滇重楼细胞培养甾体皂苷类物质积累及其代谢调控的研究

The utilization of Paris polyphylla var.yunnanensis has evolved into a major resource crisis because of huge demand, long regeneration period and long period field gathering without order. It is necessary to explore new resources and sustainable use method on the background of wild resources becoming exhausted and difficulties in cultivation with standard operation procedure. The project is to have the following researches by using new modern technologies and methods on the basis of forward research that Steroidal Saponins of Paris polyphylla var.yunnanensis callus could be synthetized: ①the research on screen of high-yield cell strain of Steroidal Saponins of Paris polyphylla var.yunnanensis by cell culture，to screen and set up cell strain with high Steroidal Saponins content and rapid growth; ②optimization of culture condition of Paris polyphylla var.yunnanensis cells,to have research on culture condition influencing cell growth and accumulation of Steroidal Saponins; ③research on accumulation and metabolic modulation of Steroidal Saponins of Paris polyphylla var.yunnanensis by cell culture, to have research on dynamic changes relation between accumulation of Steroidal Saponins and physiology and biochemistry values, protective enzymes and key enzymes in synthesis of Steroidal Saponins in the cell growth process, to explore accumulation and metabolic modulation of Steroidal Saponins in cellular and molecular levels. The research results would lay foundation on thoroughly solving the unsustainable trend and present situation of Paris polyphylla var.yunnanensis resources.

由于需求量巨大、资源再生周期长及长期无序野生挖掘，滇重楼资源的利用已经演变为严重的资源危机，在野生资源渐趋枯竭、人工规范化种植困难的背景下，探索新的资源可持续利用途径势在必行。在前期获得能够合成甾体皂苷类物质滇重楼愈伤组织的基础上，本项目拟应用现代科学新技术新方法对滇重楼植物开展以下研究：①滇重楼细胞培养甾体皂苷类物质高产细胞株筛选的研究，从优质种质资源中筛选、建立细胞生长迅速及甾体皂苷类物质含量高的细胞株；②滇重楼细胞培养条件优化的研究，对影响细胞生长和甾体皂苷类物质积累的培养条件进行研究，优选出最佳培养条件；③滇重楼细胞培养甾体皂苷类物质积累及其代谢调控的研究，在诱导子作用下研究细胞生长过程中生理生化指标、保护酶、甾体皂苷合成关键酶与甾体皂苷类物质积累的动态变化关系，从细胞和分子水平探讨甾体皂苷类物质积累及其代谢调控机制。研究结果将为从根本上解决滇重楼资源不可持续的趋势和现状奠定基础。

由于需求量巨大、资源再生周期长及长期无序野生挖掘，滇重楼资源的利用已经演变为严重的资源危机，在野生资源渐趋枯竭、人工规范化种植困难的背景下，探索新的资源可持续利用途径势在必行。项目主要获得以下研究成果：.（1）滇重楼细胞培养甾体皂苷类物质高产细胞株筛选的研究，完成2批次30个居群滇重楼样品的采集，根茎、茎、叶中重楼皂苷I、II、VI、VII含量的测定，建立指纹图谱，对甾体皂苷类物质进行定性分析和DNA条形码ITS2序列分析，滇重楼的品质及DNA条形码ITS2序列存在很大差异，以根茎中总皂苷（重楼皂I、II、VI、VII含量之和）含量大于1.20 %作为优质种质资源评价标准，筛选出永平滇重楼、云龙滇重楼、弥渡滇重楼、云龙滇重楼（多茎）4个细胞生长迅速及总皂苷含量高的细胞株；.（2）滇重楼细胞培养条件优化的研究 ，筛选出滇重楼细胞生长量和总皂苷含量的培养基及植物生长调节剂最佳组合分别为培养基∶6-BA∶NAA∶IBA =MS∶1.0 g/L∶0.1 mg/L∶0.1 mg/L和培养基∶6-BA∶NAA∶IBA =1/2MS∶1.0 g/L∶0.1 mg/L∶0.2 mg/L，培养基及培养条件的最佳组合分别为蔗糖浓度∶pH值∶温度∶光源=30 g/L∶5.6∶20/25℃∶绿光和蔗糖浓度∶pH值∶温度∶光源=45 g/L∶6.00∶20/25℃∶绿光，矿质元素最佳组合分别为CaCl2•2H2O∶MgSO4•7H2O∶MnSO4•4H2O∶ZnSO4•4H2O =88 g/L∶18.5 g/L∶4460 mg/L∶860 mg/L和CaCl2•2H2O∶MgSO4•7H2O∶MnSO4•4H2O∶ZnSO4•4H2O=44g/L∶74g/L∶2230 mg/L∶860 mg/L；.（3）滇重楼细胞培养甾体皂苷类物质积累及其代谢调控的研究 ，确定最适于滇重楼细胞甾体皂苷类物质积累的水杨酸处理的浓度为100 mg/L和茉莉酸甲酯处理的浓度为50 mg/L，水杨酸和茉莉酸甲酯处诱导子对滇重楼细胞总皂苷含量、生理生化指标（SOD、MDA、POD、CAT活性）、3-羟基-3-甲基戊二酰辅酶、鲨烯环氧酶基因表达量具有显著影响。.研究结果为滇重楼细胞培养生产甾体皂苷类物质奠定基础，为解决滇重楼资源不可持续的趋势和现状提供新思路。