革兰阴性菌基因敲除载体的构建及其应用

Construction and application of a vector for gene deletion in gram-negative bacteria

目的构建1个适用于革兰阴性菌的精确基因敲除载体并证明其有效性。方法构建的载体主要包括以下成分:π蛋白依赖性复制起点oriR6K;接合转移基因mob,使载体可以通过简单的接合方式实现转移;多克隆位点序列:EcoRⅠ-XbaⅠ-ApaⅠ-SfiⅠ-SacⅠ-NotⅠ-SpeⅠ-NdeⅠ-SacⅡ-BglⅡ;反向选择标志SacB;正向选择标志卡那霉素抗性片段。载体构建完成后,采用该载体对弗氏枸橼酸杆菌的yeeZ基因进行框架内敲除,以验证其有效性。结果成功构建了敲除载体,命名为pYG4,并对yeeZ基因进行了框架内精确敲除获得突变株。结论本研究构建的载体适用于对革兰阴性菌进行目的基因的精确敲除,将在细菌基因功能研究中得到广泛的应用。

Objective To construct a vector plasmid which facilitates generating precise deletion in the genes of gram-negative bacteria. Methods The vector was designed to encode following fragments; the replication origin of theπ protein-dependent origin of plasmid R6K （ not replicate in bacteria other than π proteinproducing bacteria and function as ＂suicide vector＂ ）, the mob region from the broad-host-range plasmid RP4 （mobilizable by conjugation into a wide range of Gram-negative bacteria） , a multiple cloning sites （EcoRⅠ-XbaⅠ-ApaⅠ-SfiⅠ-SacⅠ-NotⅠ-SpeⅠ-NdeⅠ-SacⅡ-BglⅡ ）, a kanamycin resistance marker for orthoselection, and a sacB gene for inverse selection. After the construction, the vector was used to carry out a gene dele- tion experiment by deleting yeeZ gene from Citrohacter freundii. Results The designed vector plasmid was constructed successfully and named as pYG4. It did work in the gene deletion experiment. Conclusion Our constructed vector plasmid is useful and convenient for generating gene deletion in gram-negative bacteria.