broth is subjected to nickel column affinity chromatography to obtain protein solutions of the two kinds of anti-bacterial peptides LFcinB-his and LFcinB-peptide#1-his.

[0061] To verify the fungistasis ability of LFcinB-his and LFcinB-peptide#1-his to P. sojae, the minimum fungistasis concentration of LFcinB-peptide#1-his is 1 mg / mL, and the minimum fungistasis concentration of LFcinB-his is 2 mg / mL, which determines that the fungistasis ability of LFcinB-peptide#1-his to P. sojae is stronger than that of LFcinB-his. And further through the hypocotyl inoculation experiment, it is determined that LFcinB-his and LFcinB-peptide#1-his can significantly reduce the lesion length of soybean hypocotyl and improve the disease resistance of soybean, and the effect of LFcinB-peptide#1-his on preventing and treating P. sojae is more obvious. In addition, in order to more intuitively compare the disease resistance of soybean hypocotyl after treatment of LFcinB-his and LFcinB-peptide#1-his, the expression level of PSCHS1 and PSCHS2 in soybean after treatment is further analyzed by qPCR experiment to evaluate the relative biomass of P. sojae, and the experimental results show that the transcription level of PsCHS1 and PsCHS2 after LFcinB-peptide#1-his treatment is lower, indicating that LFcinB-peptide#1-his has stronger fungistasis ability than LFcinB-his.

[0062] In summary, the fusion expression of the targeting peptide peptide#1 and LFcinB further enhances the anti-P. sojae activity of LFcinB, successfully constructs the specific antibacterial peptide of P. sojae, lays a research foundation for the disease prevention and control of P. sojae, and provides a new idea for the design of targeted P. sojae antibacterial drugs.

Claims

1. A method for preparing a fusion antibacterial peptide targeting Phytophthora sojae, characterized in that: (1) a plurality of antibacterial peptide Pichia pastoris recombinant expression vectors are constructed, and the corresponding high expression yeast transformants are screened; (2) a plurality of antibacterial peptides are expressed by using the Pichia pastoris expression system, and it is determined by the inhibition zone experiment that LFcinB is an antibacterial peptide having antibacterial ability to Phytophthora sojae; (3) Pichia pastoris recombinant expression vectors of LFcinB and LFcinB-peptide#1 are constructed, and the corresponding high expression yeast transformants are screened; (4) LFcinB and LFcinB-peptide#1 antibacterial peptides are successfully expressed by using the Pichia pastoris expression system high-density fermentation; (5) it is verified by mycelial growth inhibition experiment, hypocotyl inoculation experiment and qPCR experiment that the LFcinB-peptide#1 fusion antibacterial peptide has stronger antibacterial ability.

2. The method for preparing a fusion antibacterial peptide targeting Phytophthora sojae according to claim 1, characterized in that: the LfcinB nucleotide sequence is SEQ ID NO: 1: FKCRRWQWRMKKLGAPSITCVRRAF / TTTAAATGTCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCACCGAGCATTACCTGTGTTCGTCGCGCATTT; the LFcinB-peptide#1 nucleotide sequence is SEQ ID NO: 2: FKCRRWQWRMKKLGAPSITCVRRAFKVSDTVVEPYNATLSVHQLVENADE / TTTAAATGTCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCACCGAGCATTACCTGTGTTCGTCGCGCATTTAAGGTGTCCGACACCGTCGTGGAACCTTACAACGCTACACTGAGCGTGCACCAGCTGGTGGAGAACGCCGATGAG. The Pichia pastoris recombinant expression vectors of LFcinB and LFcinB-peptide#1 are constructed: (1) the pPIC9K-his plasmid is double digested by EcoRI and NotI, then LfcinB and LFcinB-peptide#1 are cloned into the pPIC9K-his vector by using the seamless cloning technology and transformed into the E. coli Trans T1 competent cells, and then the plasmid is extracted, i.e. pPIC9K-LfcinB-his and pPIC9K-LfcinB-A-his; (2) the pPIC9K-LfcinB-his and pPIC9K-LfcinB-A-his recombinant plasmids are transformed into GS115. ​ ​ ​ ​ ​ ​ ​ ​ 3. The method of claim 1, wherein the method of preparing a fusion antimicrobial peptide targeting Phytophthora sojae is characterized by: ​ ​ ​

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