【作者】 曹雅男；

【导师】 李景鹏；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 实验的目的是要根据国际上关于中草药的质量检测标准建立一套中药龙胆分子生物学及生物化学的指纹图谱检测技术。实验通过研究RAPD及ISSR反应体系中的一些因素对反应的影响，优化RAPD及ISSR的PCR反应，建立了适用于龙胆的RAPD及ISSR分子标记方法。并用这2种分子标记方法对4种正品龙胆的43个样品进行了DNA指纹分析。实验用改进的SDS法提取的龙胆总DNA为模板进行RAPD及ISSR的PCR扩增，对产物琼脂糖凝胶电泳结果进行数据分析。从100个RAPD引物和32个ISSR引物中分别筛选出9个RAPD引物和4个ISSR引物可用于遗传多样性分析。通过距离系数 UPGMA聚类法分析电泳产物，构建类聚关系树状图。结果显示坚龙胆与三花龙胆、龙胆、条叶龙胆的遗传距离较远，而三花龙胆、龙胆、条叶龙胆三种中三花龙胆和龙胆亲缘关系较近。而且在4个品种的内部也可见丰富的遗传多样性。在药物有效成分含量分析方面，首先建立高效毛细管电泳法测定生药龙胆中有效成分龙胆苦苷含量的新方法。实验采用高效液相色谱法和毛细管电泳法两种方法对在：不同的时期，在同一采集地采集的龙胆样品；在同一时期采集的不同品种的龙胆样品；两组同一时期采集的野生与种植龙胆样品中龙胆苦苷的含量进行了比较，以确定品质优良的龙胆品种及最佳的采收期。通过RAPD和ISSR指纹图谱的分析为正品龙胆分类及系统发育研究提供了分子生物学依据。通过高效液相色谱法和毛细管电泳色谱法对质量标准的分析为选育优良品种及选择最佳采收期提供了科学依据。更多还原

【Abstract】 The purpose of this experiment was to build a suit of molecular biological and biochemical fingerprints methods about Radix Gentiana to meet quality standards of international.This experiment established the RAPD and ISSR fingerprints methods of Radix Gentiana through optimizeing RAPD、ISSR reaction system and other factors.Those two methods were applied in analysizing forty-three samples of four species Radix Gentianae.The total DNA templates used in this experiment were extracted by improved SDS method.Among these selected primers only 9 RAPD and 4 ISSR primers could use as genetic diversity analysis.The statistic data were analyzed to construct cluster analysis tree by using genetic distance UPGMA method.Analysis showed that genetic distances of G.rigeslens Franch to G.manshurica Kitag、G.trifiora Pall and G. scarbra Bge were far,and that of G.rigeslens Franch to G.manshurica Kitag、G.trifiora Pall and G. scarbra Bge were close.There were rich genetic diversity within every species.Estiblishing HPCE method at first to determinate gentiopicroside in Radix Gentianae.HPLC and HPCE were applied to analysize gentiopicroside contents in: Samples of same species 、same period and same site; Samples of same period and different species; Wild and cultivated samples of same period.Analysis of RAPD and ISSR fingerprints supplied molecular biological evidence for Radix Gentianain classification and growth. Quality analysis of HPLC and HPCE supplied scientific theories for Radix Gentiana in cultivation and collection.Candidate:CaoYa Nan Major: Preventative Veterinary ScienceSupervisor: Prof. Li Jingpeng更多还原