A polypeptide of polar origin and its use

Abstract

The application provides a polar source polypeptide and application thereof. A first aspect of the application provides a polar source polypeptide, and the amino acid sequence of the polypeptide is PLLKWKRPILRRLLK. The application mines and identifies a new polypeptide ARC026 from a polar microorganism metagenome. The polypeptide has broad-spectrum and high-efficiency antifungal activity, can effectively inhibit and kill various plant pathogenic fungi, effectively overcomes the limitations of the prior art, and provides a reliable candidate for developing a new generation of plant disease biological control agent.

Description

Technical Field

[0001] This application relates to the field of peptide technology, and more particularly to a polar-derived peptide and its applications. Background Technology

[0002] In the field of plant fungal disease control, Cladosporium ( Cladosporium cladosporioides , C. cladosporioides ), Trichophyton mentagrophytes (also known as cucumber vine blight fungus, Plectosphaerella cucumerina , P. cucumerina ), black occulta ( Epicoccum nigrum , E. nigrum ), *Broomrum oryzae* (also known as the pathogen of rice leaf sheath rot), Sarocladium oryzae , S. oryzae ) and Aspergillus flavus ( Aspergillus flavus , A. flavus This disease causes serious damage and is difficult to control. It can trigger major diseases in many crops, leading to leaf necrosis, fruit rot, plant wilting, and even the death of the entire plant, resulting in a severe decline in crop yield and quality. Moreover, some pathogens, such as Aspergillus flavus, produce aflatoxins, which are highly carcinogenic and directly threaten the quality and safety of agricultural products.

[0003] Currently, the control of plant fungal diseases largely relies on chemical fungicides. Besides the long-term dependence on single chemical fungicides such as benomyl and flusilazole, recent years have seen research into compound fungicide formulations targeting specific pathogens to address resistance and improve efficacy. However, these formulations still have relatively simple mechanisms of action, posing a long-term risk of inducing resistance in pathogens, and their high cost hinders widespread application. In addition to chemical fungicides, some non-chemical control methods based on altering the microecological environment have also been developed, but their sterilization effects are inconsistent.

[0004] Compared to traditional methods, antimicrobial peptides are a class of naturally occurring defensive small-molecule polypeptides widely found in organisms. They selectively target negatively charged microbial cell membranes through electrostatic interactions, effectively disrupting membrane integrity and rapidly killing pathogens. However, the antimicrobial spectrum of antimicrobial peptides is typically narrow, making broad-spectrum control difficult. Furthermore, antimicrobial peptides are mainly derived from conventional habitats; for special habitats such as polar regions, systematic exploration remains insufficient due to challenges in sample collection, scarcity of culturable strains, and a lack of genomic data. Therefore, it is necessary to explore broad-spectrum and highly effective antimicrobial peptides for plant pathogens in the unique polar habitat. Summary of the Invention

[0005] The main objective of this invention is to propose a polar-derived polypeptide and its application, which is developed specifically for the polar environment to obtain a broad-spectrum and highly effective antimicrobial peptide against plant pathogens.

[0006] To achieve the above objectives, the first aspect of the present invention provides a polar-derived polypeptide (also known as an antimicrobial peptide), the amino acid sequence of which is PLLKWKRPILRRLLK (SEQ ID NO.1).

[0007] A second aspect of the present invention provides a biomaterial comprising any one of the following A1) to A4): A1) A nucleic acid molecule encoding the first aspect polypeptide; A2) Expression cassette containing A1) nucleic acid molecules; A3) A recombinant vector containing A1) nucleic acid molecules or A2) expression cassettes; A4) Recombinant cells containing A1) nucleic acid molecules or A2) expression cassettes or A3) recombinant vectors.

[0008] The term "expression cassette" refers to a construct containing regulatory elements necessary for the expression of the contained nucleic acid molecules in recombinant cells. Specific regulatory elements include one or more of the following: promoters, transcription terminators, etc.

[0009] The term "recombinant vector" refers to a molecule containing all elements necessary for the replication, integration, amplification, and / or expression of the contained nucleic acid molecule in recombinant cells. In some embodiments, all elements include, but are not limited to, promoters, transcription terminators, 5' untranslated regions, 3' untranslated regions, introns, Kozak sequences, and selectable genes. Optional types of recombinant vectors include plasmids and viruses.

