灵芝甾醇14α-脱甲基酶基因的克隆及超量表达对三萜合成的影响

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

灵芝Ganoderma lucidum是我国传统的药用真菌,三萜类物质是灵芝的主要生物活性成分,甾醇14α-脱甲基酶是三萜合成途径中的关键酶。根据已报道其他物种甾醇14α-脱甲基酶的氨基酸保守序列设计简并引物,获得灵芝甾醇14α-脱甲基酶特异基因片段,并进一步获得灵芝甾醇14α-脱甲基酶基因的全长DNA和cDNA序列。其中DNA序列长1,981bp,cDNA序列长1,635bp。结构基因编码蛋白包含544个氨基酸,分子量为61.99kDa,等电点为6.36。将甾醇14α-脱甲基酶基因的cDNA序列克隆到灵芝超量表达载体pGl-GPD中,利用农杆菌介导的转化法实现了甾醇14α-脱甲基酶基因在灵芝内的超量表达。转化子的甾醇14α-脱甲基酶基因在转录水平表达量增加,三萜含量增加。进一步研究发现,三萜合成途径的关键酶基因Gl-aact、Gl-hmgr及Gl-ls的转录表达量也有所增加。

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.