大肠杆菌巴豆甜菜碱还原酶基因缺失菌株的构建

Deletion of Crotonobetaine Reductase Gene in Escherica coli

报道了体外构建caiA基因缺失的带有卡那霉素抗性基因的5.2kb线状DNA分子,以此转化大肠杆菌JM83和BL21(DE3)株,借助于体内DNA同源重组,定向敲除了大肠杆菌中的巴豆甜菜碱还原酶编码基因caiA。经遗传稳定性实验、聚合酶链反应(PCR)以及Southern鉴定,表明所获得的JM83转化子22号和BL21(DE3)转化子4号确为caiA基因缺失突变株;酶活分析结果表明,22号和4号转化子均丧失了巴豆甜菜碱还原酶活性。

This work reports that the construction of a 5.2 kb DNA fragment in which the caiA gene was replaced by kanamycin resistant gene(Km^r), which was then transferred into JM83 and BL21(DE3). The No.4 and 22 strains from BL21(DE3) and JM83 respectively, were proved to be caiA-defective through the experiment of genetic stability, polymerase chain reaction(PCR) and southern blotting; and enzyme assays indicated that strains 4 and 22 have lost their crotonobetaine reductase activities.