重楼是名贵濒危中药材，年生物量极低，从种子成长为药用商品一般需10-15年。逐年的大量采挖，加之环境的破坏，重楼每年的消耗量远远超出了野生资源的生长量，致使重楼资源面临枯竭。甾体皂苷是重楼主要有效成分，难以通过化学分离或化学合成大量获取，通过生物途径合成重楼甾体皂苷具有重要意义。本项目首先对滇重楼采收阶段不同部位的甾体皂苷含量进行测定，并对不同组织部位进行RNA-Seq测序。将重楼甾体皂苷生物合成途径中的关键基因表达量与甾体皂苷含量变化进行关联分析，筛选表达模式与重楼甾体皂苷含量变化一致的同源共表达基因，并使用Real-time PCR技术对基因表达量进行验证。本项目为下一步进行重楼甾体皂苷生物合成研究奠定良好的基础。

Paris polyphylla as valuable scarce medicines, steroidal saponins, the bioactive components, are neither to be largely isolated from Paris polyphylla due to low level nor chemically synthesized. The annual biomass is very low, the resource depletion, in order to increase the content of steroidal saponins by means of biotechnology to solve the Paris polyphylla of resource depletion and used in raw materials have the importance of contraceptives. Their biosynthesis pathway is unclear. This study will be detected in variation of expression levels of those enzymes in the different tissues (root, leaves, stem and bulbs) and at harvesting Stage by RNA-seq technology, as well as in the relationship between genes expression level of the selected genes and contents of steroidal saponins by fingerprinting. The genes that co-express the steroidal saponins syntheses will be selected by comparing the transcirptome data of Paris polyphylla Smith var. yunnanensis (Franch.) Hand-Mazz. They will be cloned and expressed in Saccharomyces cerevisiae, and then the enzymes will be characterized through detecting the enzymatic products. Accuracy of gene expression quantity revealed by RNA-seq will be checked by Real-time PCR method. Our project will predict the molecular mechanism of the biosynthetic pathway of steroidal saponins in Paris polyphylla and help to increase the content of steroidal saponins by biotechnology.

重楼是名贵濒危中药材，年生物量极低，从种子成长为药用商品一般需10-15年。逐年的大量采挖，加之环境的破坏，重楼每年的消耗量远远超出了野生资源的生长量，致使重楼资源 面临枯竭。甾体皂苷是重楼主要有效成分，难以通过化学分离或化学合成大量获取，通过生物途径合成重楼甾体皂苷具有重要意义。本项目在重楼的收获季节采集根、茎、叶、果等不同组织部位，设置3个生物学重复。首先依照中国药典含量测定方法测定重楼皂苷I、重楼皂苷II、重楼皂苷VI和重楼皂苷VII在不同组织部分的含量差异，结果表明：4种重楼皂苷在根中均有分布，以重楼皂苷I为主；4种重楼皂苷在茎中的含量极低；叶和果中主要为重楼皂苷VII。然后提取同组织部位的RNA，对不同组织进行RNA-Seq测序分析，共获得88.29Gb Clean Data，组织拼接得到71541条Unigene，Unigene的N50为1588，共有36265条Unigene获得注释，共获得15608 个SSR标记。根与叶相比，共有2746个差异表达基因，其中1007个基因表达下调，1739个基因上调。根与果相比，共有2046个差异表达基因，其中816个基因表达下调，1230个基因表达上调。根与茎相比，共有1550个差异表达基因，610个基因表达下调，940个基因表达上调。本项目为下一步进行重楼甾体皂苷生物合成研究奠定 良好的基础。