基于ITS2序列及二级结构对紫苏与其变种、紫苏子与其混伪品的鉴别

Molecular Identification of Perilla frutescens and Its Varieties,Perillae fructus and Its Adulterants Based on ITS2 Primary Sequence and Its Secondary Structure

采用ITS2一级序列联合其二级结构对紫苏与其变种、紫苏子与其混伪品进行鉴定,为紫苏药材的使用提供更科学的分类方法。收集紫苏与其近缘种、紫苏子混伪品基源植物样本,提取DNA进行PCR扩增;并结合GenBank中的序列获得所有物种的ITS2序列。应用MEGA7.0软件进行序列分析、计算种内种间遗传距离、构建NJ树,利用ITS2数据库预测各物种的ITS2二级结构。利用4Sale软件进行一级结构联合二级结构矩阵的比对,运用ProfDistS软件构建PNJ树。ITS2序列的扩增成功率高。除白苏以外,紫苏与其他变种的ITS2序列具有明显的Barcoding gap;NJ树显示,ITS2可将紫苏、白苏与其他变种区分;紫苏和白苏在ITS2二级结构中有差异。紫苏子与其混伪品的ITS2序列差异较大,种间具有显著的遗传距离,NJ树和PNJ树显示紫苏子与各混伪品分别各自聚为一支,且有较高的自展支持率。因此,基于ITS2条形码可以准确鉴别紫苏子与其混伪品,为规范药材市场和保障临床用药安全提供可靠方法;但对于紫苏与其变种白苏的鉴别需要结合ITS2二级结构,或建议将白苏和紫苏合并为一个种,为紫苏属的植物分类提供参考。

The identification of Perilla frutescens and its varieties, Perilla fructus and its adulterants was studied based on ITS2 primary sequence combined with its secondary structure, providing a more scientific classification method for the usage of Perilla frutescens as a medicinal plant. After the collection of Perillae fructus and its adulterants, Perilla frutescens and its varieties, their DNA was isolated for PCR amplification. The ITS2 sequences of all species were obtained through the combination with the sequences in GenBank. MEGA 7.0 software was used for analysis of sequence,calculation of intra-species and inter-species genetic distances, and construction of NJ cluster tree.The prediction of ITS2 secondary structure of each species was based on ITS2 database. 4Sale software was used to compare the primary structure with the second structure matrix, and ProfDistS software was used to construct the evolutionary tree of PNJ. ITS2 sequence had high success rate of amplification. Except P. frutescens var. frutescens, ITS2 sequences of Perilla frutescens and other varieties had significant barcoding gap. NJ tree showed that ITS2 could distinguish Perilla frutescens and P. frutescens var. frutescens from other varieties. Perilla frutescens and P. frutescens var.frutescens differed in the secondary structures of ITS2. The ITS2 sequences of Perilla fructus and its adulterants were quite different, and there was significant genetic distance between various species.NJ tree and PNJ tree showed that Perilla fructus and its adulterants were clustered into separate branches with higher bootstrap values. Based on ITS2 barcode, Perillae fructus and its adulterants could be accurately identified, providing a reliable method for standardizing the pharmacopeial herbs market and ensuring the clinical medication safety. However, the identification of Perilla frutescens and its variety P. frutescens var. frutescens should be combined with the secondary structure of ITS2.It is also suggested that Perilla frutescensa and P. frutescens var. frutescens could be combined into one species to provide reference for the classification of Perilla genus.