革兰阴性菌基因敲除载体的构建及其应用.docVIP

革兰阴性菌基因敲除载体的构建及其应用 王景1a 穆媛媛2a 黎庶1 陈志瑾1 熊坤 丛延广1 （1第三军医大学基础医学部微生物学教研室，重庆400038；2遵义医学院，遵义563003） 摘 要：目的 构建一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ori R6K；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。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。 关键词：敲除；载体构建 Construction and application of a vector for gene deletion in gram-negative bacteria WANG Jing*, MU Yuan-yuan*, LI Shu, CHEN Zhi-jin, XIONG Kun, CONG Yan-guang. * Department of Microbiology, Third Military Medical University. Chongqing 400038. * Zunyi Medical College. Zunyi 563003.) Corresponding author: CONG Yan-guang Email:ygcong@tmmu.edu.cn [Abstract] Objective To construct a vector plasmid which facilitate generating precise deletion in the genes of gram-negative bacteria. Methods The vector is based on the origin of replication the ( protein-dependent origin of plasmid R6K. It will not replicate in bacteria other than ( protein-producing bacteria and function as suicide vector. The vector carry the mob region from the broad-host-range plasmid RP4 and is thus mobilizable by conjugation into a wide range of Gram-negative bacteria. A multiple cloning sites is available on the vector. The vector contains a kanamycin resistance marker for orthoselection and contains a sacB gene for inverse selection. In the present study, the vector was used to carry out gene deletion experiment in the yeeZ gene of Citrobacter freundii. Results The vector plasmid was constructed successfully and named by pYG4. It did work in the gene deletion experiment. Conclusion The vector plasmid constructed in the present study is useful and convenient for generating gene deletion in gram-negative bacteria. [Key words] Gene deletion; Vector construction; 众多细菌的全基因组测序为深入认识这些细菌的生命活动规律奠定了基础，但到目前为止，对已测序基因的功能认识还很不足[1,2]。认识细菌基因功能的基本方法就是对细菌基因进行原位的突变分析，一般采用同源重组方式。对于酵母以及少数天然感受态细