摘要
目的建立及优化青天葵样品总DNA的提取方法及RAPD反应,探讨不同品种的青天葵及其替代品、伪品在DNA分子水平上的遗传多样性及其遗传关系。方法使用高盐低pH值法提取总DNA,采用随机扩增多态性DNA,从49条随机引物中筛选能扩出清晰条带的引物,对22个青天葵样品及伪品进行RAPD分析。用统计分析软件对扩增出的条带进行聚类分析,以Shonnon’s指数和Nei’s指数对青天葵遗传多样性进行估算。结果高盐低pH值法提取的新鲜样品纯度高,药材纯度低,但都能用于RAPD。选取能扩增出清晰条带的19条引物,把鲜品与干品分别聚类分析。干药材中小叶与中叶距离较近,与大叶及毛叶距离远。新鲜叶片中三种青天葵为一类,青天葵与其组培品、毛叶与红薯叶距离稍远,与车前草、积雪草距离最远。青天葵种群遗传多样性Shonnon’s指数为0.463,Nei’s指数为0.267,种群内遗传多样性较高,基因流为0.94。结论不同品种青天葵间及伪品在分子水平上存在差异,可用此方法鉴别青天葵。青天葵的遗传多样性大多是由地理环境造成的。
Objective To optimize the method for total DNA extraction and RAPD analysis of Nervilia fordii （NF）, and to study the genetic diversity of different breeds of NF, the substitute of NF and the fake of NF at molecular level. Methods We used low -pH extraction medium with high salt to extract total DNA, and used randomly amplified polymorphic DNAs （RAPD） to select polymorphism primer from 49 random primers. Twenty - two kinds of NF samples were analyzed by RAPD, and classified by SPSS. Genetic diversity were estimated by Shonnon＇ s index and Nei＇ s index. Results A higher puritiy of DNA can be obtained from fresh HF than that from medical materials. We selected 19 polymorphism primers for the cluster analysis of fresh NF and dried medical material. For medical materials of NF, the distance of amplification band of small - leave breed is close to that of middle - size leave breed, but is far away from that of, big - leave breed and Nervilia plicata. For the fresh materials of NF, there breeds of Nervilia fordii can be classified into one kind, the distance of fresh NF band is far from that of Nervilia plicata, Pachyrhizus erosus and the cultured breed, and more far awary from that of Plantago asiatica and Centella asiatica. Shonnon＇ s genetic diversity is 0. 463, Nei＇ s genetic diversity is 0. 267. Intra - population genetic variation is obvious compared to iner- population genetic variation. The estimated gene flow from Gst （Nm） is 0.94. Conclusion The molecular difference in different breeds of Nervilia Fordii can be used to identify Nervilia Fordii. The genetic diversity of Nervilia fordii is mainly caused by the geography environment.
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2009年第6期554-557,共4页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
广东省自然科学基金项目(5004249)
广东省科技计划项目(2004B330001009
2008A030101009)
关键词
青天葵
毛唇芋兰
毛叶芋兰
遗传多样性
随机扩增多态性DNA
Nervilia fordii
Nerviliafordii （Hance） Schhr.
Nervilia plicata （Andr.） Schltr.
Genetic diversity
RAPD
