its2序列分析在白花蛇舌草鉴定中的应用研究.docVIP

ITS2序列分析在白花蛇舌草鉴定中的应用研究 　　[摘要] 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MEGA 5.1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。 　　[关键词] 白花蛇舌草；混伪品；ITS2；分子鉴定 　　[中图分类号] R282.12 [文献标识码] A [文章编号] 1673-7210（2016）11（b）-0021-04 　　[Abstract] 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. 　　[Key words] Hedyotis diffusa； Adulterants； ITS2； Molecular identification 　　白花蛇舌草?檐绮菘贫?草属植物白花蛇舌草（Oldenlandia diffusa）的全草，具有清热解毒、利尿消肿、活血止痛的功效[1]。由于白花蛇舌草在癌症的治疗中效果确切[2]，需求剧增，使其生药资源日见贫乏，而出现较多混伪品。常见混伪品伞房花耳草、纤花耳草、松叶耳草等与白花蛇舌草同科属，仅根据植物外在特点难以鉴别，经常会被当成是白花蛇舌草用于临床[3-7]。还有商家为了增加利益，掺杂其他不同科属的漆姑草（石竹科漆姑草属）、舌雀草（石竹科繁缕属）在白花蛇舌草中，导致白花蛇舌草的质量下降[8-9]。为保证白花蛇舌草的用药安全和疗效确切，它的准确鉴别是目前一项十分紧迫的任务。近年来，DNA条形码研究在物种鉴定和植物分类领域体现出较高的准确率，成为热点研究方向[10-11]。此项技术的核心在于通过比较一段通用DNA序列，对物种在基因水平进行鉴定和高效识别，同传统的显微鉴别、理化鉴别、色谱鉴别相比较，DNA条形码技术重复性好、稳定、快速、准确，更为重要的是它深入到分子水平，反映了物种的遗传特性，这种鉴别方式更加可靠[12-13]。有学者对多种