嗜热脂肪芽孢杆菌313-1启动基因在枯草杆菌中的克隆和表达

CLONING AND EXPRESSION OF Bac. stearothermophilus PROMOTER IN Bac. subtilis

以启动子探测质粒pPL603为载体,克隆了嗜热脂肪芽孢杆菌313-1染色体中有启动功能的DNA片段。经酶切、连接,转化枯草杆菌,获得了6株氯霉素抗性水平达100 μg/ml的转化子。将重组质粒pPL603-2的插入片段(1.2 kb)移到载体pPL703中,仍能表现出启动功能。经同源性分析表明,重组质粒的插入片段确实来自嗜热脂肪芽孢杆菌。EcoR I,Bgl II,Hind III等11种限制性内切酶对该片段均不能酶切。经Bal-31酶切,将此插入序列缩小到0.54 kb后仍具有启动功能。

A DNA fragment exhibiting a promoter function from B. stearothermophilus(the donor) was cloned by using the plasmid pPL603 as a promoter probing vector.Both vector and donor DNA were digested with the restriction endonuclease EcoRI, ligated and introduced into Bac. subtilis. Six transformants showed the chlora-mphenicol resistance at a high level (100μg/ml). When the insert(about 1.2 kb) ofthe recombinant plasmid pPL603-2 was sub-cloned into another plasmid pPL703, itretained the promoter function. It was concluded that the insert (1.2 kb fragment)cloned in pPL603 was a promoter-containing fragment. It was then isolated fromB. stearothermophilus, and identified by Southern blot hybridization analysis withnon-isotope labeling method. No recognition sites of 11 restriction endonucleasestested(EcoR I, Bgl II, Hind III, Pst I, Pvu II, Bcl I, BamH I, Xba I, Sma I, Xho I,Xma I)were ever found in the insert DNA. The size of the insert was further traceddown to 0.54 kb by the digestion of a nuclease Bal-31. The shortened fragment ret-ains the promoter function.