重楼中主要活性成分重楼甾体皂苷具有抗癌、清热解毒、止血止痛等药理作用，极具应用价值和市场前景。甾体皂苷在甾体母核环戊烷多氢菲基础上形成特征结构五元含氧环（呋喃E环），是由胆甾烷型结构转化为呋甾烷型甾体皂苷元的关键步骤，是螺甾烷型甾体皂苷的前体结构。该五元含氧环可能是在22-羟基胆甾醇基础上，在C-26和C-16位连续加羟基进而环化形成。本项目拟通过比较转录组数据分析、基因表达水平与次生代谢物含量检测等手段筛选并克隆参与呋喃E环形成的两个P450基因；利用VIGS体系分析目标基因体内生物学功能；在酵母中异源表达鉴定其体外催化活性；模块化途径工程策略将目标P450整合进入改造后的胆甾醇酵母工程菌，在酵母中重构胆甾醇下游P450介导的羟基化过程，准确解析产物结构。研究结果将获得该途径关键P450基因，揭示该环氧化过程，为阐释重楼皂苷生物合成通路奠定基础，为解决重楼资源问题提供思路。

Steroidal saponins are the main active ingredients in Paris polyphylla, which have the pharmacological effects of anti-cancer, heat-clearing and detoxification, hemostasis and analgesic, and have great application value and market prospect. The characteristic structure of steroidal saponins was formed on the basis of cyclopentane polyhydrophenanthrene as the steroid parent nucleus, and the quinary oxygen-containing ring (furan E-ring) was formed. Furan E-ring is the precursor structure of spirostane-type steroidal saponins, which is the key step of transformation from cholestane-type structure to furane-type steroidal saponins. The furan E-ring may be formed on the basis of 22-hydroxycholesterol by the continuous addition of hydroxyl groups at the C-26 and C-16 positions. In this project, two P450 genes involved in the formation of E-ring were screened and cloned by means of comparative transcriptome data analysis, detection of gene expression level and secondary metabolite content, and co-expression with the known key enzymes. VIGS transient silencing system was used to further analyze the biological functions of candidate genes. Heterologous protein expression in yeast was used to identify its catalytic function in vitro and identify the product structure. By modifying the cholesterol engineering yeast and providing sufficient substrates for the catalytic reaction through the modularized approach engineering strategy, the product structure can be accurately analyzed, which also lays a foundation for the research on the synthetic biology of steroidal saponins. The results of this study will obtain the key P450s of this pathway, reveal the cyclogenesis process, lay a foundation for elucidation of the biosynthesis pathway of steroidal saponins, and provide ideas for solving resource problems.