A wheat antibacterial peptide gene and application thereof

Abstract

The present application relates to the field of genetic engineering, and in particular to a wheat antibacterial peptide, a gene thereof and application. The amino acid sequence of the wheat antibacterial peptide TaThiL1 is shown as SEQ ID NO:1. The wheat antibacterial peptide TaThiL1, instead of chemical pesticides, prevents and treats plant fungal diseases, and the TaThiL1 protein has an inhibitory effect on the growth of Fusarium graminearum and Pseudofusarium graminearum.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more specifically, to a wheat antimicrobial peptide, its gene, and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of defensive bioactive peptides induced by the immune system in response to foreign stimuli. They constitute a key part of the organism's nonspecific natural defense system and are widely distributed in various organisms in nature and in the human body. As polypeptides, antimicrobial peptides are harmless to plants and do not cause pollution or residues to the environment. Therefore, they are considered alternatives to pesticides, helping to reduce the potential harm of pesticides.

[0003] Fusarium head blight outbreaks not only reduce wheat yields but also lead to the accumulation of harmful toxins in the grains. Fusarium head blight is caused by the Fusarium complex (…). F. graminearum Caused by species complex (FGSC), such as Fusarium graminearum (… F. graminearum ), Fusarium oxysporum ( F. avenaceum ), Fusarium oxysporum ( F. culmorum ), Fusarium graminearum ( F. pseudograminearum ) and Fusarium moniliformes ( F. moniliforme Among them, Fusarium graminearum is the dominant pathogen causing wheat scab.

[0004] Wheat possesses a robust immune response mechanism. When attacked by external pathogenic fungi or pests, wheat can produce antimicrobial peptides. These peptides help reduce the harm of diseases and are part of non-specific immunity.

[0005] Utilizing modern biotechnology to industrially produce targeted or specific antimicrobial peptide products is a key and efficient approach to developing novel antimicrobial peptide alternatives. This method not only provides a more environmentally friendly solution but also helps reduce reliance on traditional antibiotics while mitigating the risk of antibiotic resistance development in pathogens.

[0006] In wheat seeds, based on sequence similarity and cysteine ​​motifs, partially sequenced peptides were classified into seven families: defensins, thioproteins, lipid transfer proteins, rubber proteins, desiccant-like proteins, glycine-rich peptides, and MBP-1 homologs. Alpha-thione and β-thione isolated from common wheat endosperm have been shown to inhibit fungal and bacterial growth, exhibiting specific antibacterial activity. Furthermore, overexpression of the antimicrobial peptide lipid transfer protein TaLTP5 in wheat has been found to enhance resistance to Fusarium head blight and stem rot; overexpression of the sweet protein TaTLP1 has enhanced resistance to stripe rust and stem rot. TaArl6ip4, an ADP-ribosylation factor-like protein-6 interacting protein, has been shown to have inhibitory effects against Fusarium graminearum and Alternaria alternata. Summary of the Invention

[0007] The purpose of this invention is to provide a wheat antimicrobial peptide, TaThiL1.

[0008] Another object of the present invention is to provide a gene encoding the wheat antimicrobial peptide TaThiL1.

[0009] Another object of the present invention is to provide a recombinant vector containing the above-mentioned wheat antimicrobial peptide TaThiL1 gene.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned wheat antimicrobial peptide TaThiL1.

[0011] Another object of the present invention is to provide the application of the above-mentioned wheat antimicrobial peptide TaThiL1.

[0012] The amino acid sequence of the wheat antimicrobial peptide TaThiL1 of this invention is shown in SEQ ID NO: 1: MASGGQTKMVAVGMLLVILFAAVAHAVLVPPVPPHHQTCIHTTGEVPAASDCICSKNCACAGKCILAGGTGDHVKACFIDCVLKNDCRCNDNCFAHQPPVPIILAEPQK, This antimicrobial peptide consists of 109 amino acid residues and has a molecular weight of 11.31 kDa.

