Isolation of a ras gene from the basidiomycete Coriolus hirsutus and use of its promoter for the expression of Pleurotus ostreatus manganese(II) peroxidase cDNA in C. hirsutus

摘要

A ras gene homologue (named Ch.ras) was cloned from the basidiomycete Coriolus hirsutus. Ch.ras has a coding capacity of 215 amino acids (aa) interrupted by six small introns. The deduced Ch.Ras protein exhibited significant homology (86.5% or 86.0% identical) to the basidiomycete Coprinus cinereus Ras (215 aa) and Lentinula edodes Ras (217 aa) proteins. The 5-upstream region of Ch.ras contains two GC boxlike sequences, one TATA boxlike sequence, one CCAAT box, and three CT-rich sequences. Primer extension analysis showed the presence of three transcriptional initiation sites: one is located in the most upstream CT-sequence and the other two just after it. By using the 1.4-kb fragment containing the promoter elements and transcriptional initiation sites, we have constructed the chromosome-integrating vector pHRP, which is useful for the expression of foreign genes in C. hirsutus. The Pleurotus ostreatus manganese(II) peroxidase (MnP) cDNA (designated mnpc) was inserted into the downstream of the Ch.ras promoter elements of pHRP, yielding pHRP-mnp. We obtained, with pHRP-mnp, C. hirsutus strains that show high levels of enzymatic activity of MnP and efficiently degrade pentachlorophenol (PCP), a chlorinated aromatic toxic compound.