催化人参三萜苷元生物合成的P450基因的克隆及功能鉴定

The aglycones of ginsenosides, which are thought to be more effective than ginsenosides themself, are the most direct precursors for ginsenoside biosynthesis. Based on their structure, it is widely accepted that the dammarane-type triterpenoid sapogenin are possibly synthesized under the cytochrome of P450s. However, there are few available genetic evidence to support this speculation. Still, the function, types, and relationships of P450s remain to be clarified, leading to difficulty in studies on plant P450s as well as on the dissection of ginsenoside biosynthesis. Aiming at solving above-described problemes, we plan to clone and identify P450 genes from subtractive cDNA library constructed by secondary suppression subtractive hybridization using Panax ginseng hairy roots. The cloned genes will be identified by RNAi and heterologous expression. Furthermore, the relationships among P450s, their diversity, and specificity will be studied. The identified P450 genes will be integrated into dammarenediol-producing engineered yeast to reconstruct the biosynthesis pawthway of ginesenoside algycones. This study will clarify the biosynthesis mechanism of the ginsenoside aglycones, lead in the plant P450 researches, and enhance the knowledge about P450 roles in plant secondary metabolism.

人参苷元是人参皂苷生物合成的最直接前体，比皂苷更具药理活性。根据结构式的推测人参达玛烷型三萜苷元可能是在P450催化下合成的，但缺少有关基因方面更直接的研究证据证明该种推测，且有关P450的数量、功能和P450间的关系等问题也不清楚，成为植物P450研究的薄弱环节和破解人参皂苷生物合成机理的主要障碍。本项目从解决上述问题的角度出发，以人参发根为材料，开展以人参为代表的催化三萜苷元生物合成的P450基因的克隆与功能鉴定。采用二次差减抑制杂交研究策略筛选和克隆P450目的基因；利用RNAi与异源表达相结合的手段鉴定克隆基因的功能，探讨P450间关系及多样性和专一性机制；最后将克隆的P450有序串联转化具有达玛烯二醇合成能力的工程酵母中，重建人参苷元生物合成途径，综合验证苷元生物合成机制。将破解人参三萜苷元生物合成机理，填补达玛烷三萜P450研究的空白，丰富P450在植物次生代谢方面的理论成果。

利用RT-PCR技术克隆了原人参二醇合成酶基因（PqD12H）。通过体外异源表达的方法对PqD12H基因的功能进行了鉴定分析，利用基因重组的方法构建重组表达载体pAUR123-D12H，转染酿酒酵母菌。在重组酿酒酵母菌中进行PqD12H基因的表达，通过向酵母培养基中加入达玛烯二醇作为底物，使其生成原人参二醇。进一步经RT-PCR 和HPLC 的分析后，确定PqD12H 的功能为催化达玛烯二醇合成原人参二醇的关键酶基因，并且在GeneBank 上发表，登陆号：JX569336。利用重组PCR方法构建了PqD12H的RNAi目标载体，通过测定RNAi的遗传转化发根系中原人参二醇和原人参三醇，进一步确定PqD12H为原人参二醇合成酶基因。