摘要
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 .
出处
《四川大学学报(自然科学版)》
CAS
CSCD
1995年第2期207-212,共6页
Journal of Sichuan University(Natural Science Edition)
关键词
环状芽胞杆菌
基因启动子
DNA重组
Bacillus circulans ,promoter, kan gene,DNA recombination technique