阅读说明：本技术 一种羊毛硫肽前体肽amyA6及其制备方法和应用 (Lanthionine precursor peptide amyA6, and preparation method and application thereof ) 是由 刘洪伟 张丽萍 赵雯雅 程辉彩 张飞燕 王雅娜 崔冠慧 段普凡 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种羊毛硫肽前体肽amyA6及其制备方法和应用,涉及生物技术领域。羊毛硫肽前体肽amyA6的氨基酸序列如SEQ ID NO:1所示。在解淀粉芽孢杆菌发酵过程中添加本发明羊毛硫肽前体肽amyA6,可以显著提高解淀粉芽孢杆菌代谢产物抗菌活性,抗菌活性持续时间是原来的4倍以上。(The invention discloses a lantibiotic peptide precursor peptide amyA6, a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the lantibiotic precursor peptide amyA6 is shown in SEQ ID NO 1. The lantibiotic peptide precursor amyA6 is added in the fermentation process of the bacillus amyloliquefaciens, so that the antibacterial activity of the metabolite of the bacillus amyloliquefaciens can be obviously improved, and the duration of the antibacterial activity is more than 4 times of the original antibacterial activity.)

1. A lantibiotic precursor peptide, amyA6, comprising: the amino acid sequence of the lantibiotic precursor peptide amyA6 is shown in SEQ ID NO: 1.

2. The lantibiotic peptide precursor amyA6 as claimed in claim 1, wherein the gene sequence of the lantibiotic peptide precursor amyA6 is shown in SEQ ID NO 2.

3. A process for the preparation of the lantibiotic precursor peptide amyA6 according to claim 1 or 2, wherein: the lantibiotic precursor peptide amyA6 was obtained by heterologous expression in E.coli or by artificial synthesis.

4. Use of the lantibiotic precursor peptide amyA6 according to claim 1 or 2, wherein: application of lantibiotic precursor peptide amyA6 in the process of improving the antibacterial activity of a bacillus amyloliquefaciens metabolite.

5. The use of the lantibiotic precursor peptide amyA6 according to claim 4, wherein: the addition period of the lantibiotic precursor peptide amyA6 was: the fermentation of the bacillus amyloliquefaciens starts to the end of the logarithmic phase of growth.

6. The use of the lantibiotic precursor peptide amyA6 according to claim 4, wherein: the addition amounts of the lantibiotic precursor peptide amyA6 were: 0.001-10mg/L of lanthionine precursor peptide amyA6 is added into the fermentation liquor of bacillus amyloliquefaciens.

Technical Field

The invention relates to the technical field of biology, in particular to a lantibiotic peptide precursor amyA6, a preparation method thereof and application thereof in the process of improving the antibacterial activity of a bacillus amyloliquefaciens metabolite.

Background

In agricultural production today, plant diseases are one of the major causes affecting crop yield. The traditional method of spreading chemical pesticides is mostly adopted at present to solve the problem of agricultural diseases. The method not only causes pollution to the environment, but also harms other animals, plants and human health. In recent years, researchers begin to adopt biological control methods to treat plant diseases, and biological pesticides have a series of advantages of high efficiency, environmental protection, no drug resistance and the like. Therefore, the search for new highly effective biological control means to solve plant diseases has been largely delayed.

The current research on biocontrol strains mainly includes biocontrol bacteria (bacillus, agrobacterium, pseudomonas) and biocontrol fungi (trichoderma). Wherein, the bacillus amyloliquefaciens of the bacillus has better inhibiting effect on various plant diseases and has broad-spectrum antibacterial activity. A large number of researches show that the antibacterial substance derived from the bacillus amyloliquefaciens has good control effect on fusarium dry rot, early blight, gray mold and other plant diseases.

At present, the drug resistance of germs is a big problem in the process of preventing and treating plant diseases or medical clinical treatment. The bacillus can generate various metabolites, the lantibiotic peptide serving as an antibacterial peptide has a dual action mechanism compared with the traditional antibiotics, can inhibit various bacteria and fungi (multidrug-resistant bacteria) and is not easy to generate drug resistance, and the lantibiotic peptide is expected to be applied to the aspects of agricultural production, clinical medicine and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing a lantibiotic peptide precursor amyA6 and a preparation method and application thereof, wherein the lantibiotic peptide precursor amyA6 is added in the fermentation process of bacillus amyloliquefaciens, so that the antibacterial activity of the metabolite of the bacillus amyloliquefaciens can be obviously improved, and the duration of the antibacterial activity is more than 4 times of the original antibacterial activity.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a lantibiotic precursor peptide, amyA6, having the amino acid sequence of amyA6 shown in SEQ ID NO 1.

