Functinal Analysis Of The Promoter Of Penicillium Chrysogenum C-4 Methyl Oxidase Gene

Ergosterol is an important component of fungal cell membrane.The amount of ergosterol in cell membrane is considered an important factor affecting the secretion of penicillin in penicillin-producing fungus Penicillium chrysogenum. In previous studies in our laboratory, two C4-methyl sterol oxidase genes (Pcerg25A and Pcerg25B) involved in ergosterol biosynthesis had been first identification from Penicillium chrysogenum and anaylzed the 5’flanking region of Pcerg25B gene.To elucidate the regulation mechanism of the Pcerg25 genes, we also anaylzed the 5’ flanking region of Pcerg25A gene.Transformation vector containing 5’flanking region (1196bp) of Pcerg25A gene was constructed by using E. coliβ-galactosidase gene lacZ and transformed into Penicillium chrysogenum Wis54-1255. The result indicated that the fragment had the function of promoter. Deletion mutant analysis showed 5’flanking region (-1196bp to-725bp) in Pcerg25A gene contain some negative regulatory element as well as some positive regulatory elements in the sequence between-725bp to-196bp. The result was similar with 5’flanking region of Pcerg25B gene. But the promoter of Pcerg25A gene only have basic transcription capacity, the regulation ofβ-galactosidase activity was only one-sixth than the promoter of Pcerg25B. Bioinformatics analysis revealed that 5’flanking region of Pcerg25A gene lacked some cis-elements associated with the regulation of sterol, for example SRE,E-box and SRE-1 in 5’flanking region of Pcerg25B gene.We constructed the hybrid promoter containing the 5’flanking region of Pcerg2’5A gene and Pcerg25B gene and confirmed the sequence between-779bp to-725bp in 5’flanking region of Pcerg25B gene had positive function of regulation. Point mutation in the region and deletion mutation analysis further localized the enhanced cis-elements in the 38bp sequence.The study laid the foundation for clarifying regulatory mechanism of Pcerg25 gene.