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N-acyl homoserine lactone acyltransferase coding gene aigA and application thereof

A technology based on acyl homoserine and lactone acyl, which can be used in applications, genetic engineering, plant genetic improvement, etc. It can solve problems such as microbial resistance, pesticide and antibiotic environmental safety, and human and animal health threats, and achieve good quenching activity , good degradation effect, broad-spectrum quenching effect

Active Publication Date: 2021-07-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The use of pesticides and antibiotics is currently the most common method for preventing and controlling pathogenic bacteria. However, the long-term abuse of pesticides and antibiotics has caused threats to environmental safety and human and animal health, and even caused microbial resistance

Method used

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  • N-acyl homoserine lactone acyltransferase coding gene aigA and application thereof
  • N-acyl homoserine lactone acyltransferase coding gene aigA and application thereof
  • N-acyl homoserine lactone acyltransferase coding gene aigA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Acquisition and identification of AHLs degradation genes

[0054] According to the whole genome sequencing results of Pseudomonas nitroreduction strain HS-18 in the previous work, the gene aigA, which may encode N-acyl homoserine lactone acyltransferase, was found by genome annotation and bioinformatics comparison. The software MEGA 5.10 was used to align the amino acid sequences of AigA and the currently known N-acylhomoserine lactone acyltransferases, and ClustalX1.8.3 and neighbor-joining method were used to conduct phylogenetic analysis and construct an evolutionary tree. The result is as figure 2 It was shown that AigA belongs to AHLs acylase of Penicillin G acylase family, and has the highest similarity (82.29%) with the amino acid sequence of QuiP in known AHLs acylase.

[0055] The primers were designed to amplify and construct the primers for aigA to be inserted into the broad host vector pBBR1 and the protein prokaryotic expression vector pET32a re...

Embodiment 2

[0060] Example 2 Detection of AHLs quenching activity by recombinant bacteria DH5α(aigA)

[0061] AHLs degradation system setting: the DH5α(aigA) successfully constructed overnight was cultured in LB liquid medium to obtain OD 600 = 1.0 seed solution, add an equal volume of fresh LB liquid medium and exogenous AHLs with different carbon chain lengths and substituents with a final concentration of 10-50 μM and MOPS with a final concentration of 50 mM to prepare a degradation system (different AHLs signal According to the different intensity of the reporter strain color development, the appropriate concentration was selected), cultured at 37 ° C, 200 rpm incubator for 36 h, with DH5α (pBBR1) bacterial liquid and LB liquid medium without bacterial liquid as the control. Next, the culture solution was extracted with an equal volume of ethyl acetate, and the content of AHLs remaining in 10 μl of the ethyl acetate extract was detected by the reporter strain.

[0062] Quantitative d...

Embodiment 3

[0065] Example 3 Prokaryotic expression of AigA protein

[0066] The successfully constructed recombinant bacteria BL21(DE3)(pET32a-aigA) was cultured overnight in LB liquid medium supplemented with a final concentration of 100 μg / ml ampicillin, and the seed solution was obtained by culturing at 37°C and 200rpm in a constant temperature shaker. Then, the seed solution was added to fresh LB liquid medium containing a final concentration of 100 μg / ml ampicillin at a ratio of 1:100, and cultivated to OD in a constant temperature shaker at 37 °C and 200 rpm. 600 = 0.6-0.8, add IPTG at a final concentration of 0.5 mM for induction, and culture overnight at 18°C ​​in a constant temperature shaker at 200 rpm. The cells cultured overnight were collected and disrupted, and the expression of AigA was identified by SDS-PAGE electrophoresis. according to image 3The results showed that AigA carrying His tag could be expressed in normal prokaryotic cells, and its size was about 112.27kDa...

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Abstract

The invention belongs to the technical field of molecular biological prevention and treatment, and finds that an N-acyl homoserine lactone acyltransferase coding gene is cloned through a molecular biological technology on the basis that pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the acyl chain length of C4-C14 in early-stage research. The N-acyl homoserine lactone acyltransferase coding gene has broad-spectrum quenching activity on AHLs, and has a good degradation effect on N-acyl homoserine lactones with different carbon chain lengths and modified with different substituent groups. The previous research results show that most of the N-acyl homoserine lactone acyltransferase coding genes have good quenching activity on long-chain AHLs. The N-acyl homoserine lactone acyltransferase coding gene lays an important foundation for enriching N-acyl homoserine lactone acyltransferase coding gene resources.

Description

technical field [0001] The present invention relates to the technical field of molecular biological control, and more particularly, to aigA encoding gene aigA of N-acyl homoserine lactone acyltransferase and its application. Background technique [0002] The use of pesticides and antibiotics is the most common method for the prevention and control of pathogenic bacteria. However, the long-term abuse of pesticides and antibiotics has caused threats to environmental safety and human and animal health, and even caused microbial resistance. Therefore, a new and environmentally friendly antimicrobial method needs to be developed urgently. [0003] Quorum sensing is a communication mechanism between cells. Cells synthesize and secrete a signal of a small molecule compound. Cells determine the number of populations by sensing the concentration of signal molecules diffused outside the cell. When the concentration of signal molecules reaches a certain threshold, cells will Activates...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/80C12N15/70C12N1/21A01N63/50A01P1/00C12R1/19
CPCC12N9/80C12N15/70C12Y305/01097A01N63/50
Inventor 张炼辉王惠杉廖立胜董玲玲
Owner SOUTH CHINA AGRI UNIV
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