The preparation method of the lantibiotic precursor peptide amyA6 comprises the following steps: the lantibiotic peptide precursor amyA6 is obtained by heterologous expression or artificial synthesis of the lantibiotic peptide precursor amyA6 gene with a specific gene sequence in Escherichia coli.

The above-mentioned lantibiotic precursor peptide amyA6 gene was derived from Bacillus amyloliquefaciens S499. Extracting genome of Bacillus amyloliquefaciens S499 as a template, taking a primer A6-S, A6-A as an upstream primer and a downstream primer (SEQ ID NO: 3; SEQ ID NO:4), and carrying out PCR amplification to obtain the lanthionine precursor peptide amyA6 gene fragment.

The gene sequence of the above-mentioned lantibiotic precursor peptide amyA6, amyA6, is shown in SEQ ID NO 2.

The heterologous expression method of the escherichia coli comprises the following steps:

(1) construction and transformation of the recombinant vector for the lantibiotic precursor peptide amyA 6: the lantibiotic precursor peptide amyA6 gene fragment is constructed on a PET-28a vector by a gene recombination method. The recombinant vector was transformed into E.coli BL21(DE3) by heat shock transformation.

(2) Induction of expression of the lantibiotic precursor peptide amyA 6: BL21 expression strain is cultured in liquid LB culture medium, heterologous expression of lanthionine precursor peptide amyA6 protein is induced by adding IPTG, the thalli is re-suspended by 8M urea solution, and then the thalli is broken by ultrasonic, and the lanthionine precursor peptide amyA6 is released. The supernatant was collected by centrifugation, dialyzed for three days using a dialysis bag with a pore size of 1000Da and finally freeze-dried to powder of the lantibiotic precursor peptide amyA 6.

The application of the lantibiotic precursor peptide amyA6 is as follows: application of lantibiotic precursor peptide amyA6 in the process of improving the antibacterial activity of a bacillus amyloliquefaciens metabolite.

Preferably, the lantibiotic precursor peptide amyA6 is added for the following period: the fermentation of the bacillus amyloliquefaciens starts to the end of the logarithmic phase of growth.

Preferably, the lantibiotic precursor peptide amyA6 is added in an amount of: 0.001-10mg/L of lanthionine precursor peptide amyA6 is added into the fermentation liquor of bacillus amyloliquefaciens.

Specifically, the application comprises the following steps: culturing (fermenting) Bacillus amyloliquefaciens in liquid NB culture medium for 12 hours to logarithmic phase, taking out the shaking table, adding 0.001-10mg/L lanthionine precursor peptide amyA6 into the fermentation liquid, continuing fermentation culture, centrifuging after 24 hours, and collecting supernatant.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:

firstly, the lantibiotic peptide precursor peptide amyA6 is added into the bacillus amyloliquefaciens fermentation liquor, so that the bacteriostatic activity of the lantibiotic peptide precursor peptide amyA6 can be obviously enhanced. The results show that the diameter of the inhibition zone of the experimental group is enlarged by more than 2 times compared with the control group, and the existence time of the inhibition zone can be prolonged by more than 4 times compared with the control group. Secondly, the method has low production cost, short production period, simple process and high yield. Finally, the method adopts biological control to treat plant diseases, and does not cause harm to human bodies, animals, plants and ecological environment. Therefore, the method has wide application prospect.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments.

Experimental materials and reagents required in the first and following examples

1. Strains and vectors:

bacillus amyloliquefaciens S499 was purchased from the bacterial collection center of the Council Committee for culture of microorganisms (BCCM/LMG) and designated LMG 29676. Coli BL21(DE3) was purchased from TransGen Biotech, Inc., Beijing Korea. pET-28a vectors were purchased from Tiannze Biotechnology Inc. (TIANDZ).

2. Enzymes and other biochemical reagents:

KOD-Plus polymerase was purchased from Shanghai Gentianbio Ltd (TOYOBO). The TIANGEN bacterial genome DNA extraction kit is a product of Tiangen Biochemical technology Co. The Axygen gel recovery kit is a product of American Seikagan company. The Vazyme kit is a product of Nanjing Novozam Biotech Co.

3. Unless otherwise indicated, the biochemical techniques used in the present invention are conventional in the art.

II, the formula of the culture medium required in the following examples:

LB culture medium: 10g of tryptone, 5g of NaCl and 5g of yeast extract, adding deionized water to reach the constant volume of 1000ml, and adjusting the pH value to 7.0-7.2.

NB medium: 10g of peptone, 5g of NaCl, 5g of beef extract and 10g of glucose, adding deionized water to reach the constant volume of 1000ml, and adjusting the pH value to 7.0-7.2.

PDA culture medium: 200g of potatoes and 20g of glucose, and adding deionized water to the volume of 1000 ml.

The culture medium is liquid culture medium, and 12g agar is added into corresponding solid culture medium.