苗药一枝黄花及其混伪品的DNA条形码鉴别

摘要

目的 选用DNA条形码对湖南苗药一枝黄花及其混伪品进行分子鉴定,为其药材鉴别与安全提供技术支撑.方法 利用DNA条形码方法,分别对一枝黄花及其混伪品的ITS2核基因片段和psbA-trnH叶绿体基因片段进行扩增并双向测序,使用CodonCode Aligner软件对其序列进行拼接,用MEGA软件对数据比对分析,构建邻接(NJ)系统聚类树鉴定分析,并用Koetschan等分析预测ITS2二级结构.结果 一枝黄花的ITS2序列长度为223 bp,其种内平均K2P遗传距离小于混淆品的种间K2P遗传距离;由所构建的系统NJ聚类树图表明,各物种呈现一定的区域性,又可与其它物种明显区分,而基于psbA-trnH序列构建的统聚类树图不能将一枝黄花区分;通过ITS2二级结构比对分析,一枝黄花和其混伪品的4个螺旋发出时的角度及螺旋区的茎环位置、大小、数目均有明显区别.结论 ITS2作为条形码可方便快捷地鉴别一枝黄花及其混淆品,进一步验证了DNA条形码技术鉴定中药材的有效性.%Objective To discriminate Solidago decurrens and its adulterants by DNA barcoding to supply technical support for its identification and safety in medication. Methods The ITS2 nuclear gene fragment and psbA-trnH chloroplast gene fragment from Solidago decurrens and its adulterants were amplified and bidirectionally sequenced by DNA barcoding technology. Sequence assembly and consensus sequence generation were performed by CodonCode Aligner. The genetic distances were computed by MEGA. Identification analyses were performed by neighbor-joining (NJ) Method. The ITS2 secondary structure was predicted by the ITS2 database and website which was built by Koetschan et al. Results The length of ITS2 sequence of Solidago decurrens was 223 bp. The intra-specific genetic distances of Solidago decurrens were far lower than inter-specific genetic distances of the adulterants. In the cluster dendrogram, all species showed monophyletic and distinguished from others. But Solidago decurrens and its adulterants could not be distinguished by the cluster dendrogram built on the basis of psbA-trnH sequence. By comparison of the ITS2 secondary structure, Solidago decurrens was obviously distinguished from its adulterants in the amount, size, position of loop and the angle of helix exertion. Conclusion ITS2 sequence can be used as DNA barcode to identify Solidago decurrens and its adulterants effectively and our study can further confirm the effectiveness of ITS2 for identification of traditional Chinese medicinal materials.