知母甾体皂苷生物合成关键基因的克隆及生化功能研究

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.

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。

知母是重要的常用中药，含有50余种甾体皂苷，总含量达6%，母皂苷BII对于阿尔茨海默病以及改善学习记忆能力具有明显的效果。然而，其具体的生物合成途径及关键酶基因尚不清楚。知母甾体皂苷依靠植化分离不仅成本较高，而且受制于天然药用资源，提取物的质量难以控制。不同知母植株生物合成效率的不同，也是知母药材品质差异的根源。.本研究系统挖掘知母皂苷生物合成途径上的关键酶基因（10多条）， 包括甾体骨架环化的关键酶基因，2,3-氧化鲨烯环化酶（2,3-oxidosqualene cyclases，OSCs）、植物甾醇途径相关关键，甾醇侧链甲基转移酶1（Sterol methyl transferase 1, SMT1）、甾醇侧链还原酶2（Sterol side chain reductase enzyme 2, SSR2），环桉树醇环异构酶（cyclopropyl sterol isomerase，CPI）、7-胆固醇脱氢酶（7-Dehydrocholesterol Reductase ）、甾醇侧链氧化还原酶（sterol side chain CYP450s）、甾醇糖基转移酶（Sterol glycosyltransferase）和甾体皂苷26位葡萄糖苷酶（furostanol glycoside 26-O-β-glucosidase，F26G）等，对其进行基因克隆、异源表达和生化功能验证，初步阐明知母甾体皂苷生物合成途径；通过对知母不同OSCs催化机制的解析，为不同产地知母甾体皂苷合成效率，为知母道地性提供一定的理论依据；对知母F26G基因的功能研究，为知母耐旱、抗虫的生物特性提供实验证据支持，本项研究为植物甾体皂苷生物合成和知母道地性和优良品种选育奠定基础。

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