杀鱼爱德华氏菌ETAE_1309基因的启动子研究  

A study on ETAE _ 1309 promoter in Edwardsiella piscicida

作　　者：刘鹿忆 何弯弯 朱泽亮 邓平 艾桃山 谢海侠[3] 朱文欢[4] 张立强 LIU Luyi;HE Wanwan;ZHU Zeliang;DENG Ping;AI Taoshan;XIE Haixia;ZHU Wenhuan;ZHANG Liqiang(Wuhan Academy of Agricultural Sciences,Wuhan 430207,China;Wuhan Chopper Fishery Bio-Tech Co.,Ltd.,Wuhan 430207,China;Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China;Wuhan Aquatic Technology Promotion Center,Wuhan 430000,China)

机构地区：[1]武汉市农业科学院,武汉430207 [2]武汉中博水产生物技术有限公司,武汉430207 [3]中国科学院水生生物研究所,武汉430072 [4]武汉市水产技术推广指导中心,武汉430000

出　　处：《淡水渔业》2024年第5期3-12,共10页Freshwater Fisheries

基　　金：武汉市农科院创新项目(QNCX202307);武汉市知识创新专项基础研究项目(2022020801010414)。

摘　　要：为研究杀鱼爱德华氏菌(Edwardsiella piscicida)ETAE_1309蛋白的表达调控机制,本实验利用序列截短方式和定点突变技术鉴定了ETAE_1309基因的启动子,并检测了不同培养条件下启动子的活性。结果显示:ETAE_1309上游-139~-1 bp具有启动子活性,ETAE_1309的核心启动子为-10元件TATACT和-35元件CTGGCG,该启动子属于σ38依赖型启动子。ETAE_1309转录水平受到RpoS_(Ep)(σ^(38))强烈的正调控,与毒力相关的调控蛋白EsrB、EsrC和与生理过程相关的Fur、CpxR、ETAE_2077对ETAE_1309转录水平影响不大。当杀鱼爱德华氏菌在DMEM培养基中生长时,随着培养时间延长ETAE_1309启动子活性逐渐增加,24 h(稳定期)和36 h(衰亡期)启动子活性分别为12 h(对数期)的2.4倍和2.9倍。当培养温度升高至35℃时ETAE_1309启动子活性显著下降,约为25℃时启动子活性的44.0%。本研究确证了杀鱼爱德华氏菌ETAE_1309的启动子序列及调控蛋白,随着培养时间延长该启动子活性逐渐增加,随着培养温度升高该启动子活性显著降低,研究结果为精准控制ETAE_1309蛋白表达及其介导的细菌毒力奠定了基础。The regulation of bacterial gene expression mainly occurs at the transcriptional level.A bacterial promoter acts as a core component during transcription and decides whether a gene is transcribed.The sequence truncation combined with site-specific mutagenesis was verified the promoter of ETAE_1309 to investigate the expression mechanism of ETAE_1309 in Edwardsiella piscicida.The result showed that the-139~-1 bp upstream of ETAE_1309 shared a promoter activity.The core promoter of ETAE_1309 was-10 element with a sequence TATACT,while a mutant-10 element with a sequence ACTTAT almost destroyed the promoter activity.The-10 mutant promoter activity was only 1.1%of the wild-type promoter.Another core promoter of ETAE_1309 was-35 element with a sequence CTGTCG,and the mutant promoter with TCGCTG as its-35 element showed promoter activity at a low level.The natural spacer length between-35 and^(-1)0 element of ETAE_1309 promoter was 17 bp,while promoters with shortened spacer(15-bp)or increased spacer(19-bp)retained 3.6%and 40.6%promoter activity,respectively.The sequence signature indicated that the promoter activity of ETAE_1309 was dependent onσ^(38),andβ-galactosidase assay showed that RpoS_(Ep)(σ^(38))positively regulates the transcription of ETAE_1309.Besides,the regulatory proteins relative to virulence and physiological processes played minor roles in the transcription of ETAE_1309.E.piscicida consistently activated the transcription of ETAE_1309 during in vitro culture which mimicked infection and the promoter activity was largely repressed by elevated temperature.In this study,we identified the promoter and transcriptional regulator of ETAE_1309,which laid the foundation for accurately regulating the expression of ETAE_1309 and its mediated bacterial virulence.

关 键 词：ETAE_1309 启动子 sigma因子 转录调控 杀鱼爱德华氏菌(Edwardsiella piscicida) 

分 类 号：S917.1[农业科学—水产科学]