[0010] The term "recombinant cell" refers to a cell used for the replication, integration, amplification, and / or expression of nucleic acid molecules. Recombinant cells include, but are not limited to, microorganisms (such as bacteria, fungi, etc.), plant cells, and animal cells. Examples of bacteria include *Escherichia coli*, *Bacillus subtilis*, and *Agrobacterium*; examples of fungi include yeast or *Aspergillus*; examples of plant cells include *Arabidopsis thaliana* or tobacco cells; and examples of animal cells include mammalian cells such as those from mice, rats, pigs, sheep, monkeys, chimpanzees, or humans, specifically 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc., or insect cells, such as S2 Drosophila cells or Sf9 cells. It is understood that these recombinant cells can all be non-reproductive materials.

[0011] In addition, the aforementioned expression cassettes, recombinant vectors, or recombinant cells can be expression cassettes, recombinant vectors, or recombinant cells used for in vitro biosynthesis of peptides, or expression cassettes, recombinant vectors, or recombinant cells used for direct expression of peptides in target plants, etc.

[0012] A third aspect of the present invention provides a composition comprising the polypeptide of the first aspect, or the biomaterial of the second aspect.

[0013] In some embodiments, the composition is any one of antibacterial agents, fungicides, preservatives and mildew inhibitors, pesticides, fertilizers, pesticide additives, and fertilizer additives.

[0014] In some embodiments, the composition further includes pharmaceutically acceptable excipients, such as at least one of fillers, lubricants, wetting agents, dispersants, binders, emulsifiers, stabilizers, disintegrants, adsorbents, solubilizers, preservatives, etc.

[0015] In some embodiments, the dosage form of the composition may be any one of the following: powder, wettable powder, granules, oil, emulsion, suspension, aqueous solution, film.

[0016] In some embodiments, the composition also includes other active antibacterial ingredients.

[0017] In some embodiments, other active antibacterial components in the composition include other peptides. The synergistic effect of multiple peptides can enhance the antibacterial effect and broaden its antibacterial spectrum.

[0018] In some embodiments, other active antibacterial components in the composition include antibiotics, such as at least one of antibacterial antibiotics and antifungal antibiotics. In some embodiments, other active antibacterial components in the composition include triazoles (such as flutriafol, tricyclazole, cyproconazole, triadimefon, cyproconazole, flusilazole, difenoconazole, etc.), methoxyacrylates (such as azoxystrobin, pyraclostrobin, azoxystrobin, oxadiazon), aniline pyrimidines (such as pyrimethanil, azoxystrobin), dicarboxyimide fungicides (such as ethoxysulfuron, iprodione, procymidone), and others such as shenqinmycin, chlorothalonil, thiram, and isoprothiolane.

[0019] In some implementations, the preservative and antifungal agent can be a natural preservative and antifungal agent or an artificial preservative and antifungal agent, which can play a role of preservation alone, or antifungal alone, or both preservation and antifungal.

[0020] In some embodiments, the pesticide can be any one of a biological pesticide or a compound product of a biological pesticide and a chemical pesticide. Correspondingly, the pesticide additive can be an additive of any one of a biological pesticide, a chemical pesticide, or a compound product of a biological pesticide and a chemical pesticide.

[0021] In some embodiments, the fertilizer can be any one of bio-fertilizer or a compound product of bio-fertilizer and chemical fertilizer. Correspondingly, the fertilizer additive can be an additive of any one of bio-fertilizer, chemical fertilizer, or a compound product of bio-fertilizer and chemical fertilizer.

[0022] A fourth aspect of the present invention provides the use of the polypeptide of the first aspect, the biomaterial of the second aspect, or the composition of the third aspect in at least one of the following B1) to B3): B1) Prepare products that inhibit or kill fungi; B2) Prepare products for the prevention and / or treatment of plant diseases caused by fungal infections; B3) Prevention and / or treatment of plant diseases caused by fungal infection.

[0023] In some embodiments, the fungus includes at least one of Cladosporium, Trichophyton, Acanthopanax senticosus, Cladosporium oryzae, and Aspergillus flavus.

[0024] In some implementations, the disease includes at least one of the following: fruit rot, flower blight, wilt, root rot, phylloxera leaf blight, leaf spot, stem rot, and rice leaf sheath rot.

