革兰阴性菌基因快速失活载体的构建及其应用

Construction and application of a vector system for rapid gene disruption in gram-negative bacteria

目的构建适用于革兰阴性菌的快速基因失活载体系统并进行初步应用。方法应用分子克隆技术构建快速基因失活载体系统。载体成分主要包括:π蛋白依赖性复制起点ori R6K;编码接合转移的mob片段;多克隆位点序列;含有2个XcmⅠ酶切位点的T载体形成片段以及多种耐药片段,包括Chl(氯霉素抗性)、Tet(四环素抗性)、Km(卡那霉素抗性),获得的载体分别命名为EK3-Chl、EK3-Tet、EK3-Km。采用构建的EK3-Tet载体对弗氏枸橼酸杆菌的yeeZ基因进行插入突变,以验证其有效性。通过XcmⅠ酶切EK3-Tet质粒形成T载体状态,将PCR扩增得到的两端截短的yeeZ基因与该T载体直接连接,获得的质粒采用接合方式转移到弗氏枸橼酸杆菌ATCC8090中以实现整合,双抗培养基筛选整合突变株,对获得的突变株进行鉴定和形态观察。结果成功构建了EK3-Chl、EK3-Tet和EK3-Km,并对yeeZ基因进行插入突变获得突变株,步骤简便,工作周期短。结论上述构建的载体系统能够对革兰阴性菌进行目的基因的快速插入失活,该载体可在细菌基因功能研究中得到一定的应用。

Objective To construct a set of vector plasmids which facilitates rapid gene disruption in gram-negative bacteria and to perform a primary application.Methods A rapid gene inactivation vector system was constructed by using molecular cloning technology.The vectors were based on the protein-dependent origin of plasmid R6K.And they carried the mob region which encoded conjugative transfer,multiple cloning sites,an XcmⅠ-containing vector for direct cloning of PCR products and 3 convenient antibiotic-resistance markers （chloromycetin, tetracycline and kanamycin resistance）. The vectors were named as EK3-Chl, EK-Tet and EK-Km according to the antibiotic-resistance markers they contained. In order to verify its effectiveness, the vector EK3-Tet was used to carry out gene disruption experiments in the yeeZ gene of Citrobacterfreundii, The plasmid was digested by Xcm I to form the state of T-vector. The yeeZ gene truncated at both ends was amplified by PCR and was then ligated to the T-vector. The plasmid obtained was integrated into the yeeZ gene of Citrobacter freundii with conjugation method. The integration mutants were screened using double-antibiotic medium and were then identified by PCR and morphological observation. Results Three plasmids EK3-Chl, EK- Tet and EK-Krn were successfully constructed. They did work in the yeeZ gene disruption experiments in a convenient way. Conclusion The vector plasmids constructed in the present study are useful and convenient in gene disruption experiments of gram-negative bacteria and may have a certain application in the study of bacterial gene function.