Gene of wheat antibacterial peptide and application thereof

Abstract

The invention relates to the field of gene engineering, in particular to a wheat antibacterial peptide and a gene and application thereof. The amino acid sequence of the wheat antibacterial peptide TaThiL1 is as shown in SEQ ID NO: 1. The wheat antibacterial peptide TaThiL1 is used for replacing chemical pesticides and preventing and treating plant fungal diseases, and the TaThiL1 protein has an inhibiting effect on the growth of fusarium graminearum and pseudofusarium graminearum.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a wheat antimicrobial peptide and a gene and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs) are a class of defensive bioactive peptides induced by the immune system when an organism encounters a foreign stimulus. They constitute one of the key parts of the organism's nonspecific natural defense system and are widely distributed in various organisms in nature and in the human body. As a polypeptide substance, antimicrobial peptides are harmless to plants and will not cause pollution or residue to the environment. Therefore, they are regarded as an alternative to pesticides, which helps to reduce the potential harm of pesticides.

[0003] The outbreak of fusarium head blight not only reduces wheat yields, but also causes the accumulation of harmful toxins in the grains. Wheat fusarium head blight is caused by the Fusarium graminearum species complex (FGSC), such as F. graminearum, F. avenaceum, F. culmorum, F. pseudograminearum and F. moniliforme, among which F. graminearum is the dominant pathogen causing wheat fusarium head blight.

[0004] Wheat has a strict immune response mechanism. When attacked by pathogenic fungi or pests, wheat can produce antimicrobial peptides. These peptides help reduce the harm of the disease and are part of nonspecific immunity.

[0005] The use of modern biotechnology to industrially produce targeted or specific antimicrobial peptide products is a key and efficient way to develop new antimicrobial peptide alternatives. This approach not only provides a more environmentally friendly solution, but also helps reduce dependence on traditional antibiotics and reduces the risk of the development of pathogen resistance.

[0006] In wheat seeds, partially sequenced peptides were divided into seven families based on sequence similarity and cysteine ​​motifs: defensins, thioproteins, lipid transfer proteins, rubber proteins, knot proteins, glycine-rich peptides, and MBP-1 homologs. α-thionine and β-thionine isolated from common wheat endosperm can inhibit the growth of fungi and bacteria, and their antibacterial function is specific. In addition, it has been found that overexpression of the antimicrobial peptide lipid transfer protein TaLTP5 in wheat can enhance wheat resistance to fusarium head blight and stem base rot; overexpression of the sweet protein TaTLP1 can enhance wheat resistance to stripe rust and stem base rot. TaArl6ip4 is an ADP-ribosylation factor-like protein 6-interacting protein. Studies have shown that this protein has an inhibitory effect on Fusarium graminearum and Alternaria alternata. Summary of the invention

[0007] The purpose of the 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 comprising the wheat antimicrobial peptide TaThiL1 gene.

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

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

[0012] The amino acid sequence of the wheat antimicrobial peptide TaThiL1 of the present invention is shown in SEQ ID NO: 1:

[0013] MASGGQTKMVAVGMLLVILFAAVAHAVLVPPVPPHHQTCIHTTGEVPAASDCI CSKNCACAGKCILAGGTGDHVKACFIDCVLKNDCRCNDNCFAHQPPVPIILAEPQ K,

[0014] The antimicrobial peptide is composed of 109 amino acid residues and has a molecular weight of 11.31 kDa.

[0015] The nucleotide sequence of the gene encoding the wheat antimicrobial peptide TaThiL1 of the present invention is shown in SEQ ID NO: 2:

[0016] 5'-ATGGCTTCCGGCGGCCAAACGAAGATGGTTGCCGTTGGCATGCTGCTGGTCA TCCTGTTCGCCGCGGTGGCTCACGCCGTCCTAGTGCCGCCGGTGCCGCCGCACCACCAAACCTGCATCCACACGACGGGCGAGGTTCCCGCCGCCAGTGACTGCATCTGCTCCAAGAACTGCGCATGCGCCGGAAAGTGCA TCTTAGCAGGCGGCACGGGCGACCATGTCAAGGCCTGCTTCATCGATTGTGTGCTCAAGAACGACTGCAGGTGCAACGATAACTGCTTCGCCCACCAGCCCCCGGTGCCCATCATCCTAGCAGAGCCCCAAAAGTGA-3'.

