环状芽胞杆菌基因启动子的分离与鉴定

ISOLATION AND CHARACTE4RIZATION OF GENE PROMOTERS FROM BACILLUS CIRCULANS

环状芽胞杆菌（Ｂａｃｉｌｌｕｓｃｉｒｃｕｌａｎｓ）总ＤＮＡ经Ｓａｕ３ＡＩ酶切后插入到启动子探针型载体ｐＳＵＰＶ１的ＢａｍＨＩ位点，转化大肠杆菌后，在卡那霉素的平板上筛选到５０个抗性菌落。从随机挑取的２９个抗住菌落所分离到的质粒ＤＮＡ经限制酶切和琼脂糖凝胶电泳后表明，各质粒均有ＤＮＡ插入片段，对２９个样品进行卡那霉素抗性试验显示，抗性最高的可超过１０００μｇ／ｍＬ这表明来自环状芽胞杆菌的某些基因启动子能在大肠杆菌中十分有效地启动基因表达，选取两个最大的克隆ＤＮＡ片段ＢＣ３和ＢＣ６作为探针与Ｂ．ｃｉｒｃｕｌａｎｓｃ－２．总ＤＮＡ作Ｓｏｕｔｈｅｒｎ杂交，均获得杂交带。斑点杂交结果表明，这两个ＤＮＡ片段来自不同的基因启动子。对ＢＣ６和ＢＣ３分别进行了限制酶谱分析，并绘制了限制酶图。

DNA fragments obtained from Sau 3AI partially digested total DNA of Bacillus circulans c- 2 were cloned into Bam HI site of pSUPV1, a promoter- probe vector.The recombinantDNA was transformed into Escherichia coli cells and 50 Kan clones were obtained,from which 29clones were randomly picked up for plasmid DNA isolation,The results from agarose gelelectrophoresis and kanamycin-resistant experiment showed that all plasmids contained insertedfragments,and that the highest kanamycin- resistant level was beyond 1000μg/mL, indicating thatsome gene promoters of B. circulans c-2 could initiate gene expression very efficiently in E. coli cells .Southern hybridization with total DNA of B.circulans c-2 using BC3 and BD6 as probes clearlyshowed hybrid DNA bands. Dot hybridiziation showed that BC3 and BC6 DNA fragments might befrom different genes. The restriction mapping of BC6, the largest inserted fragment, was finishedaccording to the analysis of agarose gel electrophoresis .