嗜碱芽孢杆菌NTT89启动子的克隆及其表达

报导了以启动子探测质粒pKK232-8为载体克隆嗜碱芽孢杆菌NTT89启动子的研究.用限制性内切酶Hindl分别消化嗜碱芽孢杆菌NTT89染色体DNA和质粒pKK232-8,在体外重组,转化大肠杆菌DH5a感受态细胞,在含氨苄青霉素和氯霉素的选择性平板上筛选转化子,结果从NTT89染色体DNA中克隆到一系列带有启动子活性的DNA片段,并在大肠杆菌中得到表达,这些转化子抗氯霉素水平在34～500mg/L不等,随机挑选10个转化子,提取其重组质粒进行限制性酶切,表明转化子中的重组质粒外源片段插入的效率很高,插入片段大小在500～5500bp之间,通过地高辛标记的点杂交和Southern杂交均证明重组质粒上插入的具启动子功能的DNA片段来自于嗜碱芽孢杆菌的染色体DNA.

Cloning and Expression of Promoter from An Alkalophilic Bacillus sp. NTT89 in Escherichia coli

Promoter function fragmants from Alkalophilic Bacillus sp.NTT89 were cloned in Escherichia coli using a promoter\|probe plasmid pKK232\|8.Forty\|tow clones were obtained,whose level of resistance to Cm ranged from 34 to 500 mg/L.Restriction enzyme analysis revealed that the size of inserted fragments ranged from 500\|5500 bp.By using Dot blotting and Southern blotting,we found that the inserted fragments were indeed from Alkalophilic Bacillus sp.NTT89.