[0013] The nucleotide sequence of the gene encoding the wheat antimicrobial peptide TaThiL1 of the present invention is shown in SEQ ID NO:2: 5'-ATGGCTTCCGGCGGCCAAACGAAGATGGTTGCCGTTGGCATGCTGCTGGTCATCCTGTTCGCCGCGGTGGCTCACGCCGTCCTAGTGCCGCCGGTGCCGCCGCACCACCAAACCTGCATCCACACGACGGGCGAGGTTCCCGCCGCCAGTGACTGCATCTGCTCC AAGAACTGCGCATGCGCCGGAAAGTGCATCTTAGCAGGCGGCACGGGCGACCATGTCAAGGCCTGCTTCATCGATTGTGTGCTCAAGAACGACTGCAGGTGCAACGATAACTGCTTCGCCCACCAGCCCCGGTGCCCATCATCCTAGCAGAGCCCCAAAAGTGA-3'.

[0014] Because this gene contains a signal peptide region, the gene sequence of the mature region of this gene is shown in SEQ ID NO:3: 5'-GTCCTAGTGCCGCCGGTGCCGCCGCACCACCAAACCTGCATCCACACGACGGGCGAGGTTCCCGCCGCCAGTGACTGCATCTGCTCCAAGAACTGCGCATGCGCCGGAAAGTGCATCTTAGCAGGC GGCACGGGCGACCATGTCAAGGCCTGCTTCATCGATTGTGTGCTCAAGAACGACTGCAGGTGCAACGATAACTGCTTCGCCCACCAGCCCCGGTGCCCATCATCCTAGCAGAGCCCCAAAAGTGA-3'.

[0015] This invention provides a recombinant vector containing the wheat antimicrobial peptide TaThiL1, preferably pGEX-6p-1. TaThiL1 is amplified using a homologous arm containing BamHI and EcoRI restriction sites, and then inserted into the pGEX-6p-1 vector obtained by the same double restriction enzyme digestion, so that the nucleotide sequence is located downstream of the Tac promoter and regulated by it, preferably obtaining the recombinant vector TaThiL1-pGEX-6p-1.

[0016] The present invention also provides a recombinant strain containing the above-mentioned recombinant vector TaThiL1-pGEX-6p-1, and the preferred expression host is BL21(DE3) Chemically Competent Cell.

[0017] This invention also provides a method for preparing the wheat antimicrobial peptide TaThiL1, comprising the following steps: The host cells were transformed using the recombinant vector described above to obtain recombinant bacterial strains; Culture recombinant strains to induce expression of the wheat antimicrobial peptide TaThiL1; and The final product is obtained by post-processing with ultrasonic incubation.

[0018] The present invention also provides the application of the above-mentioned wheat antimicrobial peptide TaThiL1, preferably its application as a biological pesticide in the agricultural planting industry.

[0019] This invention amplifies the mature region CDS sequence of the TaThiL1 gene in wheat Roblin material, constructs it into the pGEX-6p-1 vector, and prepares the wheat antimicrobial peptide TaThiL1 through low-temperature induction expression. This peptide can replace chemical pesticides to control plant fungal diseases and overcome the adverse consequences of chemical pesticide overuse. The potency test results of the wheat antimicrobial peptide TaThiL1 show that TaThiL1 protein has an inhibitory effect on the growth of *Fusarium graminearum* and *Fusarium pseudograminearum*. Attached Figure Description

[0020] Figure 1 The results of SDS assay are shown below: 1: pGEX-6p-1 before induction; 2: pGEX-6p-1 after induction; 3: pGEX-6p-1 supernatant after sonication; 4: pGEX-6p-1 protein; 5: TaThiL1-pGEX-6p-1 before induction; 6: TaThiL1-pGEX-6p-1 after induction; 7: TaThiL1-pGEX-6p-1 supernatant after sonication; 8: TaThiL1-pGEX-6p-1 protein. Figure 2 The TaThiL1 protein showed an inhibitory effect on Fusarium graminearum. Figure 3 The addition of TaThiL1 protein for 12 h and 24 h showed that Fusarium graminearum growth was slowed compared to the control. Figure 4 The addition of TaThiL1 protein showed that Fusarium graminearum conidia were damaged. Figure 5 The TaThiL1 protein showed an inhibitory effect on Fusarium graminearum. Figure 6 The addition of TaThiL1 protein for 12 h showed that the growth of Fusarium pseudograss slowed down compared to the control. Detailed Implementation

[0021] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions; the reagents and biological materials mentioned are commercially available unless otherwise specified.

