基于ITS2序列的积雪草及其混伪品的分子鉴定.docVIP

  基于 ITS2 序列的积雪草及其混伪品的分子 鉴定# 陈晓辰1，陈倩2，韩建萍1** 5 10 （1. 中国医学科学院，北京协和医学院药用植物研究所，北京 100193； 2. 湖北中医药大学，武汉 430061） 摘要：目的 快速准确的鉴别中药积雪草及其混伪品。方法 采用 ITS2 序列片段，对其进行 PCR 扩增、测序，运用 CodonCode Aligner、MEGA4.1 等软件进行序列拼接和分析，构建积 雪草及其混伪品的 NJ 树。应用 Koetschan 等建立的 ITS2 数据库及其网站预测 ITS2 二级 结构。结果 积雪草及其混伪品 ITS2 序列之间存在明显差异，种间遗传距离大于种内遗传距 离，不同积雪草样品在 NJ 树上独立聚为一支。结论 运用 ITS2 序列可以准确鉴定积雪草及 其伪品。 关键词：积雪草；ITS2；混伪品；分子鉴定 中图分类号：R282.5 15 Molecular identification of Centella asiatica and its adulterants using ITS2 DNA barcode CHEN Xiaochen1, CHEN Qian2, HAN Jianping1 (1. Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, 20 25 30 35 40 Beijing 100193; 2. Huibei Univercity of Chinese Medicine, WuHan 430061) Abstract: Objective To identify Centella asiatica and its adulterants in a fast and specific way. Methods ITS2 regions as DNA Barcode were amplified and sequensed. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. Sequence analyses were carried out by MEGA4.1. The phylogenetic tree was constructed using the neighbor-joining and Kimura 2-parameter methods. Results A significant difference was observed in the ITS2 sequences between Centella asiatica and its adulterants. Inter-specific genetic distance is higher than intra-specific one，ITS2 sequences and secondary structure of ITS2 can distinguish Centella asiatica from its adulterants. Conclusion ITS2 can serve as a valuable marker for identifying Centella asiatica and its adulterants. Keywords: Centella asiatica; ITS2; adulterants; molecular identification 0 引言 积雪草为伞形科植物积雪草(Centella asiatica)的干燥全草。具有清热利湿、解毒消肿的 作用，临床上主要用于治疗湿热黄疸、中暑腹泻、石淋血淋、痈肿疮毒、跌扑损伤[1]。文献 已报道过的混伪品主要有天胡荽 (Hydrocotyle sibthorpioides Lam)，连钱草 (Glechoma longituba （Nakai）Kupr.)， 过路黄(Lysimachia christinae Hance)，马蹄金 (Dichondra repens Forst) 等[2-3]。目前已有文献报道的鉴别积雪草及其混伪品的方法主要有性状鉴别、显微鉴 [ DNA 条形码是指用短的、标准的 DNA 片段作为物种标记而建立的一种新的生物鉴定方 法。ITS2 序列作为条形码序列之一，以其序列片段较短，物种水平变异较快等优势被广泛 应用于药用植物及生药鉴定[5-8]。本文选取 ITS2 序列鉴定积雪草及其混伪品，以期为