基于ITS2序列的丹参及其混伪品分子鉴定研究

目的]采用DNA条形码及特异性引物PCR技术对中药材丹参及其混伪品进行分子鉴定研究。方法]以核基因ITS2序列作为DNA条形码,对研究材料进行PCR扩增并双向测序,将所得序列构建NJ系统发育树。利用Koetschan等建立的ITS2数据库及其网站预测ITS2二级结构,同时采用自设引物进行特异性引物PCR鉴别研究。结果]ITS2序列长度为470 bp左右;从系统聚类树图可以看出,丹参及其伪品分别聚在不同支,表现出单系性;比较二级结构发现,丹参与甘西鼠尾差异甚微,与牛蒡在螺旋茎环的数目、大小、位置以及螺旋臂由中心环伸出时的转角等方面具有明显区别;通过特异性引物PCR技术可将丹参及其伪品进行区分。结论]DNA条形码技术和特异性引物PCR技术均能够有效地区别丹参及其混伪品,在中药材的鉴定中具有重要的应用前景。

Molecular Identification of Salvia Miltiorrhiza Bge.and Its Adulterants Based on ITS2 Sequences

Objective] The research aimed to study the molecular identification of Salvia miltiorrhiza and its adulterants by DNA barcode and specific primers PCR. Method] The ITS2 sequence was used as DNA barcoding, and the materials were amplified by PCR and sequenced, and the NJ phylogenetic tree was constructed.The secondary structure of ITS2 was predicted by database established and its website by Koetschan et al.,and the self-designed primers were used to carry out specific primer PCR identification. Result] ITS2 sequence length was around 470 bp.The results of cluster analysis showed that Salvia miltiorrhiza and its adulterants were clustered in different branches and showed single character.Compared with secondary structure, Salvia miltiorrhiza and Ganxi rat tail had little difference, and there were signifi-cant difference on the number, size, location of burdock in the spiral stem, and the rotation angle of the spiral arm from the central ring with burdock.The specific primers could distinguish the Salvia miltiorrhiza and its counterfeit by PCR technique.Conclusion]DNA barcoding and specific primers PCR are effective in distinguishing Salviae miltiorrhiza and its adulterants, and it has an important application foreground in the identification of Chinese herbal medicines.