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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 is a traditional medicinal fungus in China. Triterpenoids are the main bioactive components of Ganoderma lucidum. Sterol 14α-demethylase is a key enzyme in triterpene synthesis pathway. Degenerate primers were designed according to the reported conserved amino acid sequence of sterol 14α-demethylase of other species, and the specific gene fragment of ghrelin steroid 14α-demethylase was obtained. The full length DNA of ghrelin sterol 14α-demethylase gene was further obtained And cDNA sequence. The DNA sequence was 1,981bp in length and the cDNA sequence was 1,635bp in length. Structural gene encoding protein contains 544 amino acids, a molecular weight of 61.99kDa, isoelectric point of 6.36. The cDNA sequence of sterol 14α-demethylase gene was cloned into Ganoderma Lucidum overexpression vector pGl-GPD, and the over-expression of sterol 14α-demethylase gene in Ganoderma lucidum was achieved by Agrobacterium-mediated transformation. Transformant sterol 14α-demethylase gene expression at the transcriptional level increased, triterpene content increased. Further study found that transcriptional expression of the key enzyme genes Gl-aact, Gl-hmgr and Gl-ls of triterpene synthesis pathway also increased.