[0025] In some embodiments, the disease includes at least one of the following: fruit rot, flower blight, wilt, root rot, leaf blight, leaf spot, stem rot, and rice leaf sheath rot caused by at least one of Cladosporium, Trichophyton, Acanthopanax, Cladosporium oryzae, and Aspergillus flavus.

[0026] In some implementations, the plant individual can be a dicotyledonous plant, a monocotyledonous plant, or a gymnosperm, as well as algae, lichens, mosses, ferns, etc.

[0027] In some embodiments, the plant includes at least one plant from the following families: legumes (such as peanuts, cowpeas, soybeans, kidney beans, etc.), rose family (such as strawberries, pears, apples), vitiaceae family (such as grapes), araliaceae family (such as ginseng, notoginseng), grass family (such as rice, wheat, corn, bamboo), cruciferous family (such as rapeseed, cabbage), rutaceae family (such as citrus), cucurbitaceae family (such as cucumbers, watermelons, cantaloupes, gourds, pumpkins, etc.), solanaceae family (such as eggplants, potatoes, tomatoes), and malvaceae family (such as cotton).

[0028] A fifth aspect of the present invention provides a method for preventing and controlling plant diseases caused by fungal infection, comprising the step of contacting the polypeptide of the first aspect with the plant.

[0029] In some embodiments, the fungus includes at least one of Cladosporium, Trichophyton, Acanthopanax senticosus, Cladosporium oryzae, and Aspergillus flavus.

[0030] In some embodiments, the disease includes at least one of the following: fruit rot, flower blight, wilt, root rot, *Aspergillus oryzae* leaf blight, leaf spot, stem rot, and rice leaf sheath rot. Among these, *Cladosporium* can cause a variety of diseases, including fruit rot and flower blight; *Trichophyton* can cause a variety of diseases, including wilt and root rot; *Aspergillus niger* can cause a variety of diseases, including *Aspergillus oryzae* leaf blight, leaf spot, and stem rot; and *Cladosporium oryzae* can cause diseases, including rice leaf sheath rot. Therefore, the polypeptide provided in this application can specifically prevent or resolve diseases caused by the aforementioned fungi.

[0031] In some embodiments, the plant includes at least one plant from the following families: legumes (such as peanuts, cowpeas, soybeans, kidney beans, etc.), rose family (such as strawberries, pears, apples), vitiaceae family (such as grapes), araliaceae family (such as ginseng, notoginseng), grass family (such as rice, wheat, corn, bamboo), cruciferous family (such as rapeseed, cabbage), rutaceae family (such as citrus), cucurbitaceae family (such as cucumbers, watermelons, cantaloupes, gourds, pumpkins, etc.), solanaceae family (such as eggplants, potatoes, tomatoes), and malvaceae family (such as cotton).

[0032] In some embodiments, in order to further improve the activity, stability or reduce the toxicity of the peptide, the peptide may also be modified by at least one of the following C1) to C4): C1) introduction of hydrophobic amino acids; C2) D-type amino acid substitution; C3) C-terminal amidation modification; C4) N-terminal fatty acid modification. The above modifications can be performed using conventional methods in the art. For example, hydrophobic amino acid modifications can be introduced into the peptide by referring to "Yang Y, Wang C, Gao N, Lyu Y, Zhang L, Zhang S, Wang J and Shan A (2020) ANovel Dual-Targeted α-Helical Peptide With Potent Antifungal Activity Against Fluconazole-Resistant Candida albicans Clinical Isolates. Front. Microbiol.11:548620. doi: 10.3389 / fmicb.2020.548620"; D-type amino acid substitution modifications can be performed on the peptide by referring to patent CN106632600A or "Wang Jiayi. Study on the Influence of D-type Amino Acid Replacement and Lysine Side Chain Micro-modification on the Bioactivity and Stability of Antimicrobial Peptide polybia-CP [D]. Lanzhou. Lanzhou University, 2018". C-terminal amidation modifications can be performed on the peptide by referring to patent CN111518187A; and "Yang P, Mao W, et al. A novel antimicrobial peptide with broad-spectrum and exceptionalstability derived from the natural peptide Brevicidine. European Journal of Medicinal Chemistry. 2024;269:116337” The peptide can be modified with N-terminal fatty acids; or other methods in the art can be used for the above modification.

