知母是重要的常用中药，含有50余种甾体皂苷，总含量达6%，预示它具有高效的生物合成体系。知母植株生物合成效率的不同，是知母药材品质差异的根源。目前，甾体皂苷的生物合成途径还知之甚少。Science 2013和Nature Plants 2016发现了番茄中甾体生物碱来源于胆固醇，与动物胆固醇合成途径完全不同。甾体皂苷与甾体生物碱结构相似，合成途径是否也相似？前期对知母根茎和叶进行转录组分析，克隆并测序了3个候选OSC基因、12个植物甾醇或胆固醇合成基因、2个糖苷酶基因，并进行qPCR分析，GC-MS检测到知母中存在胆固醇，推测知母甾体皂苷很可能来源于胆固醇途径。为了解析知母甾体皂苷的生物合成途径，本研究拟采用基因克隆，酵母和烟草外源表达验证关键基因的生化功能，解释知母呋甾烷型甾体皂苷向螺甾烷型甾体皂苷转化的机理，为知母药材的品质控制和甾体皂苷的生物合成提供依据。

Zhimu (Anemarrhena asphodeloides Bge.) is an important traditional Chinese herb, and riches more than 50 kinds of different steroidal saponis with the total content of 6%, that indicates it is a highly efficient biosynthetic system for steroidal saponins. The differences in the biosynthesis efficiency of Zhimu plants are the key cause of the differences in the quality of Zhimu material. At present, the biosynthetic pathway of steroidal saponins is still poorly understood. Two articles published in Science 2013 and Nature Plants 2016 revealed steroid alkaloids in tomato are derived from cholesterol, and discovered a new pathway for cholesterol biosynthesis in plant distinct from animals. Steroidal saponins are structurally similar with steroid alkaloids, whether has steroidal saponins in Zhimu the same metabolic pathway with steroid alkaloids in tomato. We have finished the transcriptome for the rhizome and leaves of Zhimu. Three OSC genes, 12 candidate Unigenes (including homologous genes) involoving the pathways for phytosterols or cholesterol and two glycosyltransferase genes were cloned and sequenced. GC-MS confirmed the existence of cholesterol in Zhimu. So we hypothesize steroidal saponis are also derived from cholesterol. Aim to confirm clear this hypothesis pathway of steroidal saponis in Zhimu, we will clone the candidate genes, construct the expression vectors to yeast and tobacco and assay the biochemical functions of these key genes. Finally, we will decipher the mechanism of the transformation of furostanol-type steroidal saponin to spirostane-type steroidal saponins. The project will contribute to the quality control and biosynthesis of steroidal saponins of Zhimu medicinal materials.