[0017] Since the gene contains a signal peptide region, the gene sequence of the mature region of the gene is shown in SEQ ID NO: 3:

[0018] 5'-GTCCTAGTGCCGCCGGTGCCGCCGCACCACCAAACCTGCATCCACACG ACGGGCGAGGTTCCCGCCGCCAGTGACTGCATCTGCTCCAAGAACTGCGCATGCGCCGGAAAGTGCATCTTAGCAGGCGGCACGGGCGACCATGTCAAGGCCTGCTTCATCGATTGTGTGCTCAAGAACGACTGCAGGTGCAACGATAACTGCTTCGCCCACCAGCCCCCGGTGCCCATCATCCTAGCAGAGCCCCAAAAGTGA-3'.

[0019] The present invention provides a recombinant vector comprising the above-mentioned wheat antimicrobial peptide TaThiL1, wherein the vector is preferably pGEX-6p-1, TaThiL1 is amplified using homologous arms containing BamHI and EcoRI restriction sites, and then inserted into the pGEX-6p-1 vector obtained by the same double restriction digestion, so that the nucleotide sequence is located downstream of the Tac promoter and is regulated thereby, and preferably the recombinant vector TaThiL1-pGEX-6p-1 is obtained.

[0020] The present invention also provides a recombinant strain comprising the above recombinant vector TaThiL1-pGEX-6p-1, and the expression host is preferably a BL21 (DE3) Chemically Competent Cell competent cell.

[0021] The present invention also provides a method for preparing wheat antimicrobial peptide TaThiL1, comprising the following steps:

[0022] Transform host cells with the above recombinant vector to obtain a recombinant strain;

[0023] Cultivating the recombinant strain to induce expression of the wheat antimicrobial peptide TaThiL1; and

[0024] Ultrasonic incubation is used for post-treatment to obtain the final product.

[0025] The present invention also provides the application of the wheat antimicrobial peptide TaThiL1, which is preferably used as a biological pesticide in the agricultural planting industry.

[0026] The present invention obtains the CDS sequence of the mature region of the TaThiL1 gene by amplifying it in the wheat Roblin material, constructs it on the pGEX-6p-1 vector, and prepares the wheat antimicrobial peptide TaThiL1 by low-temperature induced expression, thereby replacing chemical pesticides, preventing and controlling plant fungal diseases, and overcoming the adverse consequences of the abuse of chemical pesticides. The titer test results of the wheat antimicrobial peptide TaThiL1 show that the TaThiL1 protein has an inhibitory effect on the growth of Fusarium graminearum and Pseudomonas graminearum. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of SDS detection are shown, among which, 1: before induction of pGEX-6p-1; 2: after induction of pGEX-6p-1; 3: supernatant after ultrasonication of pGEX-6p-1; 4: pGEX-6p-1 protein; 5: before induction of TaThiL1-pGEX-6p-1; 6: after induction of TaThiL1-pGEX-6p-1; 7: supernatant after ultrasonication of TaThiL1-pGEX-6p-1; 8: TaThiL1-pGEX-6p-1 protein;

[0028] Figure 2 It was shown that TaThiL1 protein has an inhibitory effect on Fusarium graminearum;

[0029] Figure 3 It showed that after adding TaThiL1 protein for 12h and 24h, the growth of Fusarium graminearum was slowed down compared with the control;

[0030] Figure 4 It was shown that after adding TaThiL1 protein, conidia of Fusarium graminearum were damaged;

[0031] Figure 5 It was shown that TaThiL1 protein has an inhibitory effect on F. graminearum ;

[0032] Figure 6It showed that 12 hours after adding TaThiL1 protein, the growth of Fusarium graminearum slowed down compared with the control. DETAILED DESCRIPTION

[0033] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions; the reagents and biological materials described above can be obtained from commercial channels unless otherwise specified.

[0034] Experimental materials and reagents:

[0035] 1. Strains and vectors: (1) Fusarium graminearum strain PH-1; (2) Escherichia coli cloning competent DH5α; (3) Escherichia coli expression competent cells BL21 (DE3).

