ITS2序列分析在白花蛇舌草鉴定中的应用研究

Application study on identification of Hedyotis diffusa by ITS2 sequence analysis

目的对白花蛇舌草和它的混伪品进行分子生物鉴定,保证该药材的临床药效和使用安全。方法提取白花蛇舌草及其混伪品的DNA,使用引物进行PCR扩增,获取ITS2序列的片段,将扩增得到的产物进行双向测序,运用Condon Code Aligner软件进行校对拼接,去除低质量区。用HMMer注释法保留ITS2序列全长。运用MEGA5.1软件对真伪品序列进行分析,计算真伪品的遗传距离,构建邻接(NJ)树评估真伪品之间的亲缘关系。采用ITS2数据库预测ITS2二级结构。结果白花蛇舌草与混伪品的ITS2序列存在明显差异。结论运用ITS2序列能准确有效鉴别出白花蛇舌草和混淆品。

Objective To ensure the clinical efficacy and safety of Hedyotis diffusa, identify Hedyotis diffusa and its mix adulterants by molecular biological assay. Methods The polymerase chain reaction（PCR） technique which used DNA templates of Hedyotis diffusa and its mix adulterants and ITS2 sequence primers was performed to obtain the ITS2 regions. The amplification products were sequenced bi-directionally. Sequences were assembled by the CodonCode Aligner. ITS2 sequences of plants were obtained integrally by HMMer annotation method and analyzed by MEGA5.1 to calculate genetic distance among Hedyotis diffusa and its mix adulterants. The neighbor-joining（NJ） phylogenetic tree was constructed to evaluate genetic relationship of plants. ITS2 secondary structures were predicted by ITS2 database. Results There was distinct difference on ITS2 sequences between Hedyotis diffusa and its adulterants.Conclusion ITS2 sequence can be used as a effective marker for identifying Hedyotis diffusa and its adulterants.