Cloning of a sterol 14α-demethylase gene and the effects of over-expression of the gene on biological synthesis of triterpenes in Ganoderma lucidum

Ganoderma lucidum has been used for centuries to cure various human diseases in our country,and triterpenoids are the most important pharmacologically active constituents of the fungus.Sterol 14α-demethylase(CYP51) is one of the key enzymes involved in the biological synthesis processes of triterpenes.Degenerate primers were designed according to conservative sites of protein sequences from related species and a specific DNA fragment was obtained,then full length of Gl-cyp51 was obtained using traditional methods.Genomic DNA was 1,981bp and cDNA was 1,635bp.The ORF encoded a 544-amino acid polypeptide with a theoretical pI of 6.36 and a theoretical molecular mass of 61.99kDa.The Gl-cyp51 complete cDNA was ligated to the plasmid pGl-GPD.By successful Agrobacterium tumefaciens mediated transformation to G.lucidum,we realized Gl-cyp51 over expression transforments.We found that the transcript level of Gl-cyp51 was over expressed and triterpenes production was incrased.Further more,the transcript level of genes(Gl-aact,Gl-hmgr and Gl-ls) involved in the biosynthesis of triterpenes were also increased.