Antibacterial peptide and application thereof in prevention and treatment of citrus huanglongbing disease

Abstract

This invention discloses a class of antimicrobial peptides and their application in the control of citrus Huanglongbing (HLB). The antimicrobial peptide is any one of the following A1)-A4): A1) a polypeptide with the amino acid sequence shown in sequence 6; A2) a polypeptide with the amino acid sequence shown in sequence 5; A3) a polypeptide with the amino acid sequence shown in sequence 4; and A4) a polypeptide with the amino acid sequence shown in sequence 3. Experiments have demonstrated that the antimicrobial peptides provided by this invention can reduce the number of pathogens in HLB-infected citrus and promote the lysis of pathogens in HLB-infected citrus, ultimately killing the pathogens. Therefore, it has excellent potential application value in the control of citrus HLB.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control, specifically involving a class of antimicrobial peptides and their application in the control of citrus Huanglongbing (HLB). Background Technology

[0002] Citrus Huanglongbing (HLB), also known as citrus greening, is the most destructive disease affecting the citrus industry worldwide, causing billions of dollars in economic losses annually. HLB primarily affects the phloem, leading to leaf mottling, poor fruit quality, fruit drop, and increased twig dieback. HLB is caused by phloem-restricted Gram-negative, unculturable phloem bacilli, such as the candidate phloem bacilli species *Phyllostachys asiaticus* (Phyllostachys asiaticus). Candidatus Liberibacter asiaticus, C Las). Most commercial citrus varieties are susceptible to HLB-associated bacteria. C The effects of Las can lead to varying degrees of symptoms. C Las is mainly transmitted by the Asian citrus psyllid ( Diaphorina citri Kuwayama) spread.

[0003] Currently, many strategies for treating HLB have been developed, including controlling insect vectors, applying antimicrobial agents, chemotherapy and nutritional therapy, plant defense inducers, heat therapy, biological control, quarantine programs, and eradicating HLB-infected trees. However, there is still no cure for HLB disease, so there is an urgent need for innovative HLB treatment and prevention strategies to save the citrus industry.

[0004] Antimicrobial peptides are a class of small peptides with antimicrobial activity, targeting bacteria, fungi, viruses, and parasites. They inhibit pathogenic microorganisms through various mechanisms, the most common being binding to the cell membrane of pathogens, disrupting its structure, or directly creating micropores in the cell membrane to allow cell contents to leak out, ultimately killing the pathogens. With the widespread ban on antibiotics, antimicrobial peptides, as a next-generation antimicrobial agent capable of replacing traditional antibiotics, have a very broad application value and promising future. Summary of the Invention

[0005] The purpose of this invention is to provide a class of antimicrobial peptides and their application in the prevention and control of citrus Huanglongbing (HLB).

[0006] To achieve the above objectives, the present invention first provides a class of antimicrobial peptides.

[0007] The amino acid sequence of the antimicrobial peptide provided by the present invention includes the amino acid sequence shown in Sequence 6.

[0008] In a specific embodiment of the present invention, the antimicrobial peptide may be any one of the following A1)-A4):

[0009] A1) A polypeptide with an amino acid sequence as shown in Sequence 6;

[0010] A2) A polypeptide with an amino acid sequence as shown in Sequence 5;

[0011] A3) A polypeptide with an amino acid sequence as shown in Sequence 4;

[0012] A4) A polypeptide with an amino acid sequence as shown in Sequence 3.

[0013] Any of the antimicrobial peptides mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] To achieve the above objectives, the present invention also provides biomaterials related to the aforementioned antimicrobial peptides.

[0015] The biomaterials related to the above-mentioned antimicrobial peptides provided by this invention are any one of the following C1) to C8):

[0016] C1) The nucleic acid molecule encoding the above-mentioned antimicrobial peptide;

[0017] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0018] C3) A recombinant vector containing the nucleic acid molecule described in C1);

[0019] C4) A recombinant vector containing the expression cassette described in C2);

[0020] C5) Recombinant microorganisms containing the nucleic acid molecules described in C1);

[0021] C6) Recombinant microorganisms containing the expression cassette described in C2);

[0022] C7) Recombinant microorganisms containing the recombinant vector described in C3);

[0023] C8) Recombinant microorganisms containing the recombinant vector described in C4).

