一种温敏复制缺陷T载体的构建及在鸡白痢沙门氏菌基因敲除中的应用

Construction of a temperature-sensitive and replication-defective T-vector and its application for gene knockout in Salmonella pullorum

摘要:

基因敲除是基因功能研究的重要手段，载体是基因敲除的工具和核心。为获得有效的基因敲除载体以快速构建基因突变株及鉴定相应基因的必需性，在已有温敏复制缺陷pIDM1质粒的基础上，于EcoRⅠ和PstⅠ位点间插入串联的XcmⅠ酶切位点接头，构建了pIDM-T质粒；该质粒经XcmⅠ酶切可获得末端突出T的线性化pIDM-T载体。在验证了pIDM-T质粒复制的温敏特性基础上，应用构建的T载体克隆鸡白痢沙门氏菌CVCC527菌株的eno和ybdr两个基因，鉴定获得pIDM-T_eno和pIDM-T_ybdr两个重组质粒；将重组质粒转化527菌株，经IPC (Integration rate per cell) 值计算，鉴定eno为必需基因，ybdr为非必需基因。挑取非必需ybdr基因527菌株重组菌(SalΔybdr)，经PCR和测序，确认突变菌株重组位点的正确性。pIDM-T载体可快速克隆PCR产物，用于沙门氏菌的基因敲除及必需性鉴定，为沙门氏菌基因功能研究提供了一种有效快速的手段。

Abstract:

Knockout is an important method for gene function study, while vector is the core of gene knockout. In order to obtain an effective vector for rapid construction of mutant and essentiality identification of the corresponding gene, we constructed a recombinant plasmid named pIDM-T based on the temperature-sensitive and replication- defective plasmid pIDM1 by inserting an Xcm I adapter into the EcoR I and Pst I sites of pIDM1. Digesting with Xcm I, pIDM-T can be prepared as a linear T-vector for PCR products cloning and be used to knockout the corresponding gene in the genome with insertion duplication mutagenesis. After the verification of temperature sensitivity of the replication of the plasmid, we cloned two Salmonella pullorum genes eno and ybdr into the constructed pIDM-T, and two recombinant plasmids pIDM-T_eno and pIDM-T_ybdr were identified. The recombinant plasmids were then transformed into S. pullorum strain CVCC527 and the IPC (Integration rate per cell) values were calculated. As a result, we identified the eno gene as an essential gene and the ybdr as a non-essential gene in CVCC527. We verified the correctness of recombination site in ybdr recombinant 527 clones (SalΔybdr) by PCR and sequencing. The pIDM-T vector can be used for gene knockout in S. pullorum, as well as the identification of essentiality of the corresponding genes, which offers an effective and rapid tool for gene function study in Salmonella.