[0033] In some embodiments, the composition can be loaded using a delivery system, which may include hydrogels, nanocarriers, etc. For example, the composition can be loaded with reference to patent CN202511679812.8 or the literature “Campos JV, Pontes JTC, Canales CSC, et al. Advancing Nanotechnology: Targeting Biofilm-Forming Bacteria with Antimicrobial Peptides. BME Front. 2025;6:0104.Published 2025 Mar 4. doi:10.34133 / bmef.0104”.

[0034] In some embodiments, the composition can be loaded with engineered bacteria. For example, a corresponding commercial bacterial agent can be prepared by introducing a polypeptide sequence gene into engineered bacteria.

[0035] In some implementations, the prevention and control method includes at least one of the prevention method and the treatment method.

[0036] The fungi used in the above applications primarily use plants as hosts, and therefore do not involve treatment methods for diseases that directly target humans or living animals.

[0037] The present invention also relates to a method for inhibiting or killing fungi, comprising the step of contacting the polypeptide of the first aspect with the product to be treated.

[0038] In some embodiments, the fungus includes at least one of Cladosporium, Trichophyton, Acanthopanax senticosus, Cladosporium oryzae, and Aspergillus flavus.

[0039] In some embodiments, the product to be processed includes plants, such as individual plants or plant parts (e.g., roots, stems, leaves, flowers, fruits, seeds, etc.). In some embodiments, the plants include at least one plant from the following families: Leguminosae (e.g., peanuts, cowpeas, soybeans, kidney beans, etc.), Rosaceae (e.g., strawberries, pears, apples), Vitaceae (e.g., grapes), Araliaceae (e.g., ginseng, Panax notoginseng), Poaceae (e.g., rice, wheat, corn, bamboo), Brassicaceae (e.g., rapeseed, cabbage), Rutaceae (e.g., citrus), Cucurbitaceae (e.g., cucumbers, watermelons, melons, gourds, pumpkins, etc.), Solanaceae (e.g., eggplants, potatoes, tomatoes), and Malvaceae (e.g., cotton).

[0040] The present invention also provides a method for preparing a polypeptide of the first aspect, comprising expressing a nucleic acid molecule encoding the polypeptide in recombinant cells and collecting the culture product after culturing.

[0041] In some embodiments, the preparation method further includes separating and purifying the polypeptide from the culture product, and the specific separation and purification methods include one or more of the following: centrifugation, filtration, chromatography, chromatography, freeze drying, etc.

[0042] In some implementations, the nucleic acid molecule encoding the polypeptide is fused with a tag molecule for isolation and purification during expression, such as a His tag, a glutathione S-transferase (GST) tag, a maltose-binding protein (MBP) tag, an influenza virus hemagglutinin (HA) tag, a Flag tag, a Myc tag, etc.

[0043] Understandably, peptides can also be synthesized chemically, such as through solid-phase or liquid-phase chemical synthesis. Additionally, they can be synthesized using enzyme-catalyzed methods.

[0044] The beneficial effects of this invention are: This invention mines and identifies a novel polypeptide ARC026, also known as an antimicrobial peptide, from the metagenomics of polar microorganisms. This antimicrobial peptide has broad-spectrum and highly efficient antifungal activity, which can effectively inhibit and kill a variety of plant pathogenic fungi, effectively overcoming the limitations of existing technologies and providing a reliable candidate for the development of a new generation of plant disease biological control agents.

[0045] In addition, this antimicrobial peptide is derived from natural microorganisms and has an extremely short sequence, making it easy to degrade in the natural environment without forming harmful residues, which meets the requirements of green agriculture and sustainable development.

[0046] Moreover, unlike chemical drugs, antimicrobial peptides are less likely to induce genetic resistance in pathogens, which helps extend the lifespan of the pesticide and makes it less likely to induce resistance. This provides a new way to replace traditional chemical pesticides that are prone to causing resistance.

[0047] In addition, a novel polypeptide, ARC026, was successfully identified from polar metagenomics, breaking through the limitation that existing antimicrobial peptide resources are mostly concentrated in conventional habitats, and laying a resource foundation for the prevention and control of crop fungal diseases. Attached Figure Description

[0048] Figure 1 This is an HPLC purity detection chromatogram of the antimicrobial peptide ARC026 in an embodiment of the present invention. The horizontal axis of the figure represents the retention time (minutes), and the vertical axis represents the voltage (μV) of the original electrical signal reflecting the absorbance.

