深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定

Cloning and Identification of Gene Promoter for Gliotoxin Biosynthesis from Deep-Sea-Derived Fungus Dichotomomyces cejpii

利用染色体步移技术对深海真菌Dichotomomyces cejpii中胶霉毒素的生物合成基因Gli G、Gli I和Gli O启动子进行克隆,并将其核心区域导入p GL3-basic荧光素酶报告基因载体,通过荧光强度分析发现Gli G的启动子转录活性最强。进一步将Gli G启动子核心区域导入含潮霉素抗性标记的p AN7-1载体,以替换原有启动子pgpd A,并将重组质粒导入酿酒酵母,用潮霉素抗性平板进行筛选,发现Gli G启动子能在酿酒酵母中启动潮霉素抗性基因的表达。

The promoters of gliotoxins biosynthesis genes including Gli G,Gli I and Gli O from deep-sea-derived fungus Dichotomomyces cejpii were cloned by the method of genome walking,and the core regions of these promoters were inserted into the reporter gene vector ofluciferase p GL-3-basic,and the transcriptional activities of Gli G promoter was the highest according to its fluorescence intensity. Further,thecore region of Gli G promoter was further inserted into the p AN7-1 vector with hygromycin resistance marker to replace original pgpd A promoter,then the recombinant plasmid was transferred into Saccharomyces cerevisiae by electronic transformation. The positive clones were screened byplate with hygromycin resistance,and the results demonstrated that the Gli G promoter initiated the expression of hygromycin resistance gene.