质粒pUC19-CM-D的构建及应用

Construction and Application of Plasmid pUC19-CM-D

[目的]用克隆载体pUC19构建乳杆菌自杀质粒及乳杆菌基因缺失工程菌[方法]在质粒pUC19的多克隆位点插入氯霉素抗性基因构建pUC19-CM载体;在pUC19-CM载体氯霉素抗性基因的两侧均添加1个用于同源重组的同源臂,再构建成自杀质粒pUC19-CM-D。将自杀质粒pUC19-CM-D转化乳杆菌进行抗性筛选即可得到目标基因被抗性基因替换的突变株。[结论]pUC19-CM-D质粒的构建及应用为乳杆菌基因缺失工程菌的构建提供了一个快速有效的手段,也为乳杆菌基因功能研究奠定了基础。

[Objective] The aim was to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector.[Method] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D.A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening.[Comclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.