| Turbot is an important aquaculture economic fish in China.It is favored by consumers due to the high protein content,low fat content and delicious taste.However,it is easy to be spoiled and deteriorated during storage,resulting in a significant reduction in food quality and economic value.Many studies have found that the growth and metabolism of microorganisms are the main cause for the spoilage of aquatic products,and Quorum-sensing(QS)system,as a way of information exchange between bacteria,can regulate the spoilage phenotype of microorganisms.Based on the interference of QS system,the spoilage characteristics of spoilage microorganisms in aquatic products were inhibited by Quorum sensing inhibitors(QSIs),which provides a new way for the preservation of aquatic products.However,the traditional method of searching for QSIs is random,time-consuming,and inefficient,and the inhibitory mechanisms of some reported QSIs are still unclear.Therefore,the QS regulatory genes luxI/R in A.sobria isolated from turbot were mainly studied in this paper.The potential QSIs were efficiently obtained by homologous modeling,virtual screening,and molecular docking method,and the inhibitory effect and mechanism of QSIs was further determined.This paper aims to investigate the QS mechanism of spoilage bacteria in aquatic products,and the inhibitory mechanism of methyl anthranilate,and provide the experimental evidence for the establishment of a new preservation technology for aquatic products based on the control of QS system.The main conclusions are as follows:1.The luxI/R genes regulated by QS in A.sobria were screened and verified by molecular biology method.Bioinformatics analysis of protein sequence encoded by the genes showed that the relative molecular mass of LuxI protein was 24585.16,theoretical pI was 6.80,the relative molecular mass of LuxR protein was 29371.72,and theoretical pI was5.51.Functional region analysis indicated that LuxI/R proteins had the characteristic structure of autoinducer synthetase and HTH structure,respectively.The results of secondary structures prediction suggested that two proteins had obvious conformational differences,and LuxR protein might have more complex advanced structures and functions.2.The effect of different environmental conditions on the expression of luxI/R genes in A.sobria isolated from turbot was investigated by RTq-PCR.The results showed that low temperature(15℃)and weak alkalinity(pH=8)conditions were beneficial to the expression of luxI/R genes,while high temperature(37 ℃)and strong acid and alkalinity(pH=5 or 9)inhibited the expression of two genes.In addition,with the raise of NaCl concentration,the expression levels of two genes showed a downward trend.3.The 3D model of LuxI/R receptor protein was obtained by homologous modeling,and evaluated using QMEAN scores and Ramachandran plot.With LuxI/R proteins of A.sobria as the receptor target,potential QSIs were screened from the traditional Chinese medicine database by virtual screening technology,and the QS inhibitory activity was verified.The results showed that 14 and 16 small molecules were screened for LuxI and LuxR protein,respectively.Analysis of anti-QS activity suggested that methyl anthranilate,vanillin,capsaicin and 1,10-decanediol had strong QS inhibitory activity.4.The inhibitory effect of methyl anthranilate,a low-cost,safe and efficient,on the QS phenomenon and spoilage activity in A.sobria was investigated by GC-MS,RTq-PCR and molecular docking.The results showed that methyl anthranilate significantly inhibited the QS regulatory phenotype in A.sobria,such as biofilm formation,bacterial motility and protease activity.RTq-PCR and GC-MS results indicated that the expression level of QS-related genes was down-regulated,and the level ofAHL biosynthesis was reduced when treated with different sub-minimal inhibitory concentration(sub-MIC)of methyl anthranilate.Molecular docking results suggested that methyl anthranilate might inhibit QS system in A.sobria by competitively binding with receptor protein. |
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