银杏内酯生物合成途径相关P450基因的克隆与功能研究

Ginkgolides are diterpenes which have beneficial effects on cardiovascular systems and Alzheimer’s disease. However, the ginkgolides biosynthetic pathway is still unclear, which results in the impossibility of ginkgolides' production by the method of synthetic biology. Cytochrome P450 had already been indirectly verified that they were the key enzymes which had the oxidation functions in the ginkgolides biosynthetic pathway. Previous studies had shown that although the accumulation and storage of ginkgolides are in leaves of Ginkgo biloba, the biosynthesis of ginkgolides are carried out in roots. In this study, the high-expression in root or root-specific expression cytochrome P450 genes, which are responsive to methyl jasmonate (MeJA), will be screening by the transcriptome analysis and expression pattern analysis in roots and leaves of Ginkgo biloba. Furthermore, the function of the target gene will be verified by enzymatic activity assay and yeast engineering strain experiment in vivo. The project will clone and identify the first cytochrome P450 gene in ginkgolides biosynthetic pathway, which will be the important basis on industrial production of ginkgolides by the method of synthetic biology.

银杏内酯作为一种二萜类次生代谢物质是治疗心脑血管疾病和老年痴呆症的重要药物和保健品。然而，银杏内酯生物合成途径的研究至今仍然停滞不前，这给利用合成生物学方法工业化生产银杏内酯带来了极大的困难。细胞色素P450已被间接证明参与银杏内酯的生物合成，是该途径下游中间产物氧化反应的关键酶。前人研究表明，尽管银杏内酯在银杏叶片中积累和储存，然而其生物合成却是在银杏根部进行。本课题拟通过对银杏根部和叶片转录组以及甲基茉莉酸诱导前后银杏根部转录组的差异比较，筛选出受甲基茉莉酸诱导的银杏根部高表达或特异性表达的细胞色素P450基因。在此基础上，拟通过体外酶活实验和构建酿酒酵母工程菌进行真核体内实验相结合的方法验证目标基因的功能。该项目的启动有望克隆和鉴定银杏内酯生物合成途径中的第一个细胞色素P450基因，将为今后采用合成生物学的方法合成银杏内酯奠定重要基础，具有深远的理论意义和现实意义。

银杏内酯是银杏提取物的主要药效成分，但其生物合成途径还不清晰。我们采用UPLC-QQQ-MS定量分析了银杏幼苗不同组织部位中银杏黄酮和内酯的分布情况，结果表明银杏内酯是在银杏的须根及主根的根周皮中合成，通过根的皮层和茎的皮层来进行向上运输至叶片中。采用高通量转录组差异分析的方法，对茉莉酸甲酯诱导后银杏根部高表达或特异性表达的细胞色素P450基因进行筛选，得到了12个既受茉莉酸甲酯诱导又在根中高表达的CYP450类基因。采用模块化改造的方法提高酿酒酵母中银杏内酯前体物质的含量，将BTS1-ERG20融合基因和来源于嗜酸热硫化叶菌GGPPS基因整合入酿酒酵母染色体上，并将来源与银杏的GbLPS基因和来源于拟南芥的AtCPR1基因整合到酿酒酵母Gal80位点，完成了酿酒酵母菌株二萜前体的改造。经GC-MS检测，在酿酒酵母菌体中有明显的银杏内酯前体物质脱氢松香烷的合成。将筛选到的差异基因GbCYPs构建到过表达载体上，转化改造后的酿酒酵母工程菌，并进行目标基因的功能验证，以上研究为银杏内酯生物合成途径的解析奠定了良好的基础。