[0036] 2. Enzymes and kits: BeyoGold GST-tag Purification Resin, high-fidelity PCR enzyme Phanta Max Super-Fidelity DNA Polymerase, gel recovery kit FastPure Gel DNA Extraction Mini Kit, restriction endonucleases BamHI, EcoRI, etc., recombination cloning kit 2×Hieff Plus Multi One Step Cloning Kit, Plasmid Mini Kit I.

[0037] 3. Culture medium:

[0038] LB (Luria Bertani) medium: weigh 5 g of yeast powder, 10 g of peptone, and 10 g of NaCl, add double distilled water to 1 L. For solid culture medium, add 1.5% agar powder and sterilize at 121°C for 15 min.

[0039] NB (Nutrient Broth) medium: 10 g of peptone, 3 g of beef extract, 5 g of NaCl, add double distilled water to 1 L, and sterilize at 121°C for 15 min.

[0040] CMC (Carboxymethyl Cellulose) medium: KH2PO4 1 g, NH4NO3 1 g, yeast powder 1 g, MgSO4.7H2O 0.5 g and CMC 15 g, add ultrapure water to 1 L, sterilize at 121℃ for 15 min.

[0041] PBS (Phosphate Buffered Saline) buffer: NaCl 8g, KCl 0.2g, Na2HPO4.12H2O3.58g, KH2PO40.24g, add double distilled water to 1L, adjust pH to 7.3

[0042] Example 1. Cloning of TaThiL1 encoding gene and expression of recombinant protein

[0043] Using the cDNA of wheat Roblin material ears at flowering stage as a template, primers (F: 5'-TCCAGGGGCCCCTGGGATCCATGGTCCTAGTGCCGCCGGTGC-3', R: 5'-TCGAGTCGACCCGGGAATTCCTTTTGGGGCTCTGCTAGGATG-3') were designed, and appropriate restriction sites and homologous sequences at both ends of the linearized vector were introduced at the 5' end of the forward and reverse amplification primers, so that the 5' and 3' ends of the amplified insert fragments were respectively provided with homologous sequences (15-20 bp, excluding restriction sites) corresponding to both 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.

[0044] According to different vectors, appropriate restriction sites were selected, and the vectors were single-digested or double-digested with restriction endonucleases. The resulting digestion products were purified and recovered. 10 μL of the recovered products were separated and detected by 1.5% agarose gel electrophoresis to construct the ampicillin-resistant vector TaThiL1-pGEX-6p-1 and the empty control pGEX-6p-1.

[0045] According to the number of bases of the inserted fragment and the linearized vector, refer to the recombination cloning kit 2×Hieff The instructions for the Plus Multi One Step Cloning Kit configure 10 μL of the recombination reaction system and incubate at 50°C in a PCR instrument for 30 minutes. Add 10 μL of the recombinant product to 50 μL of DH5α competent cells for transformation. Design appropriate universal detection primers based on the vector, pick several clones on the recombination reaction transformation plate for colony PCR identification, pick positive colonies and inoculate them into liquid LB medium containing antibiotics, culture overnight, extract the plasmid and sequence to verify whether it contains the target gene. The gene sequence is shown in SEQ ID NO: 3. The plasmid with correct sequencing is used for further experiments.

[0046] The above bacterial solution was cultured in LB culture medium containing antibiotics at 37°C overnight for activation; the bacterial solution cultured overnight was taken at a ratio of 1:100 and inoculated into the LB culture medium containing antibiotics. The culture was cultured at 37°C for 4 hours until the OD of the bacterial solution reached 600When the concentration reaches 0.4-0.6 (the bacterial solution is turbid and opaque), collect 1 mL of the bacterial solution after shaking, centrifuge at 12000 rpm / 2 min, remove the supernatant, and use it for spotting samples before induction.

[0047] Add IPTG to a final concentration of 0.3 mM, maintain 16 °C and 200 rpm for 24 h to induce the expression of the target protein, take 1 mL of the induced bacterial solution, centrifuge and remove the supernatant for post-induced sample spot detection.