[0022] Experimental materials and reagents: 1. Strains and vectors: (1) Fusarium graminearum strain PH-1; (2) Escherichia coli ( Escherichia coli (3) Escherichia coli expressing competent cells BL21 (DE3).

[0023] 2. Enzymes and kits: BeyoGoldGST-tag Purification Resin, PhantaMax Super-Fidelity DNA Polymerase (high-fidelity PCR enzyme), FastPure Gel DNA Extraction Mini Kit, restriction endonucleases BamHI, EcoRI, etc., recombinant cloning kit 2 × Hieff Clone® Plus MultiOne Step Cloning Kit, Plasmid Mini Kit I (plasmid extraction kit).

[0024] 3. Culture medium: LB (Luria Bertani) medium: Weigh 5 g yeast powder, 10 g peptone, and 10 g NaCl, add double-distilled water to 1 L. For solid medium, add 1.5% agar powder. Sterilize at 121℃ for 15 min.

[0025] NB (Nutrient Broth) medium: 10 g peptone, 3 g beef extract, 5 g NaCl, add double-distilled water to 1 L, sterilize at 121℃ for 15 min.

[0026] CMC (Carboxymethyl Cellulose) medium: 1 g KH2PO4, 1 g NH4NO3, 1 g yeast extract, 0.5 g MgSO4·7H2O and 15 g CMC, add ultrapure water to 1 L, sterilize at 121℃ for 15 min.

[0027] PBS (Phosphate Buffered Saline) buffer: 8g NaCl, 0.2g KCl, 3.58g Na₂HPO₄·12H₂O, 0.24g KH₂PO₄, plus double-distilled water to a final volume of 1 L. Adjust the pH to 7.3.

[0028] Example 1: Cloning of the TaThiL1 encoding gene and expression of recombinant protein

[0029] Using cDNA from wheat roblin spikelets at flowering stage as a template, primers were designed (F: 5'-TCCAGGGGCCCCTGGGATCCATGGTCCTAGTGCCGCCGGTGC-3', R: 5'-TCGAGTCGACCCGGGAATTCCTTTTGGGGCTCTGCTAGGATG-3'). Appropriate restriction enzyme sites and homologous sequences at both ends of the linearized vector were introduced into the 5' end of both the forward and reverse amplification primers, ensuring that the amplified insert fragments contained homologous sequences (15-20 bp, excluding restriction enzyme sites) at both the 5' and 3' ends, corresponding to the ends of the linearized cloning vector. The amplified insert fragments were purified and recovered, and 4 μL of the recovered product was separated and detected by 1.2% agarose gel electrophoresis.

[0030] Select appropriate restriction enzyme sites according to different vectors, and perform single or double restriction enzyme digestion on the vectors. Purify and recover the obtained digestion products, and separate and detect 10 μL of the recovered products by 1.5% agarose gel electrophoresis. The constructed ampicillin resistance vector TaThiL1-pGEX-6p-1 and the empty control pGEX-6p-1 were obtained.

[0031] Based on the number of bases in the inserted fragment and the linearized vector, a 10 µL recombination reaction system was prepared according to the instructions of the 2 × HieffClone® Plus Multi One Step Cloning Kit and incubated at 50 °C for 30 min in a PCR instrument. 10 µL of the recombination product was added to 50 µL of DH5α competent cells for transformation. Suitable universal detection primers were designed based on the vector. Several clones from the recombination reaction transformation plate were picked for colony PCR identification. Positive colonies were inoculated into liquid LB medium containing antibiotics and cultured overnight. Plasmids were extracted and sequenced to verify the presence of the target gene. The gene sequence is shown in SEQ ID NO:3. Plasmids with correct sequencing were used for further experiments.

