《与灵芝三萜合成相关的细胞色素P450单加氧酶基因的克隆》-毕业论文（设计）.docVIP

PAGE 1 与灵芝三萜合成相关的细胞色素P450单加氧酶基因的克隆 摘要：灵芝(Ganoderma lucidum)是我国最著名和最具经济价值的药用真菌，灵芝三萜是灵芝的主要有效成分之一，具有抗肿瘤和免疫调节等多种功效，是新药研发的重要源泉。但是其单体难以通过化学分离或合成方法大量获得已成为灵芝三萜类药物研发的主要瓶颈，采用合成生物学思路在微生物中构建异源次生代谢途径是解决这一问题的有效途径。这一策略首先要解决的问题是灵芝三萜次生代谢途径中关键酶基因的获得及功能描述，细胞色素P450单加氧酶便是该途径中最为重要的关键酶之一。本实验根据由灵芝全基因组序列分析获得的细胞色素P450候选基因序列设计引物，以灵芝菌丝cDNA 为模板进行PCR 扩增反应，成功获得单一的扩增产物，且其大小与预期相一致。进一步通过T - A 克隆、大肠杆菌转化、菌落PCR 及测序等工作验证其序列正确性，最终挑选出一条与原始序列完全一致的单克隆，可用于后续表达及功能验证等相关研究。鉴于细胞色素P450 在灵芝三萜生物合成途径中发挥的巨大作用，本实验所获之克隆可以为灵芝三萜合成生物学研究提供重要的数据参考和借鉴。 关键词：灵芝三萜；细胞色素P450；基因克隆；合成生物学 Cloning of Cytochrome P450 HYPERLINK app:ds:monooxygenasemonooxygenase involved in the biosynthesis of triterpenoids from Ganoderma lucidum Abstract：Ganoderma lucidum is the most famous medicinal fungus with the most economic value in China. Triterpenoids are one of the major group of bioactive components found in G. lucidum. Modern pharmacological research has demonstrated that G. lucidum triterpenoids have multiple therapeutic actions, including antitumor and immunomodulatory activities, suggesting they are important source for RD of novel drugs. However, the difficulties in separation and chemical synthesis in large scale have greatly limited RD of G. lucidum triterpenoid-derived drugs, and one useful method to solve this problem is to apply the principle of synthetic biology to constructing heterologous secondary metabolic pathway in the microorganism. The primary problem of this strategy is obtain and characteristic describe of the key enzyme involved in the secondary metabolic pathway of G. lucidum triterpenoids, and cytochrome P450 HYPERLINK app:ds:monooxygenasemonooxygenase is one of the most important key enzyme of this pathway. In this experiment we design primer according to candidate P450 gene sequence obtained from the analysis of G. lucidum genome, and do PCR amplification use G. lucidum hypha cDNA as the template. As the result we successfully get a unique amplification product which has the same size as long as we expected. Furth