Bacillus licheniformis source antibacterial peptide and application thereof

Abstract

The invention relates to the field of polypeptides, in particular to a bacillus licheniformis source antibacterial peptide and application thereof. The bacillus licheniformis source antibacterial peptide is selected from at least one of the following components: A1) the amino acid sequence of the bacillus licheniformis source antibacterial peptide is MVFFKYKKKIRIRKR (SEQ ID NO.1); a2) the bacillus licheniformis source antibacterial peptide with the same function is obtained by connecting tag protein to the N end and / or the C end of A1), and the bacillus licheniformis source antibacterial peptide has good antibacterial activity and can be widely applied to preparation of crop antibacterial products or screening of new antibacterial plant varieties.

Description

Technical Field

[0001] This invention relates to the field of polypeptides, specifically to an antimicrobial peptide derived from Bacillus licheniformis and its applications. Background Technology

[0002] In the agricultural field, research on crop antimicrobial agents has always been a key research area. Because existing chemical fungicides can easily lead to problems such as pesticide residues in the soil, increased drug resistance in pathogens, and ecosystem imbalance, scientists no longer rely solely on traditional chemical pesticides. Instead, they are committed to developing more efficient, environmentally friendly, and long-lasting antimicrobial strategies through a variety of cutting-edge technologies such as molecular biology, microbiome, and gene editing.

[0003] Existing studies have shown that plants have scarce resources of antibacterial genes, and existing resistance genes (such as some NB-LRR genes) have limitations such as narrow resistance spectrum and susceptibility to being overcome by pathogens. With the development of bioinformatics technology, antimicrobial peptides have gradually come into view. For example, Mohamed et al. screened a 24-amino acid α-helical antimicrobial peptide HR2-7 from Burkholderia strain HR2, which has inhibitory effects on a variety of plant pathogenic fungi and bacteria. Its activity was verified by in vitro experiments, providing a new approach to crop antimicrobial treatment. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention employs a progressive strategy of "AntiBP Server high-throughput prediction (narrowing the range to 210 entries) - PEP-FOLD3 secondary structure screening (focusing on 20 entries) - constraint design (locking in 3 entries)" to screen for an antimicrobial peptide derived from Bacillus licheniformis. This antimicrobial peptide, by optimizing its sequence, enhances its bipolar characteristics (hydrophobicity / hydrophilicity), enabling its wide application in plant antimicrobial treatment. The specific details of this invention are as follows: First aspect of the present invention. A Bacillus licheniformis-derived antimicrobial peptide is provided, wherein the Bacillus licheniformis-derived antimicrobial peptide is selected from at least one of the following: A1) The amino acid sequence of the antimicrobial peptide derived from Bacillus licheniformis is MVFFKYKKKIRIRKR (SEQ ID NO.1). A2) Obtain an antimicrobial peptide derived from Bacillus licheniformis with the same function by attaching a tag protein to the N-terminus and / or C-terminus of A1).

[0005] Furthermore, a Bacillus licheniformis-derived antimicrobial peptide derivative is also provided, which is obtained by acetylation of the N-terminus and / or amidation of the Bacillus licheniformis-derived antimicrobial peptide.

[0006] In a second aspect, the present invention provides a biomaterial selected from at least one of the following: B1) Contains a nucleic acid molecule encoding the Bacillus licheniformis-derived antimicrobial peptide described above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant cells containing the nucleic acid molecule described in B1), or recombinant cells containing the expression cassette described in B2), or recombinant cells containing the recombinant vector described in B3); B5) Plants containing nucleic acid molecules of B1), or plants containing recombinant vectors of expression cassettes of B2), or plants containing recombinant vectors of B3), or plants containing recombinant cells of B4).

[0007] Furthermore, the nucleic acid molecules described in B1) include publicly available nucleic acid molecules and nucleic acid molecules that have undergone codon optimization according to actual needs. Preferably, the nucleic acid molecules are nucleic acid molecules obtained after optimization based on the codon preferences of the host plant.

