外源激素诱导鹅掌草皂苷积累机制与关键调控基因挖掘的代谢组学与转录组学整合分析研究

Anemone flaccida is a commonly used medicinal plant, with saponins as its principal effective antitumor component. The monomer saponins have different biological activities according to the link way of sugar chain. However, this medical plant is lack of study on the level of systems biology. Additionally, the molecular mechanism of effective component biosynthesis is still poorly understood, which severely impedes the study on metabolic regulation. We have carried out the transcriptome sequencing, monomer saponins bioactivity testing of A. flaccida, and developed a LC-MS method for the component analyzing of saponins. This project was designed to characterize the accumulation mechanism.of anemone flaccida saponins induced by exogenous hormone using Illumina high throughput RNA sequencing technology in association with metabonomic methods. The profiles of digital gene expression and metabolites will be established to screen the key gene for effective component biosynthesis. Finally, RACE, bacterial expression system, and qRT-PCR will be employed to characterize whole gene sequence, gene function and expression profile,respectively. The results will provide provide a scientific basis for the elucidating of effective component.biosynthesis and regulation. Furthermore, this project will facilitate the metabolic engineering and directed breeding of A. flaccida with higher quantity of saponins.

鹅掌草为祛风湿、消肿解毒药用植物。其所含齐墩果烷型三萜皂苷具有较好的抗肿瘤作用。鹅掌草皂苷糖链数量和链接方式产生的生物活性各异。目前尚缺乏系统生物学水平的鹅掌草皂苷生物合成研究，萜类合成及糖基化机制尚不清楚，限制了鹅掌草资源开发利用。课题组已开展了高通量转录组测序、单体皂苷提取分离、活性筛选和皂苷LC/MS 分析工作，本项目拟采用转录组测序结合基于LC/MS的代谢组学方法，系统研究茉莉酸甲酯这种明确具有调控皂苷生物合成作用的植物激素对鹅掌草皂苷积累的分子机制；通过构建基因表达谱和代谢物谱，筛选负责萜类合成及糖基化的关键基因，再利用RACE 及原核表达系统进行基因克隆及功能鉴定；为阐明鹅掌草有效成分合成和调控的分子机制供科学依据，为深入开发打下良好的理论基础。

鹅掌草源自毛茛科银莲花属植物，具祛风湿、消肿解毒等功效。该属植物含多种药物植物，具有悠久的民间药用历史，所含次生代谢产物三萜皂苷为生物活性成分。在鹅掌草所含齐墩果烷型三萜皂苷中，其生物活性因糖链数量和链接方式的不同而各异。但是，目前尚缺乏系统生物学水平的鹅掌草皂苷生物合成研究，萜类合成及糖基化机制更是鲜见，而且鹅掌草对生长环境要求严苛，自然资源十分有限，使鹅掌草资源开发利用更受限制。对于鹅掌草，课题组前期已开展单体皂苷提取分离、活性筛选、皂苷LC/MS分析及高通量转录组测序工作，分离鉴定了13种皂苷类成分，并对三萜皂苷单体开展抗肝癌潜力的实验研究。本课题在植物激素茉莉酸甲酯具有调控皂苷生物合成作用的基础上，采用转录组测序结合基于LC/MS代谢组学的方法，分析鹅掌草的基因表达、KEGG代谢通路、三萜合成通路及相关酶、三萜皂苷结构及含量、细胞色素p450与糖基转移酶的茉莉酸甲酯响应性，系统研究茉莉酸甲酯对鹅掌草皂苷积累的作用及其分子机制，为阐明鹅掌草有效成分合成和调控的分子机制提供科学依据，为深入开发打下良好的理论基础。