摘要
用大肠杆菌启动基因探针质粒pHE5克隆了解淀粉芽胞杆菌的启动基因。供体菌DNA的HindⅢ片段与pHE5重组后转化大肠杆菌,获得一批带启动基因的抗四环素转化子,其中四株的抗性达到200μg/mL,从这四株高抗性转化子中提取质粒,并对其中的一个重组质粒pAE23的插入片段进行限制性图谱分析和缺失研究,获得了一个缺失了部份片段,四环素抗性仍达到200μg/mL的衍生质粒pAED23,经酶切分析证明其上的启动基因位于0.8kb的EcoR Ⅰ—HindⅢ片段上。点杂交分析证实该片段来自解淀粉芽胞杆菌的DNA。将地衣杆菌的α-淀粉酶基因亚克隆至pAED23启动基因下游的HindⅢ位点上能增强该基因在大肠杆菌中的表达。
The B. amyloliquefaciens promoters were cloned in E.coli by using the promoter probe plasmid pH E5.156 tetracycline resistant transformanfs carrying promoters were obtained. 52% of them have tetracycline resistance over 100μg/mL.Plasmids were isolated from 4 tranaformants with high level of resistance (over 200μg/mL) , one of them (pAE23) was characterized. After deletion and physical mapping, the cloned promoter was localized on a 0.8kb EcoRI-HindⅢ fragment on the derived plasmid pAED23. Dot blotting analysis shows that the 0.8kb fragment was originated from B.amyloliquefaciens chromosome. The B. licheniformis amylase gene was removed from plasmid pJ07 and inserted downstream of the cloned promoter on pAED23, the derived plasmid pPA222 was introduced into E. coli. the gene under the control of cloned promoter was expressed at a level 1.7 times higher than in original plasmid pJ07.
