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 Citrobacter freundii. The plasmid was digested by XcmⅠto 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-Km 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.