[0049] Figure 2 This is a mass spectrometry molecular weight identification diagram of the antimicrobial peptide ARC026 in an embodiment of the present invention.

[0050] Figure 3 This is an example of the colony growth of several test bacteria on plates after treatment with different concentrations of the antimicrobial peptide ARC026 in the embodiments of the present invention. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used herein is for the purpose of describing embodiments of this application only and is not intended to limit the application. Those skilled in the art will recognize that, with the evolution of technology and the emergence of new application scenarios, the technical solutions provided in the embodiments of this application are equally applicable to similar technical problems.

[0052] Example 1: Antimicrobial peptide mining 1. Data source: Metagenomic data obtained by sequencing and assembling sediment samples from different regions and environments in Antarctica from the CNSA database (accession number CNP0002256) of the National Gene Bank Life Big Data Platform.

[0053] 2. Gene cluster identification: Microbial genome data were preprocessed using EMBOSS software to predict small open reading frames (sORFs) and their length was controlled within the range of 5-50 amino acids to cover potential small peptides.

[0054] 3. Deep Learning Prediction: A multi-model collaborative antimicrobial peptide prediction system was constructed by integrating multiple machine learning and deep learning models, including Convolutional Neural Network (CNN), Long Short-Term Memory (LSTM) network, pre-trained Bidirectional Encoder Representation (BERT), and a hybrid model of CNN-BiLSTM-Attention (CNN-BiLSTM-Attention). sORFs (short-term Oriented Representations) with high confidence (prediction probability >99%) across multiple models were selected as candidate antimicrobial peptide sequences. This step is crucial in distinguishing it from traditional experimental screening; by using computational model ensemble prediction, the scope of experimental validation was significantly narrowed, improving the efficiency and accuracy of novel antimicrobial peptide discovery.

[0055] 4. Filtering: Subsequently, the candidate antimicrobial peptide sequences are compared with known antimicrobial peptide databases (such as APD3, dbAMP 3.0, dramp4.0, DBAASP, etc.) to remove sequences that are highly homologous to known sequences in the database, ensuring that the final obtained antimicrobial peptide is a novel one.

[0056] The final identified ARC026 (PLLKWKRPILRRLLK, SEQ ID NO.1) is a novel antimicrobial peptide sequence with high predictive activity selected through the above process.

[0057] Example 2: Synthesis and Quality Control of Antimicrobial Peptide ARC026 1. Synthesis: The antimicrobial peptide ARC026 was commercially synthesized by Shanghai Sangon Biotech Co., Ltd. using the solid-phase peptide synthesis method.

[0058] 2. Purity Identification: Analysis was performed using high-performance liquid chromatography (HPLC, column: SHIMADZU Inertsil ODS-SP (4.6 × 250 mm, 5 μm)). Conditions: Mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution, mobile phase B was 0.1% (v / v) trifluoroacetic acid acetonitrile solution, gradient elution (0%-44% B, 20 min), flow rate 1.0 mL / min, detection wavelength 214 nm, injection volume 30 µL.

[0059] The results are as follows Figure 1 As shown, the chromatographic peak of the antimicrobial peptide ARC026 is single, and its purity is greater than 98%.

[0060] 3. Molecular weight confirmation: Mass spectrometry (MS) analysis was performed, and the results are shown below. Figure 2 The observed molecular weight of the antimicrobial peptide ARC026 was 1931.25 Da, which is basically consistent with the theoretical molecular weight of 1930.481 Da. This indicates that the molecular weight of the peptide obtained by mass spectrometry analysis matches the expected theoretical value, verifying the accuracy of the peptide sequence.

[0061] Example 3: In vitro antibacterial activity assay of antimicrobial peptide ARC026 1. Strains and culture medium: Strains include Cladosporium (Cladosporium spp.) C. cladosporioides ), Tinea cysticercosis ( P. cucumerina ), black occulta ( E. nigrum ), rice broom mold ( S. oryzae ) and Aspergillus flavus ( A. flavus All bacterial strains were provided by Shenzhen BGI Genomics Co., Ltd. Potato dextrose agar (PDA) medium was used for strain activation and spore preparation, while cationic-regulated Mueller-Hinton broth (CaMHB) liquid medium was used uniformly for the antibacterial activity test to ensure the standardization and comparability of experimental conditions.

