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Garlic antimicrobial peptide AR117 and its application

An antibacterial peptide, garlic technology, applied in the biological field, can solve problems such as environmental pollution

Inactive Publication Date: 2018-05-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because it is difficult to control soil-borne diseases with chemical drugs, and chemical control is easy to produce resistance and cause environmental pollution, it is of far-reaching significance to explore the resistance mechanism of plants to this disease source and discover new resistance genes

Method used

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  • Garlic antimicrobial peptide AR117 and its application
  • Garlic antimicrobial peptide AR117 and its application
  • Garlic antimicrobial peptide AR117 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Acquisition of garlic antimicrobial peptide AR117:

[0031] 1) Construct a high-quality garlic cDNA library, and preliminarily screen for genes capable of autolyzing host cells (WB800): take 1,700 strains of the Bacillus subtilis garlic cDNA library stored at -70°C and thaw the WB800 strain for later use, and store them in kana-containing Streak on the LB plate, observe the results every 12 hours, and record the numbers of the strains that were originally growing well but showed autolysis in the later stage.

[0032] 2) Re-screening: For the effective strains of the primary screening, streaking is carried out again on the LB plate containing kana, and each strain is repeated 3 times, and the growth on the plate is observed every 12 hours, and finally the strains with stable autolysis effect are determined, such as figure 1 As shown in middle A, the AR117 strain and the AS118 strain grew well and had the same phenotype when they were grown for 12 hours, while at 60 hours...

Embodiment 2

[0039] The preparation method of antimicrobial peptide AR117 comprises the following steps:

[0040] 1) Construction of fusion expression vector PBE-S-AR117

[0041] The AR117 gene was ligated with the linearized PBE-S vector containing Nde1 and Xba1 restriction sites with T4 ligase to obtain the target gene fusion expression vector PBE-S-AR117.

[0042] 2) Expression, isolation and purification of antimicrobial peptide AR117

[0043] The correctly sequenced fusion expression vector PBE-S-AR117 plasmid was transformed into Escherichia coli HST08 high-efficiency competent cells by heat shock method for amplification, and the plasmid was extracted and then transferred into WB800 competent cells to obtain the PBE-S-AR117 fusion expression strain WB800 / AR117. Utilizing the secretory expression characteristics of the constitutive expression vector PBE-S, shake culture LB (containing kana) liquid medium for 72 hours to obtain WB800 / AR117 fermentation broth, centrifuge to obtain th...

Embodiment 3

[0045] Antibacterial peptide AR117 antibacterial effect:

[0046] 1) Antibacterial peptide AR117 inhibits bacteria:

[0047] Bacterial canker of tomato, B. solanacearum, Bacillus anthracis, Bacillus cereus, Bacteria spp. of wheat, B. subtilis of potato ring rot, B. subtilis SCK6, B. subtilis 330-2 were shaken with 1% inoculation amount (Ahmad Z, Wu J, ChenL, Dong W. Isolated Bacillus subtilis strain 330-2 and its antagonistic genes identified by the removing PCR. Scientific reports. 2017; 7(1):1777.), B. subtilis168, respectively in 37 ℃ and 28 ℃ shaker Medium 170r / min, shaking culture for 8-10h; after shaking culture, take 300μL indicator bacteria liquid in a 10mL shaking tube, mix into 4mL 50℃ semi-solid beef extract medium, mix quickly, and quickly pour into solid LB medium In the flat plate, spread the whole dish and place it for 5 minutes to make the medium fully solidified and dry; divide the culture dish into four areas, place a sterile filter paper in the center of ea...

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Abstract

The invention belongs to the field of biotechnology and specifically discloses a garlic antimicrobial peptide AR117 and its application. The applicant builds a garlic cDNA library and finally isolatesthe garlic antimicrobial peptide AR117. The garlic antimicrobial peptide AR117 has a sequence shown in the formula of SEQ ID NO. 2. The applicant finds that the AR117 has a strong inhibitory effect on the growth of tomato canker, bacterial wilt, wheat seedlings and phytophthora capsici, which are difficult to be controlled by chemical drugs, can also inhibit bacillus cereus causing food poisoning, abdominal pain, vomiting and diarrhea and bacillus anthracis causing human anthracnose, and can effectively influence caenorhabditis elegans feeding. The hemolysis experiment result shows that the gene can inhibit nematodes and does not damage the mammals and human body. The antibacterial peptide gene is screened from garlic and can be used for biological control in the prevention and control ofplant diseases and insect pests in the later research, or can be used as an antibacterial and anti-nematode drug.

Description

technical field [0001] This field belongs to the field of biotechnology, and specifically relates to a garlic antimicrobial peptide AR117 and its application. Background technique [0002] Pathogenic bacteria can cause serious diseases including humans, livestock, crops and other organisms, causing serious economic losses. There are five types of pathogenic organisms that cause plant diseases: fungi, bacteria, viruses, nematodes and parasitic seed plants, among which plant diseases caused by bacteria are second only to those caused by fungi and viruses, ranking third. Among the more than 1600 kinds of bacteria identified so far, about 300 kinds cause plant bacterial diseases. It is known that there are more than 500 kinds of plant diseases caused by bacteria, among which bacterial wilt, soft rot and canker are important diseases worldwide (Liao Yongmei, Zhang Juncheng, Wang Zhongwen. The taxonomic status of plant pathogenic bacteria and their role in agricultural Applicati...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29A01N47/44A01P1/00A01P3/00A01P5/00
CPCA01N47/44C07K14/415
Inventor 孔熹杨梅吴佳董五辈
Owner HUAZHONG AGRI UNIV
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