[0032] The above bacterial culture was activated by incubating overnight at 37°C in LB broth containing antibiotics. The overnight culture was then inoculated into LB broth containing antibiotics at a ratio of 1:100. The culture was incubated at 37°C for 4 hours until the OD of the bacterial culture reached [value missing]. 600 The bacterial concentration should reach 0.4-0.6 (the bacterial solution should be turbid and opaque). Collect 1 mL of the bacterial solution after vigorous shaking, centrifuge at 12000 rpm / 2 min, discard the supernatant, and use it for sample spotting before induction.

[0033] IPTG was added to a final concentration of 0.3 mM, and the target protein was induced to express at 16℃ and 200 rpm for 24 h. 1 mL of the induced bacterial culture was taken, centrifuged to remove the supernatant, and used for sample spotting and detection after induction.

[0034] Collect the precipitate, discarding as much of the bacterial culture as possible. Resuspend in pre-cooled 1× PBS, then sonicate. Centrifuge the sonicated liquid, and collect 20 µL of the supernatant and precipitate for electrophoresis to determine whether the protein will form inclusion bodies. Use the sonicated supernatant for subsequent experiments. Incubate the protein supernatant with magnetic beads overnight, collect the beads, and wash away unbound protein. Add elution buffer (10 mM glutathione solution) and incubate with the magnetic beads for 20 min to elute thoroughly. Collect 10 µL of each eluent for SDS-PAGE electrophoresis. Figure 1 ).

[0035] Example 2: Determination of the properties of wheat antimicrobial peptide TaThiL1

[0036] The activity of wheat antimicrobial peptide TaThiL1 in inhibiting Fusarium graminearum was determined.

[0037] In the experiment, different concentrations of antimicrobial peptides were mixed with Fusarium graminearum spores, and the OD values ​​of each bacterial solution were measured after cultivation. The lower the OD value, the better the antibacterial effect. Freshly prepared Fusarium graminearum PH-1 conidia were taken, and after filtering out mycelia with sterile gauze, the spore concentration was adjusted to 1×10⁻⁶ using NB medium. 5 500 spores / mL were added to a sterile 96-well cell culture plate, along with 50 µL of a target protein solution diluted 2-fold. The plate was incubated at 28°C in the dark for 36 h, and the OD value of the bacterial culture was measured using a microplate reader. The OD value decreased with increasing protein concentration. Figure 2 Observation of Fusarium graminearum growth revealed that the growth of Fusarium graminearum slowed down after 12 h and 24 h of TaThiL1 protein application. Figure 3 Transmission electron microscopy revealed that the application of TaThiL1 protein damaged the conidia of Fusarium graminearum. Figure 4 ).

[0038] The activity of wheat antimicrobial peptide TaThiL1 in inhibiting Fusarium graminearum was determined.

[0039] In the experiment, different concentrations of antimicrobial peptides were mixed with Fusarium graminearum spores, and the OD values ​​of each bacterial solution were measured after cultivation. The lower the OD value, the better the antibacterial effect. Freshly prepared Fusarium graminearum conidia were taken, and after filtering out the mycelium with sterile gauze, the spore concentration was adjusted to 1×10⁻⁶ using NB medium. 5500 spores / mL were added to a sterile 96-well cell culture plate, along with 50 µL of a target protein solution diluted 2-fold. The plate was incubated at 28°C in the dark for 36 h, and the OD value of the bacterial culture was measured using a microplate reader. The OD value decreased with increasing protein concentration. Figure 5 Observation of the growth status of *Fusarium graminearum* revealed that the growth of *Fusarium graminearum* slowed down after 12 h of application of TaThiL1 protein. Figure 6 ).

[0040] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. The application of wheat antimicrobial peptide TaThiL1 in inhibiting the growth of Fusarium graminearum or Fusarium pseudograminearum, characterized in that the amino acid sequence of the wheat antimicrobial peptide TaThiL1 is as shown in SEQ ID NO:1.

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