[0024] In the aforementioned biological materials, the expression cassette may be DNA capable of expressing the aforementioned antimicrobial peptides in host cells.

[0025] The recombinant vector may be a vector containing a nucleic acid molecule encoding the aforementioned antimicrobial peptide.

[0026] The recombinant microorganism may be yeast, bacteria, algae, or fungi containing the above-mentioned nucleic acid molecules, expression cassettes, or recombinant vectors.

[0027] To achieve the above objectives, the present invention also provides new uses for the aforementioned antimicrobial peptides or biomaterials.

[0028] This invention provides the application of the above-mentioned antimicrobial peptides or biological materials in the prevention and control of Huanglongbing (HLB) in plants.

[0029] This invention also provides the application of the above-mentioned antimicrobial peptides or biomaterials in the preparation of products for the prevention and control of Huanglongbing (HLB) in plants.

[0030] In the above applications, the control of Huanglongbing (HLB) in plants is manifested in reducing the number of pathogens in infected plants and / or promoting the lysis of pathogen cells in infected plants. The pathogen is the Huanglongbing pathogen. C Las.

[0031] To achieve the above objectives, the present invention also provides a method for preventing and controlling Huanglongbing (HLB) in plants.

[0032] The method for preventing and controlling Huanglongbing (HLB) in plants provided by the present invention includes the step of applying the above-mentioned antimicrobial peptides or the above-mentioned biological materials to the plants.

[0033] In the above method, the antimicrobial peptide can be an antimicrobial peptide solution with a concentration of 1 μM.

[0034] The solvent for the antimicrobial peptide solution can be water.

[0035] The application method may be vacuum injection.

[0036] To achieve the above objectives, the present invention provides a product whose function is to prevent and control Huanglongbing (HLB) in plants.

[0037] The active ingredient of the product provided by this invention is the above-mentioned antimicrobial peptide or the above-mentioned biomaterial.

[0038] In the above products, the antimicrobial peptide can be an antimicrobial peptide solution with a concentration of 1 μM.

[0039] The solvent for the antimicrobial peptide solution can be water.

[0040] In any of the above-described applications, methods, or products, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0041] The dicotyledonous plants mentioned may be plants of the Rutales order.

[0042] The plants mentioned in the Rutales order can be plants of the Rutaceae family.

[0043] The Rutaceae plants mentioned can be citrus plants.

[0044] The Citrus species mentioned can be citrus.

[0045] This invention provides a short antimicrobial peptide AMP3 and its truncated form for the prevention and control of Huanglongbing (HLB) in citrus. The antimicrobial peptide AMP3 provided by this invention is derived from the human gut microbiome and possesses an α-helix and a β-sheet structure. Its amino acid sequence is highly similar to polypeptides secreted by several human intestinal bacteria of the Bacteroidetes family. Experiments have demonstrated that both the antimicrobial peptide AMP3 and its truncated polypeptide can reduce the number of pathogens in HLB-infected citrus and promote the lysis of HLB pathogens, ultimately effectively inhibiting or killing the candidate Asian species of *Bacillus phloem* infecting citrus. C Las. The antimicrobial peptides provided by this invention have excellent potential application value in the prevention and control of citrus Huanglongbing (HLB). Attached Figure Description

[0046] Figure 1 To screen for antimicrobial peptides that inhibit the E3 ligase activity of the citrus CsPUB21 gene, a susceptibility gene for Huanglongbing (HLB). Figure 1 A shows that the antimicrobial peptides AMP3, AMP9, and AMP20 can reduce the ubiquitination level of CsPUB21 when the Huanglongbing bacteria effector SDE3 and the citrus protein CsPUB21 are present simultaneously. These peptides are potential novel antimicrobial peptides synthesized through AI-enabled mining of the human gut microbiome and citrus soil microbiome. Figure 1 B represents the protein docking model structure of CsPUB21-AMP3. The complex shows key amino acids at the interaction interface between the CsPUB21 and AMP3 antimicrobial peptides. The structures of the CsPUB21 and AMP3 antimicrobial peptides were predicted using the Phyre2 network. The modeling structure of the CsPUB21-AMP3 complex was based on the docking algorithm ZDOCK, and the structural data were processed using the PyMOL software. Green represents CsPUB21; purple represents the AMP3 antimicrobial peptide.

