基于ITS2条形码的中药材赤芍及其易混伪品的DNA分子鉴定

Molecular Identification of Paeoniae Radix Rubra and Its Adulterants based on ITS2 DNA Barcode

目的:对中药材赤芍及其易混伪品进行分子鉴定,以确保该药材的质量以及临床疗效。方法:以核基因ITS2片段作为DNA条形码,对研究材料进行PCR扩增并双向测序,所得序列经CodonCode Aligner拼接后,用软件MEGA4.0进行相关数据分析,并构建NJ(邻接)树。利用Koetschan等建立的ITS2数据库及其网站预测ITS2二级结构。结果:赤芍两基源植物ITS2序列长度均为227bp;由所构建的系统聚类树图可以看出,无论芍药还是川赤芍,它们的不同来源样本均分别聚在一起,表现出单系性;比较赤芍及其易混伪品的二级结构发现,赤芍的正品来源芍药与川赤芍彼此间差异甚微,而与其它物种在螺旋区茎环的数目、大小、位置以及螺旋臂由中心环伸出时的转角等方面具有较明显区别。结论:ITS2作为DNA条形码序列能够有效的区别中药材赤芍基源植物与其易混伪品,在中药材的鉴定中具有重要的应用前景。

This study aimed to discriminate between Paeoniae Radix Rubra and its adulterants in order to guarantee the quality and clinical curative effect of this medical material. In this study, the second internal transcribed spacer （ITS2） of ribosomal DNA were amplified and sequenced. Sequence assembly and consensus sequence generation were performed by using the CodonCode Aligner. To perform a phylogenetic study, sequences of DNA regions were aligned using Clustal W and the genetic distances were computed using MEGA 4.0 in accordance with the Kimura 2- parameter （K2P） model. And the phylogenetic tree was constructed using the neighbor-joining and Kimura 2-parameter methods. The ITS2 secondary structure was predicted using the ITS2 database and website which was found by Koetschan et al. The results showed that the length of ITS2 sequence of the origin plant of Paeoniae Radix Rubra were both 227 bp. In the cluster dendrogram, both Paeonia lactiflora and P. veitchii showed monophyletic. To compare the ITS2 secondary structure of the origin plants of Paeoniae Radix Rubra and its adulterants, we noticed that the difference of Paeonia lactiflora and P. veitchii was slight. But they were obviously distinguished from other species in the amount, size, position of loop and the angle of helix exertion. It is concluded that ITS2 can be used to correctly identify Paeoniae Radix Rubra from its adulterants. Hence, the application of ITS2 in the identification of traditional Chinese medicine has an important prospect.