河弧菌AL536_RS29525基因功能研究  

Functional study on gene AL536_RS29525 of Vibrio fluvialis

作　　者：张安冉 刘笑舒 黄元铭[1] 韩雨 刘平 李杰[1] 阚飙[1] 梁未丽[1] Zhang Anran;Liu Xiaoshu;Huang Yuanming;Han Yu;Liu Ping;Li Jie;Kan Biao;Liang Weili(State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《疾病监测》2021年第5期481-488,共8页Disease Surveillance

基　　金：国家自然科学基金(No.81772242);国家科技重大专项(No.2018ZX10713003-002-009)。

摘　　要：目的探讨河弧菌基因组中VflT6SS2核心基因簇上游paar基因簇中AL536_RS29525的序列特征、对VflT6SS2分泌功能和杀菌活性的影响以及可能的调控机制。方法利用Muscle、GeneDoc软件进行AL536_RS29525基因编码产物的序列特征分析;同源重组技术构建AL536_RS29525的缺失株,PCR扩增AL536_RS29525编码序列克隆于pSRKTc获得回补质粒pSR-29525,继而导入ΔAL536_RS29525得到回补株;Western Blot(WB)检测相应菌株中VflT6SS2 Hcp蛋白的表达和分泌,以大肠埃希菌为prey的细菌竞争/杀菌实验检测VflT6SS2介导的菌株杀菌能力;基于Lux报告基因的启动子融合系统冷光检测确定AL536_RS29525缺失对VflT6SS2核心基因簇启动子和hcp启动子活性的影响;荧光定量反转录聚合酶链式反应(qRT-PCR)检测hcp(tssD2)和vipA(tssB2)mRNA的相对表达水平。结果AL536_RS29525同源基因编码蛋白的结构和功能较为保守;缺失AL536_RS29525负性影响VflT6SS2 Hcp蛋白的表达及分泌,降低VflT6SS2介导的菌株杀菌能力,回补AL536_RS29525可恢复缺失株Hcp的表达、分泌和杀菌能力;缺失株ΔAL536_RS29525中hcp(tssD2)和vipA(tssB2)的mRNA表达量均低于野生株;而VflT6SS2的核心基因簇启动子和hcp启动子活性在ΔAL536_RS29525缺失株和野生株之间无显著差异。结论河弧菌AL536_RS29525基因较为保守,与霍乱弧菌以及弗尼斯弧菌的同源基因均具有较高的同源性。AL536_RS29525基因是河弧菌VflT6SS2的重要组成部分,为其Hcp效应子的正常表达、分泌功能以及VflT6SS2介导的杀菌活性所必需。AL536_RS29525可能转录后水平影响VflT6SS2的功能表达。Objective To investigate the sequence characteristics of gene AL536_RS29525 located upstream of VflT6SS2 major cluster and downstream of paar gene of Vibrio fluvialis,its influence on VflT6SS2 secretion function and bactericidal activity as well as possible regulatory mechanism.Methods Muscle and GeneDoc softwares were used to compare and analyze AL536_RS29525 and homologs.Deletion mutant of AL536_RS29525 was constructed using suicide plasmid based on the homologous recombination technology.Trans-complementation plasmid was constructed by cloning the coding sequence of AL536_RS29525 into plasmid pSRKTc,which was mobilized into the deletion mutant by conjugation.The expression and secretion of Hcp protein were analyzed by Western Blot.Standard bactericidal assay with E.coli as prey was used to measure the bactericidal ability of derivative strains.Luminescence activity of Lux based promoter fusion was used to test the promoter activities of VflT6SS2 major cluster and hcp orphan cluster inΔAL536_RS29525 mutant strains and wild strains.Quantitative reverse transcription PCR(qRT-PCR)was used to detect the hcp(tssD2)and vipA(tssB2)mRNA level.Results The genomic structure and sequence of AL536_RS29525 homologs were conserved in Vibrio species.The deletion of AL536_RS29525 greatly decreased the expression and secretion of Hcp,and also significantly reduced the bactericidal ability.These defects can be restored by introducing the trans-complementation plasmid PSR-29525 intoΔAL536_RS29525 mutant.The mRNA expression levels of hcp(tssD2)and vipA(tssB2)ofΔAL536_RS29525 were lower than those of wild strains.However,the promoter activities of VflT6SS2 major cluster and hcp orphan were not significantly different between the mutant strains and wild strains.Conclusion AL536_RS29525 is an important component required for the expression and secretion of VflT6SS2 with a critical role in bactericidal activity of V.fluvialis.

关 键 词：河弧菌 Ⅵ型分泌系统 AL536_RS29525 HCP 表达调控 

分 类 号：R211[医药卫生—中医学] R378.2