[0047] Figure 2 The AMP3 short peptide, containing 14 amino acids, can inhibit the activity of CsPUB21 enzyme. Figure 2 A shows the amino acid sequence alignment of the AMP3 antimicrobial peptide in the microbiome from an AI-mined antimicrobial peptide database and the NCBI database. The AMP3 antimicrobial peptide contains an α-helix and a β-sheet structure. The amino acid sequence of the AMP3 antimicrobial peptide shows high similarity to peptides secreted by several bacteria of the Bacteroidetes family. The amino acid sequence of AMP9 belongs to a peptide encoded by Bacteroidetes serratifolia. Figure 2B shows the effects of the antimicrobial peptides AMP3, AMP9, and AMP20, as well as the truncated AMP3 peptide, on the self-ubiquitination of CsPUB21 protein. These peptides were not effective in inhibiting the activity of CsPUB21 enzyme when it was alone. However, when the Huanglongbing Fungicide effector SDE3 and the citrus protein CsPUB21 were co-present, AMP3 and AMP9 significantly inhibited CsPUB21 enzyme activity. The smallest peptide sequence that inhibited CsPUB21 enzyme activity was AMP3-14, containing 14 amino acids.

[0048] Figure 3 To identify Huanglongbing infection in citrus leaves. Figure 3 A shows the phenotypes of healthy red orange leaves and red orange leaves infected with Huanglongbing (HLB). Figure 3 B shows the molecular identification results of healthy red orange leaves and red orange leaves infected with Huanglongbing (HLB).

[0049] Figure 4 Antimicrobial peptides can reduce the levels of bacteria in the leaves of infected citrus trees. C Las titer. Figure 4 A shows the relative efficacy of 1 μM AMP3 antimicrobial peptide and its truncated peptide, AMP9 antimicrobial peptide, on citrus leaves infected with Huanglongbing (HLB) 48 hours after treatment. C Las concentration. Values ​​are mean ± SEM (n=12). Based on one-way ANOVA and Duncan's multiple range test, lowercase letters indicate significant differences between different columns. P <0.05). Figure 4 B shows the effects of AMP3 and AMP3-14 antimicrobial peptide treatment for 24 hours and 48 hours. C Las half-inhibitory concentration (IC50).

[0050] Figure 5 AMP3-14 peptide was found to be an effective treatment for infected citrus plants. Figure A shows the disease status of infected citrus plants 60 days after treatment with AMP3-14 peptide and BSA via trunk injection, with BSA treatment serving as a control group. Figure B shows the detection of Huanglongbing fungus in AMP3-14 peptide and BSA-treated infected citrus plants every 30 days. C Las titer, with day 0 representing the pathogen titer in plants before treatment. Values ​​are average ± SEM (n=12) (**, P<0.01; Student's t -test).

[0051] Figure 6 Treatment with 1 μM AMP3 and AMP3-14 antimicrobial peptides C Transmission electron microscopy images of Las cells, with BSA or water treatment as negative controls. Red arrows indicate cells that have been damaged. C Las cells. Detailed Implementation

[0052] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0054] The citrus variety used in the following examples is the Lianjiang Red Orange from Guangdong ( Citrus sinensis L.).

[0055] The vectors pACYCDuet-CDS-Myc-AtUBC8-S, pGEX-DC, pCDFDuet-AtUBA1-S, and pET-28a-FLAG-UBQ involved in the Escherichia coli ubiquitination detection system in the following examples are all described in the literature "Yufang Han, Jianhang Sun, Jun Yang, Zhaoyun Tan, Jing Luo, Dongping Lu. Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach. The Plant Journal 91, 766-776 (2017)."