[0062] 2. Spore suspension preparation and concentration adjustment: The activated test strains were inoculated onto PDA agar plates and incubated at 37 ℃ until good sporulation occurred (incubation time is usually 5-10 days, depending on the specific strain). The spore surface was gently rinsed with sterile physiological saline, and spores were gently scraped off with a sterile cotton swab or inoculation loop and transferred to the appropriate CaMHB medium to prepare a homogeneous spore suspension. The spore suspension was then centrifuged to remove residual hyphal fragments. Spores were counted under a microscope using a hemocytometer, and the concentration of the spore suspension was adjusted to 5.5 × 10⁻⁶ using the appropriate CaMHB medium. 4 ~5.5×10 6 Within the CFU / mL range.

[0063] 3. Determination of minimum inhibitory concentration (MIC): To systematically evaluate antibacterial activity, the experiment was set up with the following three groups. All experimental groups were replicated three times to ensure data reliability and reproducibility.

[0064] (1) Negative control group: 200 μL of CaMHB medium was added to each well; (2) Positive control group: 100 μL CaMHB medium, 80 μL spore suspension and 20 μL sterile water were added to each well; (3) Experimental group: 100 μL CaMHB medium, 80 μL spore suspension and 20 μL of antimicrobial peptide ARC026 solution were added to each well. The final concentration range of antimicrobial peptide in the 96-well plate was 1024 μM to 1 μM.

[0065] After incubating the 96-well plate at 37 °C for 72 hours, remove it and gently tap or shake to mix the contents of each well. Against a black background, compare each experimental well with the positive control well (which should show obvious turbidity or visible hyphal clumps) and the negative control well (which should be clear and transparent). Record the lowest concentration of antimicrobial peptide corresponding to the well that completely inhibits fungal growth, i.e., the well is as clear as the negative control well with no visible growth, and determine it as the minimum inhibitory concentration (MIC) of the strain. The results are shown in Table 1.

[0066] 4. Minimum bactericidal concentration (MBC) determination: After completing the MIC detection, 10 μL samples were taken from the wells with 2×, 1×, and 0.5× MIC concentrations, diluted in 100 μL buffer, and then 20 μL of each sample was evenly spread onto the corresponding agar plates. Based on the growth characteristics of different strains, the plates were incubated at 37 ℃ according to the incubation times listed in Table 1. The areas on the plates without visible colony growth were observed (colony growth after sampling in each well is shown in Table 1). Figure 3The minimum antimicrobial peptide concentration (MBC) required to completely kill the strain was determined. All MBC assays were independently repeated three times. Results are shown below. Figure 3 See Table 1.

[0067] The antimicrobial peptide ARC026 exhibits the following antibacterial and bactericidal effects against these pathogens: [The text abruptly ends here, so the translation also ends here.] C. cladosporioides Both the MIC and MBC are 4 μM; P. cucumerina The MIC and MBC were 32 μM and 64 μM, respectively; E. nigrum The MIC and MBC were 128 μM and 256 μM, respectively; S. oryzae Both the MIC and MBC are 256 μM; A. flavus The MIC and MBC were 512 μM and 1024 μM, respectively. Therefore, Example 1 provides a broad-spectrum, highly effective, and environmentally friendly antimicrobial peptide for plant pathogens based on the unique polar habitat.

[0068] Table 1. MIC and MBC values ​​of ARC026 activity against various plant pathogenic fungi

[0069] Example 4: Modification of antimicrobial peptides 1. Drainage modification Aromatic hydrophobic amino acids (such as tryptophan and phenylalanine) were introduced into the amino acid sequence of ARC026 in Example 1 to enhance hydrophobicity, thereby improving its antibacterial activity (such as against Candida albicans) (refer to Yang Y, Wang C, Gao N, Lyu Y, Zhang L, Zhang S, Wang J and Shan A (2020) A Novel Dual-Targeted α-Helical Peptide With Potent Antifungal Activity Against Fluconazole-Resistant Candida albicans Clinical Isolates. Front. Microbiol. 11:548620. doi:10.3389 / fmicb.2020.548620; Liu Quan, Lü Yanxue. Identification and Development of Multifunctional Protein Biopesticide AMEP. Jilin Science and Technology Press, 2022.03, p.159).

