大肠杆菌诺氟沙星超敏感多基因敲除株KYAH06构建与功能验证  

Construction and functional verification of norfloxacin hypersensitive multi-gene knockout mutant Escherichia coli KYAH06

作　　者：姜巨全[1] 宋阳 Heba Abdel-Motaal 邹俏 JIANG Juquan;SONG Yang;HEBA Abdel-Motaal;ZOU Qiao(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2023年第2期49-55,86,共8页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(32070031,31770051)。

摘　　要：以E.coli DH5α为出发菌株采用pKD46质粒提供λRed重组系统完成以下三步基因敲除:大肠杆菌中RND超家族多药物抗性蛋白acrAB基因并借助其与pKD3质粒的氯霉素抗性片段同源重组被敲除;大肠杆菌中MATE家族多药物抗性蛋白ydhE基因通过卡那霉素抗性筛选和蔗糖反向筛选被无痕敲除;大肠杆菌中主要负责诺氟沙星外排的MFS超家族转运蛋白mdtH基因通过与pK18mobSacB质粒中的抗性片段的同源重组被敲除。以上基因敲除不同程度导致大肠杆菌对诺氟沙星的敏感性变化,最终获得诺氟沙星敏感性为0.0125μg·mL^(-1)的E.coli KYAH06,为大肠杆菌MdtH详细转运机制以及其他诺氟沙星外排转运蛋白鉴定和功能分析奠定试验基础。E.coli DH5αwas used as an original strain for the following three-step gene knockout experiments with the aid of theλRed recombination system of pKD46 plasmid.First,acrAB genes encoding RND(resistance-nodulation-cell division)family multidrug resistance transporters were knocked out through its homologous recombination with the chloramphenicol resistance fragment derived from the pKD3 plasmid.Secondly,E.coli ydhE gene encoding a MATE(multidrug and toxic compound extrusion)family multidrug resistance transporter was completely knocked out using a markerless deletion technique of kanamycin resistance screening combined with sucrose reverse screening.Finally,E.coli mdtH gene,one key determinant for norfloxacin resistance,encoding a major facilitator superfamily(MFS)transporter was knocked out through its homologous recombination with the kanamycin-resistance fragment in the pK18mobSacB plasmid.Knockout of the above-mentioned genes led to the change in norfloxacin sensitivity of E.coli,to different extent.The finally constructed norfloxacin hypersensitive mutant E.coli KYAH06 was 0.0125μg·mL^(-1)in norfloxacin sensitivity,which could provide an effective experimental basis for the detailed analysis of transporting mechanism of E.coli MdtH and the identification and functional analysis of other norfloxacin efflux transporters.

关 键 词：诺氟沙星 λRed重组系统 多药物逆向转运蛋白 AcrAB YdhE MdtH 

分 类 号：Q933[生物学—微生物学] Q784