[0056] The main reagents and their sources in the following examples are as follows: SYBR qPCR Mix is ​​a product of TOYOBO; Cocktail protease inhibitor is a product of Roche; IPTG is a product of Inolco; 40% Acrylamide is a product of Sigma; the primary and secondary antibodies are both products of Beijing TransGen Biotech Co., Ltd.; the pre-stained protein molecular weight marker is a product of Bio-Edge Biotechnology Co., Ltd.; and the ECL chemiluminescence solution is a product of GE Healthcare.

[0057] The primers used in the following examples were synthesized by Kinsun Biotech Ltd. and the relevant sequencing work was performed. The antimicrobial peptides were synthesized by Genscript Biotech Ltd.

[0058] The amino acid sequence of the CsPUB21 protein in the following examples is shown in Sequence 1. The SDE3 protein (SDE3 protein is the pathogen of Huanglongbing) C The amino acid sequence of a secretory protein of Las is shown in Sequence 2.

[0059] Example 1: Obtaining the AMP3-14 short peptide

[0060] I. AMP3 antimicrobial peptide inhibits the E3 ligase activity of the citrus Huanglongbing (HLB) susceptibility gene CsPUB21.

[0061] 1. Carrier Construction

[0062] First, vectors for in vitro ubiquitination detection in *E. coli* were constructed: the full-length CsPUB21 clone was ligated into the pACYCDuet-CDS-Myc-AtUBC8-S vector using a double enzyme digestion method, resulting in the pACYCDuet-CsPUB21-Myc-AtUBC8-S vector; the full-length SDE3 clone was ligated into the pGEX-DC vector using a double enzyme digestion method, resulting in the pGEX-SDE3 vector. The primers for vector construction are as follows:

[0063] CsPUB21-Fw(BamHI):CAAGGGATCCATGATTTTGTCATGGAAAAGAC;

[0064] CsPUB21-Rv(StuI):CAAGAGGCCTAAACGGCCTTTTCAGGTCCT;

[0065] SDE3-Fw(KpnI):CAAGGGTACCATGCTTAATTGCAACGAAAC;

[0066] SDE3-Rv(XhoI):CAAGCTCGAGCAATTATTTATAAATGGGCA.

[0067] 2. Constructing an Escherichia coli ubiquitination detection system

[0068] (1) pACYCDuet-CsPUB21-Myc-AtUBC8-S vector, pGEX-SDE3 vector, pCDFDuet-AtUBA1-S vector and pET-28a-FLAG-UBQ vector were co-transfected into BL21(DE3) competent cells. CsPUB21 was used as an E3 ubiquitin ligase, AtUBC8 was used as an E2 ubiquitin conjugating enzyme, AtUBA1 was used as an E1 ubiquitin activating enzyme, and UBQ was a small ubiquitin protein, Ubiqutin.

[0069] (2) Strains containing the expression vector were cultured at 37°C until OD600nm The concentration was 0.4-0.6. After adding 500 nM isopropyl β-D-thiogalactoside (IPTG), protein expression was induced at 28°C for 10 hours, followed by overnight reaction at 4°C.

[0070] (3) The crude protein material was separated by 8% SDS-PAGE gel and the CsPUB21 autoubiquitination level was analyzed by immunohybridization using anti-c-myc tag antibody.

[0071] 3. AMP3 and AMP9 antimicrobial peptides inhibit the E3 ligase activity of PUB21.

[0072] (1) Small molecule inhibitors targeting ubiquitination are effective in treating certain diseases. Since the E3 ligase activity of PUB21 is crucial for the sensitivity of Huanglongbing to HLB, small molecule inhibitors can be identified by targeting and inhibiting the E3 ligase activity of PUB21 to treat Huanglongbing HLB.

