摘 要 ：为寻找简单、可重复的分子鉴定方法,对绞股蓝(Gynostemma pentaphyllum(Thunb.) Makino)新鲜品、药材干品及其混淆品乌蔹莓(Cayratia japonica(Thunb.) Gagnep)进行了鉴定。首先从2000余条10碱基的随机引物中筛选出20条,并以5条为1组随机分成4组,然后采用RAPD方法对7份不同来源的绞股蓝新鲜品进行扩增,对扩增得到的绞股蓝特征条带进行基因克隆、测序,分析序列特征,再设计特异性引物,最后,用绞股蓝新鲜品、药材干品及其混淆品乌蔹莓等样品进行PCR验证。结果显示:RAPD扩增能够得到7份绞股蓝新鲜品重复的共有特征条带;经基因克隆、测序并设计特异性引物后进行PCR验证,获得了绞股蓝特异序列扩增区域标志(Sequence-characterized amplified region maker,SCAR)。初步建立了区分绞股蓝及其混淆品乌蔹莓的分子鉴定标准,并首次将SCAR应用于绞股蓝分子鉴定中。
Abstract:The aim of this study was to develope species-specific molecular marker for identification of Gynostemma pentaphyllum and that with their adulterant Cayratia japonica,in order to avoid adulteration that affects the quality and quantity of G.pentaphyllum The first,twenty 10-bp random primers were picked out from more than 2000 random primers and then divided randomly into four terms that were consist of five random primers.The second,Genomic DNAs of seven G.pentaphyllum from different places in Guangxi,China,were amplified using a PCR-based RAPD technique and the same manipulation was repeated to confirm species-specific DNA fragment which was the one of RAPD produts.The last,with getting the message of cloning and sequencing of the RAPD products,the species-specific primer was designed and synthesized to amplify fresh leaves and dried medicines of G.pentaphyllum,Cayratia japonica and so on.As anticipated,the genomic DNAs of G.pentaphyllum were only amplified a band and that of others were amplified no band,using the species-specific primer and so sequence-characterized amplified region(SCAR) marker were developed.The SCAR marker are potentially useful for the identification of G.pentaphyllum in Guangxi and their adulterant Cayratia japonica,also other plants.In addition.This is the first report of species-specific SCAR development in G.pentaphyllum.