【作者】 王大威；

【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】 本实验应用RAPD及ISSR分子标记，对来源于黑龙江、河北、陕西、四川、甘肃的五个防风及其近缘种共38份样本进行了DNA指纹分析。从100个RAPD引物和32个ISSR引物中分别筛选出21、6个扩增结果稳定、扩增条带清晰的引物。其中，8个RAPD引物和3个ISSR引物扩增出的带可以将防风及其近缘种区分开。用筛选出的引物对所有样本进行PCR扩增，得到DNA指纹图，从DNA指纹图中可见不同品种之间仅有少数引物得到可以明确区分它们的不同条带。同时对电泳条带进行基于遗传相似度的聚类分析可将5类38份材料分为三类：第一类包括竹节防风、前胡，第二类包括硬苗防风、葛缕子，第三类为正品防风。前两类之间的遗传相似度范围为0.77～0.80，正品防风和这二者之间的遗传相似度范围为0.72～0.75。不同品种之间的遗传相似度范围显示了正品防风与其他防风近缘种或混伪品之间的遗传距离，以及防风近缘种之间的遗传相似性，但也说明正品防风与其他防风近缘种或混伪品遗传相似度比较低，具有比较远的亲缘关系。在药物有效成分含量分析方面，本文采用《中华人民共和国药典》中规定的高效液相色谱法对所有样本进行了防风升麻苷的含量测定。同时，尝试使用了先进的毛细管电泳法。《中华人民共和国药典》中规定防风中色原酮类成分的含量不可低于0.308%，产地黑龙江富裕的正品防风的有效成分升麻苷的含量为0.842%～0.864%明显高于其他如硬苗防风（河北 0.159%～0.163%）、竹节防风（四川0.350%～ 0.411%）、前胡（陕西 0.473%～0.590%）、葛缕子（甘肃 0.456%～0.501%）等防风亲缘种中的升麻苷含量，结合DNA指纹分析，引物SBS A17对正品防风扩增出一条特异带，其它产地的防风近缘种无此带。本研究筛选得到的RAPD及ISSR引物可用于防风类药材的分子鉴定。黑龙江富裕地区的防风质量最好，升麻苷含量高，是培育防风优良品种的首选引种之地。引物SBS A17扩增所得特异带可初步作为防风品质优良的特异性标记，在种苗的早期品质鉴定方面应用价值很高。更多还原

【Abstract】 38 samples of five Species Fangfeng and Its Close Relative Species collected from Heilongjiagn, Hebei, Shanxi, Sichuan, and Gansu were analysed by RAPD and ISSR molecular markers. 21 RAPD primers and 9 ISSR primers were selected from 100 and 32 respectively, and could produced qualified fragments. Among these selected primers only 8 RAPD and 3 ISSR primers could identify the Fangfeng and Its Close Relative Species. DNA fingerprints were obtained by PCR with all selected primers and analyzed using cluster analysis. The fingerprints indicated that only few primers could get the specific fragments able to identify Fangfeng and Its Close Relative Species. The cluster analysis of cluster analysis based on the genetic similarity divide 38 samples of five Species Fangfeng to 3 groups: fisrt group included P.dielsianum Fedde ex wolff and Peucedanum ledebourieuoides K.T.Fu; second group included Curum Carvil and Friocycla albescens (Franch). Wolff; last group included Fangfeng. The genetic similarity of first two groups ranged between 0.77 to 0.80, the genetic similarity of first two groups and last group Fangfeng ranged between 0.72 to 0.75. The genetic similarity range of the different varieties show that The Genetic Distance of Fangfeng and Its Close Relative Species ,The genetic similarity of Its Close Relative Species,and also illustrate that The genetic similarity of Fangfeng and Its Close Relative Species were tiny, and relatively big between them.Content of prim-O-glucosylcimifugin, the mainly effective compound was determined by high performance liquid chromatography, the method used in People’s Republic of China pharmacopeial convention. Also, the high performance capillary electrophoresis was used. People’s Republic of china pharmacopeial convention prescript the content of prim-O-glucosylcimifugin in Fangfeng and Its Close Relative Species were aove 0.308. The Content of prim-O-glucosylcimifugin in Fangfeng selected from Heilongjiang Fuyu ranged from 0.842% to 0.864%,which obviously were much more than the Content in Its Close Relative Species,such as Friocycla albescens (Franch). Wolff(Hebei 0.159%～0.163%)、 P.dielsianum Fedde ex wolff (Sichuan 0.350%～ 0.411%)、Peucedanum ledebourieuoides K.T.Fu (Shanxi 0.473%～0.590%)、Curum Carvil(Gansu 0.456%～0.501%)。In concern of the DNA fingerprints, primer SBS A17 amplified a specific fragment within Fangfeng, while not in other samples.The selected primers could be used in identifying Fangfeng selected form HeilongjianFuyu produced the best quality Fangfeng containing highest content of prim-O-glucosylcimifugin, and should be considered first while culturing new good variety. The specific fragment amplified by primer SBS A17 could be used as a potential DNA marker indicating good quality of Fangfeng, especially useful in the use of identifying good seedling in an early stage of growth.更多还原