同源重组法构建枯草芽孢杆菌224 yugS基因缺失突变株  

Construction of Bacillus Subtilis 224yugS Mutant by Homologous Recombination Method

作　　者：刘杰[1] 房春红[1] 李琬[1] 矫洪涛 喻江[1] 李景鹏[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《生物技术通报》2007年第4期148-151,共4页Biotechnology Bulletin

摘　　要：从枯草芽孢杆菌224的基因组DNA中分别扩增出溶血样基因yugS的上、下游片段,并利用携带新霉素抗性基因(neor)的重组质粒pMD18-T-neo作为骨架,构建了基于yugS基因位点的基因阻断质粒pMD18-T-neo-yugS,线性化后电转化入枯草杆菌224,从新霉素抗性平板上挑取转化子,通过对基因组的PCR鉴定和核苷酸测序证明,确定转化子yugS156为yugS基因缺失突变株。Upstream and downstream fragments of yugS gene which might be related to hemolysis were amplified by PCR using chromosomal DNA of Bacillus Subtilis 224 as a template,gene disruption plasmid pMD18-T-neo-yugS which can be integrated into yugS locus within Bacillus Subtilis 224 chromosome was constructed by using recombination plasmid pMD18-T-neo,which carried the neomycin resistance gene （neor）as a resistant marker,as a skelecton.Then BS224 was electro-transformated with linear pMD18-T-neo-yugS.The neor transformants was verified by genome PCR identification and nucleotide sequence evidence,finally transformant yugS156 was testified as a yugS gene imperfection strain.

关 键 词：枯草杆菌224 yugS基因 同源重组 

分 类 号：Q78[生物学—分子生物学]