[0073] (2) This invention uses AI-powered computational prediction to identify novel antimicrobial peptides with antimicrobial activity from human gut microbiome and citrus soil microbiome data. Thirty-two antimicrobial peptides were randomly selected for chemical synthesis (AMP1-AMP32 antimicrobial peptides, where AMP1-AMP12 are derived from the gut microbiome and AMP13-AMP32 are derived from the citrus soil microbiome) to detect whether they can inhibit the ubiquitination level of CsPUB21. The amino acid sequences of the antimicrobial peptides are as follows:

[0074] AMP1: SVIWRKLFFIFIKRSGNWIKKVEKRQNLL.

[0075] AMP2:IFFRRNKKMAVKVAINGFGRIGRLAFRQMF.

[0076] AMP3: MKKVKNIFHKIANADPMIWGYVMLSESK (sequence 4).

[0077] AMP4: GVPMGSVIKKRRKRMAKKKHRKLLRKTRHQRRNKK.

[0078] AMP5:GRYIAKINPDNKKFKTMPSGKKRKGHKMATHKRKKRLRKNRHKKK.

[0079] AMP6:RGTCYNRVGLIIRNFSKLKGKKV.

[0080] AMP7: DKLISILSLLSKRRKADGFRVKKTQKSSAYKKRF.

[0081] AMP8: KQKTLKKVWKLSEKVLIFASAFAKKAGAAEATLVL.

[0082] AMP9: MKKVKNIFHKIANADPMIWGYVMLNDRLSK (Sequence 3).

[0083] AMP10: AMTLRKRKFAWYVLSSSLKWLIKKAKKIGVQVCGFE.

[0084] AMP11: AMTSRKRKFVWYVLSSSLKWLIKKAKKIGVQVCGFE.

[0085] AMP12: DRDRPECSTMVKYEQKLPSLGKYALKRAIKIKFGRK.

[0086] AMP13: MCRAGRCSPGRCR.

[0087] AMP14: MCWSPGPGTGSPASCRYWCRRG.

[0088] AMP15: MWPGPGKGFPASCRYWCRRD.

[0089] AMP16: MSRANAKPRPWRWPRWPWR.

[0090] AMP17: MLLRRWQGGRCRSAICRCRGP.

[0091] AMP18: MTRPRPGGSCGTPGSTCSWWPCR.

[0092] AMP19: MPSTVVCRPVGARSGPCWRWGTACW.

[0093] AMP20: MSLRSGNWCVRVCFKEFCGRKCRY.

[0094] AMP21: MAIKVGINGFGRIGRNIMRAA.

[0095] AMP22: MAKHAVSEGTKAVTKYTSSK.

[0096] AMP23: MLPGELAKHAVSEGTKAVTKYTSSK.

[0097] AMP24: MDIVYALKRQGRTLYGFGG.

[0098] AMP25:MSGRGKGGKGLGKGGAKRHRKVLR.

[0099] AMP26: MGELAKHAVSEGTKAVANYSSNKP.

[0100] AMP27: MLGQAQTGTGKTAAFALPLLQRIDL.

[0101] AMP28: MCMADFSSSGLGDCTVGKLPSGVCCS.

[0102] AMP29: MDVIYAFKRQGRTLYG.

[0103] AMP30: MGELAKHAVSEGTKAVAKYSTNKT.

[0104] AMP31: MPPSGAWPRARAR.

[0105] AMP32: MCPMIFAPICGCDGKT.

[0106] (3) Using two immune activators, flg22 and chitin peptide, as controls, it was found that AMP3, AMP9 and AMP20 antimicrobial peptides had a significant inhibitory effect on CsPUB21 enzyme activity. Figure 1 A).

[0107] II. The AMP3-14 short peptide containing 14 amino acids can inhibit CsPUB21 enzyme activity.

[0108] 1. Protein structure prediction and complex structure modeling

[0109] (1) To further understand the molecular basis of the interaction between AMP3 antimicrobial peptide and CsPUB21, protein structure modeling was performed. The structures of CsPUB21 and AMP3 antimicrobial peptide were predicted using the Phyre2 network, and the modeled structure of the CsPUB21-AMP3 antimicrobial peptide complex was predicted based on the docking algorithm ZDOCK. The structural data were processed using the PyMOL software.