[0008] Further, the recombinant vector described in B3) is a plant virus vector, optionally including a geminivirus vector. Optionally, the geminivirus vector includes at least one of a complementary vector, a gene substitution vector, or a satellite DNA-derived microvector.

[0009] Preferably, the complementary vector includes at least one of pRHVnHA and pRHVcGFP.

[0010] Furthermore, the plants described in B5 include at least rice.

[0011] In a third aspect, the present invention provides a composition comprising the aforementioned Bacillus licheniformis-derived antimicrobial peptide or a derivative thereof. Optionally, the concentration of the Bacillus licheniformis-derived antimicrobial peptide in the composition is ≥ 15.625 μg / mL.

[0012] Furthermore, the composition further includes additives acceptable in the agricultural field. Optionally, the additives are common additives used in the preparation of pesticides. Optionally, the additives include at least one of fillers, surfactants, solvents, dispersants, stabilizers, and synergists.

[0013] Optionally, the additive includes at least one of alkylphenol polyoxyethylene ether, alkylbenzene sulfonate, lignin sulfonate, azone, organosilicon, polyvinyl alcohol, synergistic ether, xylene, mineral oil, clay, and kaolin.

[0014] It should be noted that this invention does not specifically limit the types of additives. Any reagent that is acceptable in the agricultural field and can be used as a filler, surfactant, solvent, dispersant, stabilizer, or synergist is protected by this invention.

[0015] In a fourth aspect, the present invention provides the use of the Bacillus licheniformis-derived antimicrobial peptide, or a derivative thereof, or the biomaterial thereof, or the composition thereof, in the preparation of antimicrobial and / or bactericidal products.

[0016] Furthermore, the use of the Bacillus licheniformis-derived antimicrobial peptide, or a derivative thereof, or the biomaterial or composition thereof, in any of the following aspects is provided: C1) Application in the preparation of biological pesticides for the prevention and control of bacterial plant diseases; Application of C2 in the breeding of new antibacterial plant varieties.

[0017] Furthermore, the biopesticide also includes additives acceptable in the agricultural field. Optionally, the additives are common additives used in the preparation of pesticides. Optionally, the additives include at least one of fillers, surfactants, solvents, dispersants, stabilizers, and synergists.

[0018] In a fifth aspect, the present invention provides a method for preparing the Bacillus licheniformis-derived antimicrobial peptide, the method comprising at least one of solid-phase peptide synthesis, liquid-phase peptide synthesis, and enzymatic hydrolysis.

[0019] Preferably, the method is a solid-phase polypeptide synthesis method.

[0020] In a sixth aspect, the present invention provides a method for cultivating new antibacterial plant varieties, the method comprising at least the step of introducing a nucleic acid molecule encoding the antimicrobial peptide derived from Bacillus licheniformis into a target plant.

[0021] Furthermore, the specific operation of the method involves introducing Bacillus licheniformis-derived antimicrobial peptide genes into the genome of plants (such as rice) to establish a technical path of "microbial-derived active peptides - plant genetic transformation - bacterial control" to solve the problem of scarce plant antibacterial gene resources.

[0022] Furthermore, the plant includes rice.

[0023] In a seventh aspect, the present invention provides a method for screening Bacillus licheniformis-derived antimicrobial peptides, the method comprising at least the following steps: Initial candidate peptides were obtained through high-throughput screening and secondary structure analysis, and molecular optimization was performed to finally obtain a polypeptide sequence with high bactericidal activity.

[0024] Furthermore, the molecular optimization specifically includes the following operations: (1) The total net charge reaches +8, which can target and bind to the negatively charged plasma membrane of bacteria through strong electrostatic action; (2) The proportion of β-fold structure is ≥75%, and the rigid conformation enhances the membrane penetration efficiency; (3) Hydrophobic amino acids (M, V, F) and hydrophilic amino acids (K, R) are regularly partitioned, and the amphiphilic characteristics are more suitable for the bacterial cell membrane structure.

[0025] The beneficial effects of the present invention include, but are not limited to: The antimicrobial peptides derived from Bacillus licheniformis exhibit the following properties: higher efficiency, better safety, and significantly enhanced bactericidal activity.