[0070] 2. D-type amino acid substitution Replacing one or more L-amino acids with D-amino acids in the amino acid sequence of ARC026 in Example 1 can improve the stability of the peptide while retaining its original antibacterial activity (refer to CN106632600A; Wang Jiayi. Study on the effects of D-amino acid substitution and lysine side chain micro-modification on the bioactivity and stability of antimicrobial peptide polybia-CP [D]. Lanzhou: Lanzhou University, 2018; Liu Quan and Lü Yanxue. Identification and development of multifunctional protein biopesticide AMEP. Jilin Science and Technology Press, 2022.03, p. 159; Hu Guixue. Veterinary Microbiology. China Agricultural University Press, 2018.08, p. 80).

[0071] 3. C-terminal amidation modification The C-terminus of the amino acid sequence of ARC026 in Example 1 was modified by amidation to improve stability and antibacterial activity (refer to CN111518187A; Wang Deyuan et al., Introduction to Vegetable Biotechnology, China Agricultural Press, 2002.01, p.144; Guo Yuming, ed. Poultry Nutrition, China Agricultural University Press, 2016.04, p.498; Hu Guixue, Veterinary Microbiology, China Agricultural University Press, 2018.08, p.80).

[0072] 4. N-terminal fatty acid modification (lipopeptidation) In Example 1, a fatty acid chain (such as decanoic acid) was added to the N-terminus of the amino acid sequence of ARC026 to enhance the stability of the antimicrobial peptide (see Yang P, Mao W, et al. A novel antimicrobial peptide with broad-spectrum and exceptional stability derived from the natural peptide Brevicidine. European Journal of Medicinal Chemistry. 2024;269:116337.; Hu Guixue. Veterinary Microbiology. China Agricultural University Press, 2018.08, p.80; Wang Dong (ed.). Biomedical Nanofiber Materials. China Textile Press Co., Ltd., 2022.12, p.99).

[0073] 5. Adjustment of the number of positive charges In Example 1, the types and numbers of positively charged amino acids in the amino acid sequence of ARC026 were adjusted to increase the amount of positive charge on the antimicrobial peptide, thereby enhancing its stability and antimicrobial activity (see Yang P, Mao W, et al. Anovel antimicrobial peptide with broad-spectrum and exceptional stability derived from the natural peptide Brevicidine. European Journal of Medicinal Chemistry. 2024;269:116337.; Liu Quan, Lü Yanxue. Identification and Development of Multifunctional Protein Biopesticide AMEP. Jilin Science and Technology Press, 2022.03, p. 159).

[0074] Example 5: Antibacterial Composition This embodiment provides an antimicrobial composition comprising a variety of antimicrobial peptides, one of which is the antimicrobial peptide in Example 1.

[0075] Example 6: Antibacterial Composition This embodiment provides an antibacterial composition comprising the antimicrobial peptide from Example 1 and an antibiotic.

[0076] Example 7: Antibacterial agents This embodiment provides an antibacterial preparation comprising a hydrogel carrier and the antibacterial peptide of Example 1 loaded in the hydrogel carrier.

[0077] Example 8: Antibacterial engineered bacteria This embodiment provides an inoculum agent of engineered bacteria (such as yeast or Escherichia coli), wherein the engineered bacteria are infused with the nucleotide sequence corresponding to the antimicrobial peptide of Example 1.

[0078] As can be seen from the above embodiments, the polar-derived antimicrobial peptides provided in this application have considerable potential in agricultural applications.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide of polar origin, characterized in that, The amino acid sequence of the polypeptide is PLLKWKRPILRRLLK.

2. A biomaterial, characterized in that, The biomaterial includes any one of the following A1) to A4): A1) A nucleic acid molecule encoding the polypeptide of claim 1; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2); A4) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), or the recombinant vector described in A3).

3. The composition, characterized in that, Includes the polypeptide of claim 1, or the biomaterial of claim 2.

4. The composition according to claim 3, characterized in that, The composition is any one of antibacterial agents, bactericides, preservatives and mildew inhibitors, pesticides, and fertilizers.

5. The use of the polypeptide of claim 1, the biomaterial of claim 2, or the composition of any one of claims 3 to 4 in the preparation of a product that inhibits or kills fungi; wherein the fungi include at least one of Cladosporium, Trichophyton, Acanthopanax senticosus, Cladosporium oryzae, and Aspergillus flavus.

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