[0110] (2) Structural modeling predicts that the AMP3 antimicrobial peptides (M17 and Y21) bind to the N-terminal U-box domains (W100 and E103) of the CsPUB21 protein. Figure 1 (B) It is speculated that the binding of SDE3 and CsPUB21 may change the direction of U-Box towards ARM, thereby inhibiting the autoubiquitination level of CsPUN21.

[0111] (3) The AMP3 antimicrobial peptide contains an α-helix and a β-sheet, and the interaction interface with CsPUB21 is located in its β-sheet structure region.

[0112] 2. The AMP3-14 short peptide can inhibit the activity of CsPUB21 enzyme.

[0113] (1) The amino acid sequences of AMP3 antimicrobial peptides were compared and analyzed using AI-mined antimicrobial peptide databases and the NCBI database. The comparison and analysis results are as follows: Figure 2 As shown in Figure A, its amino acid sequence is highly similar to polypeptides secreted by several species of Bacteroides, a class of symbiotic bacteria widely found in the intestines of humans and animals, playing an important role in maintaining host health.

[0114] (2) Based on sequence alignment and structural modeling, in order to identify the minimum peptide sequence of the AMP3 antimicrobial peptide that inhibits CsPUB21 enzyme activity, two stepwise truncated peptides, AMP3-24 (MKKVKNIFHKIANADPMIWGYVML, sequence 5) and AMP3-14 (DPMIWGYVMLSESK, sequence 6), were designed and synthesized.

[0115] (3) Screening was conducted using ubiquitination experiments to determine whether truncated peptides inhibited CsPUB21 enzyme activity. The results of the ubiquitination experiments are as follows: Figure 2 As shown in B, the smallest peptide sequence recognized by the inhibitory CsPUB21 enzyme activity is AMP3-14, which contains 14 amino acids: DPMIWGYVMLSESK.

[0116] Example 2: AMP3-14 short peptide can reduce the pathogen of Huanglongbing. C Las titer and can promote C Las cell lysis

[0117] I. Antimicrobial peptides can reduce the levels of bacteria in the leaves of infected citrus fruits. C Las titer

[0118] 1. Identification of Huanglongbing infection in citrus leaves

[0119] Leaves of citrus (red orange) infected with Huanglongbing were collected and the pathogen of Huanglongbing was used. C Las-specific detection primers A2 and J5 amplified the prlKAJL-rpoBC gene (target fragment length 703 bp) located in *Citrus citrus Huanglongbing* to identify the susceptibility of citrus leaves. Healthy citrus (red orange) leaves were used as a control.

[0120] Huanglongbing pathogen C Las-specific detection primer A2: TATAAAGGTTGACCTTTCGAGTTT;

[0121] Huanglongbing pathogen C Las-specific detection primer J5: ACAAAAGCAGAAATAGCACGAACAA.

[0122] The results of the identification are as follows Figure 3 As shown in A and 3B, infected citrus leaves turn yellow. A fragment of about 700 bp can be amplified in the citrus leaves and leaf midribs rich in bacteria, while this fragment cannot be detected in healthy citrus leaves and leaf midribs, thus confirming the susceptibility of the citrus leaves.

[0123] 2. To test the therapeutic efficacy of AMP3 and AMP9 antimicrobial peptides against HLB scab disease in Huanglongbing (HLB) and to determine whether AMP3-14 and AMP3-24 short peptides are effective variants of these antimicrobial peptides, 1 μM antimicrobial peptide solution (water solvent), 1 μM tetracycline (water solvent), and 10 μM tetracycline (water solvent) were injected into the identified infected area in step 1 using a vacuum injection method. C Citrus leaves from Las were used, with water as a control. Midrib samples were collected after 48 hours, and DNA was extracted from the leaves using the CTAB method. Subsequently, the DNA content in the midribs was detected by quantitative real-time PCR. C Las titer. COX was used as an internal reference gene. Primer sequences are as follows:

[0124] Huanglongbing pathogen C Las-specific detection primers HLBas: GTCGAGCGCGTATGCAATACG;

[0125] Huanglongbing pathogen C Las-specific detection primer HLBr: GCGTTATCCCGTAGAAAAAGGTAG;

[0126] Citrus internal reference gene detection primers COXf: GGTATGCCACGTCGCATTCCAGA;

[0127] Citrus internal reference gene detection primer COXr: GCCAAAACTGCTAAGGGCATTC.

