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Aegilops tauschii antimicrobial peptide AtR100 and application thereof

An antibacterial peptide technology, which is applied in the biological field, can solve the problems of drug resistance of pathogenic bacteria, environmental pollution, frequent chemical pesticides, etc.

Inactive Publication Date: 2019-08-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the frequent use of chemical pesticides, the problem of pesticide residues is becoming more and more serious
Chemical pesticides can quickly prevent and control plant pathogens, but using too much chemical pesticides will lead to resistance of pathogens, pesticide residues and environmental pollution and other problems

Method used

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  • Aegilops tauschii antimicrobial peptide AtR100 and application thereof
  • Aegilops tauschii antimicrobial peptide AtR100 and application thereof
  • Aegilops tauschii antimicrobial peptide AtR100 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Acquisition of the antimicrobial peptide AtR100 of jiejiemai:

[0026] 1) Construct a high-quality cDNA library of S. subtilis, and initially screen for genes capable of autolyzing host cells (SCK6): take 1205 strains of the cDNA library of Bacillus subtilis and SCK6 strains stored at -70°C and thaw them for later use. Spot the samples on the LB plates containing kana, 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.

[0027] 2) Re-screening: For the effective strains of the initial screening, spotting treatment was carried out again on the LB plate containing kana, and each strain was repeated three times, and the growth on the plate was observed every 12 hours, and finally the strains with stable autolysis effect were determined.

[0028] 3) The plasmid with the best autolysis effect was extracted from the strain, and the nucleotide sequence of AtR100 with bacteriolysis...

Embodiment 2

[0033] The preparation method of antimicrobial peptide AtR100 comprises the following steps:

[0034] 1) Construction of fusion expression vector PBE-S-AtR100

[0035] The AtR100 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-AtR100.

[0036] 2) Expression, separation and purification of antimicrobial peptide AtR100

[0037] The correctly sequenced fusion expression vector PBE-S-AtR100 plasmid was transformed into Escherichia coli HST08 high-efficiency competent cells by the heat shock method for amplification, and the plasmid was extracted and then transferred into SCK6 competent cells to obtain the PBE-S-AtR100 fusion expression strain SCK6 / AtR100. Utilizing the secretory expression characteristics of the constitutive expression vector PBE-S, shake LB (containing kana) liquid medium for 72 hours to obtain SCK6 / AtR100 fermentation broth, centrifuge to obtain...

Embodiment 3

[0039] Antibacterial peptide AtR100 antibacterial effect:

[0040] With 1% inoculum, the bacterial canker of tomato, R. solanacearum, Bacterial spot of rice, B. subtilis of wheat, B. subtilis 168, B. subtilis 330-2 (Ahmad Z, Wu J, Chen L, Dong W.IsolatedBacillus subtilis strain 330-2 and its antagonistic genes identified by theremoving PCR. Scientific reports.2017; 7(1):1777.), Bacillus K1 (Liang Xiaohua, Yang Yingying, Li Zhuojia, Chen Yongqing, Hong Minna, Yang Keng. Bacillus Study on the characteristics of K1 degradation of nitrite [J]. Marine Environmental Science, 2008 (03): 228-230+235.), in a shaker at 37°C or 28°C at 170r / min, shake culture for 10h; after shake culture , take 300 μL of the indicator bacteria solution in a 10mL shaking tube, mix it with 4mL of 50℃ semi-solid beef extract medium, mix quickly, quickly pour it into a solid LB medium plate, spread the entire plate, and place it for 5min to make the medium Fully solidify and dry; divide the petri dish into ...

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Abstract

The invention belongs to the technical field of biotechnology and specifically discloses an aegilops tauschii antimicrobial peptide AtR100 and an application thereof. The inventor finally separates the aegilops tauschii antimicrobial peptide AtR100 with a sequence shown as SEQ ID NO.2, by establishing an aegilops tauschii cDNA library. The inventor finds that AtR100 has a strong inhibiting effectto the growth of tomato ulcer bacteria, ralstonia solanacearum, bacterial leaf streak of rice and clavibacter fangii which are difficult to be controlled by chemical drugs. A hemolytic test proves that the gene is harmless to mammals and humans. A powder acquired by performing liquid fermentation and spray drying on an antimicrobial peptide strain proves that the gene has a certain inhibiting effect to botrytis cinerea. The antimicrobial peptide strain is screened from aegilops tauschii. In later research, the antimicrobial peptide strain can be produced as a biological agent in large scale and can be applied to plant disease control.

Description

technical field [0001] This field belongs to the field of biotechnology, and in particular relates to an antimicrobial peptide AtR100 of barley wheat and its application. Background technique [0002] Pathogenic bacteria can cause serious economic losses to plants, and some pathogenic bacteria can even cause serious diseases including humans, livestock, crops and other organisms. 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 and canker are important diseases in the world (Liao Yongmei, Zhang Juncheng, Wang Zhongwen. The taxonomic status of plant pathogenic...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29A01N37/46A01P1/00A01P3/00
CPCA01N37/46C07K14/415
Inventor 傅婷婷孔熹吴佳董五辈
Owner HUAZHONG AGRI UNIV
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