| 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 |
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| 中文关键词:灵芝，Agrobacterium tumefaciens介导转化，超量表达，三萜合成 |
| 英文关键词:Ganoderma lucidum, Agrobacterium tumefaciens mediated transformation, over-expression, triterpenes biosynthesis |
| 基金项目:国家自然科学基金（No. 30970042 & 30871767） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
| 方星,师亮,徐颖洁,赵明文. 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[J]. 菌物学报, 2011, 30(2): 242-248 |
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