怀牛膝根的结构发育和主要药用成分积累关系研究

Achyranthes bidentata BL. is the important species of the Chinese traditional medicine plants. The medicinal ingredients in its dried roots are mainly included three kinds of chemicals,ecdysterone, saponin and polysaccharede. Previous studies on A. bidentata mainly focus on the phytochemistry, pharmacology, clinical usage, cultivation and processingof A. bidentata . However, there is no in-depth research on the correlation between the structural development and bioactive ingredients accumulation in A. bidentata in the present time, which is the main index for the quality evaluation of A. bidentata. Based on our previous works, we will study the synthesis positions,transport route and storage sites of mainly medicinal ingredients,the phloem unloading pathway and transportation form of photoassimilates during the growth and development of A. bidentata using transmission electron microscopy,laser confocal microscope，immunity colloidal gold localization, and phytochemistry methord to reveal the the relationship between the synthesis,conversion,allocation and storage of photoassimilates and the accumulation of the medicinal ingredients.The effects of exogenous and exogenous hormones on the root development of A. bidentata and the accumulation of medicinal ingredient in root will be also investigaed to explore the regulation machanism in this project. The results of this project will be greatly beneficial for revealing the correlation between the sturctural development of A. bidentata roots and the accumulation of medicinal components. Furthermore, it also can provide scientific data for the study on the prodution improving and the quality control of A. bidentata in the future practice of A. bidentata planting.

怀牛膝的药用部位为根，其主要药用成分为蜕皮甾酮、三萜皂苷和牛膝多糖。以往的研究偏重植化、药理、临床、栽培和加工等领域，有关牛膝结构发育与药用成分积累的关系缺乏深入研究，而牛膝根的形态结构及根内主要药用成分的含量是中药牛膝质量评价的主要参考指标。本项目在前期研究基础上，应用透射电镜、激光共聚焦显微镜、免疫胶体金定位和植物化学等技术相结合，从细胞水平研究牛膝生长发育过程中主要药用成分的合成部位、转运途径和贮存场所以及同化物卸载的细胞学路径和后韧皮部运输的形式，揭示同化物的合成、转化、分配及储藏与主要药用成分积累的相关性；并研究内源激素和外源激素对其根的结构发育和药用成分积累的影响，探讨其调控机制。研究结果对探讨牛膝结构发育与主要药用成分产生和积累的关系具有重要意义，同时为提高牛膝产量及药用成分含量等生产实践问题提供科学依据。

牛膝是我国传统的道地药材，三萜皂苷和蜕皮甾酮是牛膝中的主要活性成分，主要由甲羟戊酸（MVA）和丙酮酸磷酸甘油醛（MEP）代谢途径产生。本研究以怀牛膝为材料，系统地研究了外源激素对牛膝生长发育及主要药用成分三萜皂苷和蜕皮甾酮合成的影响，明确了牛膝的根是三萜皂苷合成和储藏的主要器官，而叶是牛膝蜕皮甾酮合成的主要场所，根则是蜕皮甾酮的主要储存器官。利用高通量测序技术，成功构建了怀牛膝根与叶器官比较转录组学数据库，牛膝根与叶中共获得28,339个差异基因，有915个差异基因参与次生代谢产物合成，566个差异基因参与者三萜皂苷的生物合成，还发现有260个基因为编码MVA途径中齐墩果酸和蜕皮甾酮生物合成的酶基因，14个基因为编码MEP途径中类异戊二烯合成酶的基因。此外，还挖掘到115个P450 unigenes，92个UGT unigenes，17个GTs unigenes，4个与皂苷转运有关的PDRs基因。.对外源激素处理组的牛膝进行转录组分析，从叶及根中分别获得了20,296和9,812个差异基因。KEGG代谢途径分析显示，叶中有3,363个基因被分配到生物代谢途径中，其中包括1,108个差异基因，有15个差异基因参与甲羟戊酸途径；根中仅有631个差异基因被注释到生物代谢途径中。此外，我们还利用RT-q PCR 技术分析了8个候选基因在牛膝不同组织、不同实验条件下的的表达情况，确定了具有较高稳定性和重复性的内参基因。本项目的开展为阐明三萜皂苷和蜕皮甾酮的生物合成机制，从而为牛膝的种质和品种改良奠定了基础。