[0128] C Las concentration test results are as follows Figure 4 As shown in Figure A, 1 μM AMP3 antimicrobial peptide, its truncated peptide, and AMP9 antimicrobial peptide can significantly reduce the levels of [unclear - possibly referring to a specific substance or ingredient] in the leaves of infected citrus. C Las concentration, of which the AMP3-14 truncated peptide can significantly reduce CLas titer. Compared to the same concentration of the antibiotic tetracycline, the antimicrobial peptide is more effective, equivalent to the effect of 10 μM tetracycline. With the ban on antibiotics in agricultural production, peptide drugs have a better application prospect in combating citrus Huanglongbing (HLB).

[0129] 3. Different concentrations of antimicrobial peptides were used for testing. C Las titer analysis was performed, and the half-inhibitory concentrations (IC50 values) of the antimicrobial peptides were calculated.

[0130] The IC50 value calculation result is as follows: Figure 4 As shown in Figure B, the IC50 values ​​of the antimicrobial peptides treated for 48 hours were significantly lower than those treated for 24 hours. The IC50 values ​​for AMP3 antimicrobial peptide and AMP3-14 truncated peptide treated for 48 hours were 52.1 µM and 1 µM, respectively. These results suggest that the truncated AMP3-14 peptide may be effective in reducing... C Las titers are more effective.

[0131] II. AMP3-14 short peptide can effectively treat infected citrus plants.

[0132] To further investigate the therapeutic effect of AMP3-14 short peptide on citrus plants infected with Huanglongbing (HLB), 1 μM AMP3-14 short peptide solution (water) and 1 μM BSA solution (water) were injected into the infected plants identified in step one (1) via intravenous infusion. C Citrus plants in Las were sampled, and midrib samples of citrus leaves were collected every 30 days. DNA was extracted from the leaves using the CTAB method, and then detected in the midribs of the leaves by quantitative real-time PCR. C Las titer was measured, with COX as an internal control gene. Plant phenotypes were observed by photographing after 60 days.

[0133] Phenotypic observation results of infected plants are as follows Figure 5 As shown in Figure A, the results indicate that after 60 days, when diseased citrus plants treated with AMP3-14 short peptide and BSA were photographed and observed, the leaves of diseased plants treated with BSA showed a mottled yellow color, while the yellow color on the leaves of diseased plants treated with AMP3-14 short peptide was significantly reduced.

[0134] C Las concentration test results are as follows Figure 5 As shown in Figure B, the results indicate that treatment with the AMP3-14 short peptide for 30 days significantly reduced the pathogens in the plants. C Las titer, and even after 120 days, AMP3-14 short peptide treatment of infected plants showed low pathogen titers, indicating that AMP3-14 short peptide can effectively treat infected citrus plants.

[0135] III. Antimicrobial peptides can promote the production of antimicrobial peptides in the leaves of infected citrus fruits. CLas cell lysis

[0136] 1. To understand how antimicrobial peptides reduce pathogens in infected leaves C The mechanism of action of Las titer was observed using transmission electron microscopy after treatment with antimicrobial peptides. C Morphological changes of Las. The infected samples from step 1, section 1, were identified. C Citrus leaves from Las were co-cultured with 1 μM AMP3 antimicrobial peptide or its truncated peptide AMP3-14 in a plant growth chamber for 24 hours, while leaves incubated with water or 1 μM BSA for 24 hours served as a control treatment.