[0048] Collect the precipitate, pour out the bacterial solution as clean as possible, resuspend it with pre-cooled 1× PBS, and then perform ultrasonic treatment. Centrifuge the liquid after ultrasonic treatment. Take 20μL of supernatant and precipitate after centrifugation as supernatant after ultrasonic treatment and precipitate electrophoresis detection respectively to determine whether the protein will form inclusion bodies. Take the supernatant after ultrasonic treatment for subsequent experiments. Incubate the protein supernatant with magnetic beads overnight, collect the magnetic beads, and wash away the unbound proteins; add elution solution (10mM glutathione solution) and incubate with magnetic beads for 20min to fully elute. Collect 10μL of elution liquid each time for SDS-PAGE electrophoresis detection ( Figure 1 ).

[0049] Example 2 Determination of properties of wheat antimicrobial peptide TaThiL1

[0050] The inhibitory activity of wheat antimicrobial peptide TaThiL1 against Fusarium graminearum was determined.

[0051] Through experiments, different concentrations of antimicrobial peptides were mixed with Fusarium graminearum spores, and the OD values ​​of each bacterial solution were measured after culture. The smaller the OD value, the better the antibacterial effect. Freshly prepared Fusarium PH-1 conidia were taken, and the mycelium was filtered out with sterile gauze. The spore concentration was adjusted to 1×10 5 500 spores were added to a sterile 96-well cell culture plate, and 50 μL of the target protein solution diluted 2-fold was added at the same time. The culture was incubated at 28°C in the dark for 36 hours, and the OD value of the bacterial solution was measured using an ELISA reader. As the protein concentration increased, the OD value decreased ( Figure 2 ) Observing the growth status of Fusarium graminearum, it was found that the growth of Fusarium graminearum slowed down after applying TaThiL1 protein for 12h and 24h ( Figure 3 ), transmission electron microscopy was used to observe the conidia of Fusarium graminearum, and it was found that the conidia were damaged by the application of TaThiL1 protein ( Figure 4 ).

[0052] The inhibitory activity of wheat antimicrobial peptide TaThiL1 against Fusarium graminearum was determined.

[0053] Through experiments, different concentrations of antimicrobial peptides were mixed with spores of Pseudomonas graminearum, and the OD values ​​of each bacterial solution were measured after culture. The smaller the OD value, the better the antibacterial effect. Freshly prepared conidia of Pseudomonas graminearum were taken, and the mycelium was filtered out with sterile gauze. The spore concentration was adjusted to 1×10 5 500 spores were added to a sterile 96-well cell culture plate, and 50 μL of the target protein solution diluted 2-fold was added at the same time. The culture was incubated at 28°C in the dark for 36 hours, and the OD value of the bacterial solution was measured using an ELISA reader. As the protein concentration increased, the OD value decreased ( Figure 5 ) and observed the growth of Fusarium graminearum. It was found that the growth of Fusarium graminearum slowed down after applying TaThiL1 protein for 12 hours ( Figure 6 ).

[0054] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. Wheat antimicrobial peptide TaThiL1, characterized in that: The amino acid sequence of wheat antimicrobial peptide TaThiL1 is shown in SEQ ID NO:

1.

2. A wheat antimicrobial peptide gene, characterized in that: The wheat antimicrobial peptide gene encodes the wheat antimicrobial peptide TaThiL1 according to claim 1.

3. The wheat antimicrobial peptide gene according to claim 2, characterized in that: The nucleotide sequence of the wheat antimicrobial peptide gene is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

4. A recombinant expression vector comprising the wheat antimicrobial peptide gene according to claim 2.

5. A recombinant cell comprising the wheat antimicrobial peptide gene according to claim 2.

6. Use of the wheat antimicrobial peptide TaThiL1 according to claim 1.

7. The use according to claim 6, characterized in that: The wheat antimicrobial peptide TaThiL1 is used for inhibiting the growth of Fusarium graminearum or pseudo-Fusarium graminearum.

8. An antibacterial agent, characterized in that The active component of the antibacterial agent includes the wheat antibacterial peptide TaThiL1 according to claim 1.

9. A method for preparing wheat antimicrobial peptides, characterized in that: The method comprises the following steps: Transforming a host cell with the recombinant expression vector according to claim 4; Inducing the expression of the wheat antimicrobial peptide TaThiL1 whose amino acid sequence is shown in SEQ ID NO: 1; The wheat antimicrobial peptide TaThiL1 is isolated and purified.

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