基于转录组测序筛选岩白菜的岩白菜素生物合成关键酶基因

Bergenin has multiple pharmacological values of anti-tussive, antibacterial, anti-inflammatory, anticancer, anti-HIV, anti-arrhythmia, antiurolithic, antioxidant, α-glucosidase and α-amylase inhibitory, immunomodulatory, hepatoprotective and neuroprotective activities. It is mainly extracted from Bergenia purpurascens and has been applied in clinic. We found that the bergenin biosynthesis in Bergenia purpurascens was induced by environmental conditions (the same resource by rhizome propagation produced 12.35% of bergenin in Qiaojia County and 6.1% of bergenin in Xianggelila County, respectively), but the bergenin biosynthesis pathway and its regulation mechanism need to be explored. RNA-Seq is a new tool to study the secondary metabolism pathway of medicinal plants while the rea1-time fluorescent quantitative PCR (FQ-PCR) is the most effective means for examining the gene expression levels. In this research project the two Bergenia purpurascens samples with same origin and different bergenin contents will be used as research materials for screening out the genes encoding key enzymes in the bergenin biosynthesis pathway based on the RNA-Seq technology combined with bioinformatics approach. The expression levels of these genes in different organs and at different developing stages will be examined by FQ-PCR while their bergenin contents will be determined via high performance liquid chromatography (HPLC). Then a correlation analysis will be conducted to verify the functions of these genes. Finally the bergenin biosynthesis pathway in Bergenia purpurascens and its regulation mechanism will be explored. The research achievement of this project will provide a scientific basis for breeding new Bergenia purpurascens varieties with high bergenin contents and directly producing bergenin via biotechnology means.

岩白菜素具有止咳祛痰、抗菌消炎、抗肿瘤、抗艾滋病毒、抗心律失常、提高免疫力、保护肝脏和神经等多方面的药理价值，主要从岩白菜中提取，已被制成片剂用于临床。前期研究发现岩白菜中岩白菜素的生物合成受到环境条件的诱导（用根状茎繁殖的同一种源在巧家县和香格里拉县种植后岩白菜素含量分别为12.35%和6.1%），但其合成途径及调控机制尚不清楚。RNA-Seq是研究药用植物次生代谢途径的新工具，而荧光定量PCR技术是检测基因表达量的最有效手段。本项目以岩白菜素含量差异悬殊的2份同源岩白菜为材料，采用RNA-Seq技术结合生物信息学方法筛选出岩白菜中岩白菜素生物合成的关键酶基因，并通过基因表达量（荧光定量PCR法检测）与岩白菜素含量（HPLC法测定）的相关性分析来验证关键酶基因的功能，从而揭示岩白菜中岩白菜素的生物合成途径及其调控机制，为利用生物技术手段进行岩白菜种质创新和直接生产岩白菜素提供科学依据。

岩白菜素具有止咳祛痰、抗菌消炎、抗肿瘤、抗艾滋病毒、抗心律失常、提高免疫力、保护肝脏和神经等多方面的药理价值，主要从岩白菜中提取，已被制成片剂用于临床。前期研究发现岩白菜中岩白菜素的生物合成受到环境条件的诱导（同一种源在巧家县和香格里拉县种植后岩白菜素含量分别为12.35%和6.1%），但其合成途径及调控机制尚不清楚。筛选岩白菜中调控岩白菜素合成的关键酶候选基因并验证其功能，从而揭示岩白菜中岩白菜素的合成途径及其调控机制具有重要的理论和实践意义，研究成果可为利用生物技术手段进行岩白菜种质创新和直接生产岩白菜素提供科学依据。. 以栽培在巧家县和香格里拉县的岩白菜素含量差异悬殊的两份同源岩白菜的根状茎和叶片为材料，应用Hiseq2000平台进行了8个样本的转录组测序，构建了世界首个岩白菜转录组数据库，获得94755个岩白菜基因，丰富了非模式植物的基因数据库。. 揭示了岩白菜中岩白菜素的生物合成途径：葡萄糖经过糖酵解产生了磷酸烯醇式丙酮酸（PEP），经过磷酸戊糖途径产生了4-磷酸赤藓糖（E4P），PEP和E4P经过莽草酸途径合成了没食子酸（GA），GA与UDPG发生转糖基反应生成了异岩白菜素，异岩白菜素再经过转甲基反应生成了岩白菜素。从葡萄糖到岩白菜素共有21步反应，需20种酶催化，本研究找到了全部20种酶的610个候选基因。. 从上述候选基因中选取了14个表达量较高的包含完整阅读框序列的重点候选基因，开展了保守结构域分析和系统发育树构建等生物信息学分析，采用qRT-PCR方法检测了它们在不同地点、不同时间和不同部位的相对表达量，采用HPLC法测定了不同地点、不同时间和不同部位的岩白菜素和没食子酸的含量，通过基因表达量与活性成份含量的相关性分析确认了调控岩白菜素合成的5个关键酶基因，并完成了UDPG葡萄糖基转移酶基因的克隆和原核表达研究。