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为研究分子标记技术在成药鉴定方面的应用,本文选用连翘败毒丸为研究对象,采用改良CTAB法提取其总DNA,通过聚合酶链式反应(polymerase chain reaction,PCR)分别对psbA-trnH和rbcL两个叶绿体序列进行梯度扩增,将扩增产物与pEASYTM-T5 vector连接后,转化到大肠杆菌Trans1-T1感受态细胞中,挑取单克隆,并选择阳性克隆进行测序。所得序列校正、比对后,进行BlastN比对分析,同时采用MEGA 4.0软件构建系统聚类树。结果表明,通过psbA-trnH序列分析可以鉴定出9种原料药材,rbcL序列分析可以鉴定出6种原料药材。本研究结果说明采用分子标记技术鉴定中成药原料具有一定的可行性。
In order to study the application of molecular markers in the identification of traditional Chinese medicines, this study selected the Forsythia Bei poison pill as the research object, using the modified CTAB method to extract the total DNA, by polymerase chain reaction (PCR) to psbA-trnH And rbcL were amplified by gradient amplification. The amplified product was ligated with pEASYTM-T5 vector and transformed into E. coli Trans1-T1 competent cells. The single clones were picked out and the positive clones were selected for sequencing. The obtained sequence is calibrated and aligned, then BlastN comparison analysis is carried out, and a system clustering tree is constructed by using MEGA 4.0 software. The results showed that 9 kinds of raw medicinal materials could be identified by psbA-trnH sequence analysis, and 6 kinds of raw medicinal materials could be identified by rbcL sequence analysis. The results of this study indicate that it is feasible to use molecular markers to identify proprietary Chinese medicines.