[0137] 2. The midrib of the leaf was dissected 24 hours after treatment. The tissue was fixed in fixation buffer (1% paraformaldehyde, 2.5% glutaraldehyde and 0.1 M phosphate buffer, pH 7.2) for 1 hour at room temperature and then placed at 4°C overnight for fixation. The sample was then washed five times in 0.1 M phosphate buffer and then fixed in 1% (wt / vol) osmium tetroxide with 0.1 M phosphate buffer at 4°C for 1.5 hours. The sample was dehydrated in acetone at fractional concentrations and embedded in Spurr resin.

[0138] 3. Collect ultrathin sections on a 200-mesh nickel grid coated with fumwah and stain with 1% (w / v) lead citrate and uranyl acetate. Examine the sections using a JEM-1400 transmission electron microscope at an accelerating voltage of 80 kV.

[0139] The results are as follows Figure 6 As shown in the figure, transmission electron microscopy sections revealed that water and BSA treatments did not affect the infection of Huanglongbing pathogens. C In the midrib of Las's citrus leaves C Morphological changes of Las. Application of 1 μM antimicrobial peptide to bacteria infected with Huanglongbing (HLB). C Las's citrus leaves contain substances that cause... C Las cytoplasmic leakage and the release of extracellular vesicles ultimately lead to C Las dissolves, and the truncated peptide AMP3-14 is more effective at destroying pathogen cells.

[0140] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An antimicrobial peptide, wherein the antimicrobial peptide is any one of the following A1)-A3): A1) A polypeptide with an amino acid sequence as shown in Sequence 6; A2) A polypeptide with an amino acid sequence as shown in Sequence 5; A3) A polypeptide with an amino acid sequence as shown in Sequence 4.

2. The biomaterial related to the antimicrobial peptide of claim 1 is any one of the following C1) to C8): C1) A nucleic acid molecule encoding the antimicrobial peptide of claim 1; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1); C4) A recombinant vector containing the expression cassette described in C2); C5) Recombinant microorganisms containing the nucleic acid molecules described in C1); C6) Recombinant microorganisms containing the expression cassette described in C2); C7) Recombinant microorganisms containing the recombinant vector described in C3); C8) Recombinant microorganisms containing the recombinant vector described in C4).

3. The application of antimicrobial peptides or biomaterials related to said antimicrobial peptides in the prevention and control of Huanglongbing (HLB) in plants or in the preparation of products for the prevention and control of HLB; The amino acid sequence of the antimicrobial peptide is shown in sequence 6, sequence 4, sequence 5, or sequence 3. The biomaterial is any one of the following D1) to D8): D1) The nucleic acid molecule encoding the antimicrobial peptide; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecules described in D1); D4) A recombinant vector containing the expression cassette described in D2); D5) Recombinant microorganisms containing the nucleic acid molecules described in D1); D6) Recombinant microorganisms containing the expression cassette described in D2); D7) Recombinant microorganisms containing the recombinant vector described in D3); D8) Recombinant microorganisms containing the recombinant vector described in D4); The plant in question is a member of the Rutaceae family.

4. The application according to claim 3, characterized in that: The control of Huanglongbing in plants is manifested in reducing the number of pathogens in infected plants and / or promoting the lysis of pathogen cells in infected plants.

5. The application according to claim 3, characterized in that: The plant in question belongs to the genus Citrus.

6. The application according to claim 5, characterized in that: The Citrus species mentioned are citrus.

7. A method for controlling Huanglongbing (HLB) in plants, comprising the step of applying an antimicrobial peptide to the plant; wherein the amino acid sequence of the antimicrobial peptide is as shown in sequence 6, sequence 4, sequence 5, or sequence 3; and wherein the plant is a Rutaceae plant.

8. The method according to claim 7, characterized in that: The plant in question belongs to the genus Citrus.

9. The method according to claim 8, characterized in that: The Citrus species mentioned are citrus.

10. The method according to claim 7, characterized in that: The antimicrobial peptide is an antimicrobial peptide solution with a concentration of 1 μM.

11. A product wherein the active ingredient is the antimicrobial peptide of claim 1 or the biomaterial of claim 2.

12. The product according to claim 11, characterized in that: The antimicrobial peptide is an antimicrobial peptide solution with a concentration of 1 μM.

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