[0026] The antimicrobial peptide MVFFKYKKKIRIRKR derived from Bacillus licheniformis of the present invention has the following characteristics: (1) a total net charge of +8, which can target and bind to the negatively charged plasma membrane of bacteria through strong electrostatic interaction; (2) a β-sheet structure accounting for ≥75%, and a rigid conformation that enhances membrane penetration efficiency; (3) regular partitioning of hydrophobic amino acids (M, V, F) and hydrophilic amino acids (K, R), with amphiphilic characteristics that are more suitable for bacterial cell membrane structure. Experimental verification shows that the peptide can exhibit bactericidal activity against Xanthomonas xanthomonas at a low concentration (15.625 μg / mL). Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the secondary structure of the antimicrobial peptide derived from Bacillus licheniformis in an embodiment of the present invention.

[0028] Figure 2 These are the antimicrobial activity test results of different concentrations of Bacillus licheniformis-derived antimicrobial peptides in the embodiments of the present invention. Among them, the concentration of No. 1 is 1 mg / mL; the concentration of No. 2 is 250 μg / mL; the concentration of No. 3 is 62.5 μg / mL; the concentration of No. 4 is 15.625 μg / mL; the concentration of No. 5 is 3.906 μg / mL; the concentration of No. 6 is 0.977 μg / mL; the concentration of No. 7 is 0.244 μg / mL; and the concentration of No. 8 is 0.061 μg / mL. Detailed Implementation

[0029] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0030] Example 1: Screening of antimicrobial peptides derived from Bacillus licheniformis 1. High-throughput screening of candidate peptides A library of whole-genome encoded protein sequences of Bacillus licheniformis strains was downloaded from the NCBI database. High-throughput screening of nematicides (including peptides with potential bactericidal activity) was performed using the AntiBP Server platform. Approximately 210 candidate peptides (top 5% of predicted scores) were selected as the initial research subjects. Their secondary structures were analyzed using the PEP-FOLD3 tool to further screen for candidate peptides with α-helices or β-sheets comprising ≥70% of the structure, ensuring a stable structural basis.

[0031] 2. Molecular optimization The initial candidate peptides were molecularly designed and optimized, strictly adhering to the following constraints: both ends of the molecule exhibited distinct hydrophilic and hydrophobic characteristics, respectively, enhancing amphiphilicity to improve binding ability to bacterial cell membranes; the number of positively charged residues (such as lysine K and arginine R) was increased to improve electrostatic interaction with the negatively charged membrane of pathogens; the α-helix or β-sheet core secondary structure was maintained to preserve its membrane permeability; and methionine (M) was introduced at the amino terminus to provide a start codon for efficient subsequent expression in plants. After multi-dimensional structural verification (hydrophilicity / hydrophobicity, net charge, etc.), the optimized Bacillus licheniformis-derived antimicrobial peptide sequence MVFFKYKKKIRIRKR (SEQ ID NO.1) with two β-sheet secondary structures was finally obtained. The secondary structure of this Bacillus licheniformis-derived antimicrobial peptide is shown below. Figure 1 As shown.

[0032] Example 2: Validation of the in vitro bactericidal activity of antimicrobial peptides derived from Bacillus licheniformis 1. Peptide synthesis and concentration gradient preparation A biotechnology company was commissioned to prepare antimicrobial peptides derived from Bacillus licheniformis (HPLC purity ≥95%) using solid-phase chemical synthesis technology. The peptides were dissolved in 0.9% NaCl solution and then serially diluted 4-fold to obtain peptide solutions of different concentrations, specifically 1.0 mg / mL to 0.061 μg / mL.

