畜牧与兽医2025，Vol.57Issue(8)：61-68,8.
单增李斯特菌谷胱甘肽过氧化物酶报告系统的构建及其调控因子筛选
Construction of glutathione peroxidase GSH-Px reporting system for Listeria monocytogenes and screening of regulatory factors
摘要
Abstract
The aim of this study was to establish a green fluorescent protein(GFP)reporter system for glutathione peroxidase(GSH-Px)of Listeria monocytogenes(LM),to screen the regulatory factors of GSH-Px,and to verify the effects of the regulatory factors on the growth capacity and antioxidant stress ability of LM.The GFP reporter plasmid pFL516 carrying the gsh-px promoter region was constructed using the overlapping extended PCR(SOE-PCR)method.The success of the construction of the reporter plasmid pFL516 was verified by measur-ing the expression of the fluorescent protein in the parental strain 10403S-pFL516.The regulatory factors of GSH-Px were screened by meas-uring the transcription levels of the parental strain 10403S-pFL516 and each of the strains lacking the dual regulatory systems.The influence of the absence of the regulatory factors obtained through growth curve detection and screening on the growth ability of Listeria monocytogenes was investigated.The results of the oxidative stress survival test were used to determine the impact of the absence of the regulatory factors ob-tained through screening on the antioxidant stress ability of Listeria monocytogenes.The test results showed that the plasmid pFL516 was suc-cessfully constructed and electrically transfected into 14 dual-regulation systems.Among them,the absence of 7 pairs of dual-regulation systems led to an increase in the transcription level of the gsh-px gene,while the absence of 6 pairs of dual-regulation systems resulted in a decrease in the transcription level of the gsh-px gene.After the absence of 1 pair of dual-regulation systems,there was no significant differ-ence in the transcription level of gsh-px.Further study on the dual regulatory system lmo1508/lmo1509 revealed that,in the absence of stress and metal ion stress conditions,the gfp transcription level of the lmo1508/lmo1509 deletion strain was significantly higher than that of the pa-rental strain 10403-pFL516,and the transcription level of the gsh-px gene was upregulated,indicating that lmo1508/lmo1509 negatively regulated the transcription level of gsh-px.The results of the growth capacity assay showed that lmo1508/lmo1509 gene did not affect the growth of the 10403S strain,and the results of the stress survival test showed that lmo1508/lmo1509 deletion significantly reduced the surviv-al rate of LM under oxidative stress,indicating that lmo1508/lmo1509 had a positive regulatory effect on the antioxidant stress ability of LM.The above results suggested that the reporter system of gsh-px was successfully constructed in LM,and the regulators of gsh-px,lmo1508/lmo1509,were screened out;which provided an important research tool for understanding the regulatory relationship between the dual regula-tory system and the antioxidant enzyme GSH-Px of LM.关键词
单增李斯特菌/谷胱甘肽过氧化物酶/报告系统/调控因子Key words
Listeria monocytogenes/glutathione peroxidase/reporter system/regulator分类
农业科技引用本文复制引用
张政,肖金华,张钰,杨雨婷,洪嘉俊,方春,韩旭..单增李斯特菌谷胱甘肽过氧化物酶报告系统的构建及其调控因子筛选[J].畜牧与兽医,2025,57(8):61-68,8.基金项目
江西省教育厅科学技术研究项目(GJJ2207208) （GJJ2207208）
湖北省教育厅科学技术研究项目(Q20221302) （Q20221302）