[0033] 2. In vitro antibacterial activity assay In a sterile operating table, use an inoculation loop to collect a small amount of Xanthomonas oryzae streak pathogenic strain (…). Xanthomonas oryzae pv. oryzicolaThe bacterial culture was inoculated into 100 mL of NB liquid medium in an Erlenmeyer flask and incubated at 28°C for 24 hours. The culture was then transferred to a 1.5 mL centrifuge tube and centrifuged at 6000 rpm for 2 minutes at 24°C. The supernatant was discarded, and 1.5 mL of physiological saline was added. The mixture was shaken well to obtain the activated bacterial culture. 20 μL of the activated bacterial culture was spread onto a plate using NA solid medium. The plate was incubated at 28°C for 2 hours. Eight sterile filter paper discs were placed tangentially at equal intervals in a petri dish, with one sterile filter paper disc (0.9% NaCl solution) placed in the center as a control. The initial concentration of the target Bacillus licheniformis antimicrobial peptide was 1 mg / mL, and a 4-fold serial dilution was performed. 10 μL of the Bacillus licheniformis antimicrobial peptide solution was added dropwise to each filter paper disc according to the concentration gradient. The results were observed after incubation at 28°C for 48 hours. The results are as follows: Figure 2 As shown, by Figure 2 It can be seen that a clear inhibition zone appeared at concentrations ranging from 1 mg / mL to 15.625 μg / mL, proving that the antimicrobial peptide derived from Bacillus licheniformis has significant bactericidal activity against Xanthomonas xanthomonas.

[0034] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide derived from Bacillus licheniformis, characterized in that, The Bacillus licheniformis-derived antimicrobial peptide is selected from at least one of the following: A1) The amino acid sequence of the antimicrobial peptide derived from Bacillus licheniformis is MVFFKYKKKIRIRKR (SEQ ID NO.1). A2) Obtain an antimicrobial peptide derived from Bacillus licheniformis with the same function by attaching a tag protein to the N-terminus and / or C-terminus of A1).

2. A Bacillus licheniformis-derived antimicrobial peptide derivative, characterized in that, The Bacillus licheniformis-derived antimicrobial peptide derivative is obtained by acetylation of the N-terminus and / or amidation of the Bacillus licheniformis-derived antimicrobial peptide of claim 1.

3. A biomaterial, characterized in that, The biomaterial is selected from at least one of the following: B1) A nucleic acid molecule containing the antimicrobial peptide derived from Bacillus licheniformis as described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant cells containing the nucleic acid molecule B1), or recombinant cells containing the expression cassette described in B2), or recombinant cells containing the recombinant vector described in B3); B5) A plant containing the nucleic acid molecule described in B1), or a plant containing the recombinant vector of the expression cassette described in B2), or a plant containing the recombinant vector described in B3), or a plant containing the recombinant cell described in B4).

4. A composition, characterized in that, The composition comprises the antimicrobial peptide derived from Bacillus licheniformis according to claim 1 or the antimicrobial peptide derivative derived from Bacillus licheniformis according to claim 2.

5. The use of the antimicrobial peptide derived from Bacillus licheniformis according to claim 1, or the antimicrobial peptide derivative derived from Bacillus licheniformis according to claim 2, or the biomaterial according to claim 3, or the composition according to claim 4, in the preparation of antimicrobial and / or bactericidal products.

6. The use of the Bacillus licheniformis-derived antimicrobial peptide of claim 1, or the Bacillus licheniformis-derived antimicrobial peptide derivative of claim 2, or the biomaterial of claim 3, or the composition of claim 4, in any of the following aspects: C1) Application in the preparation of biological pesticides for the prevention and control of bacterial plant diseases; Application of C2 in the breeding of new antibacterial plant varieties.

7. The application according to claim 6, characterized in that, The biopesticides also include additives acceptable in the agricultural field.

8. A method for preparing the Bacillus licheniformis-derived antimicrobial peptide according to claim 1, characterized in that, The method includes at least one of solid-phase peptide synthesis, liquid-phase peptide synthesis, and enzymatic hydrolysis.

9. A method for cultivating new antibacterial plant varieties, characterized in that, The method includes at least the step of introducing a nucleic acid molecule encoding the antimicrobial peptide derived from Bacillus licheniformis as described in claim 1 into the target plant.

10. A method for screening Bacillus licheniformis-derived antimicrobial peptides as described in claim 1, characterized in that, The method includes at least the following steps: Initial candidate peptides were obtained through high-throughput screening and secondary structure analysis, and molecular optimization was performed to finally obtain a polypeptide sequence with high bactericidal activity.

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