基于ITS2序列及二级结构对易混淆药材牛尾独活、当归、独活和羌活的鉴别研究

Molecular authentication of the medicinal species of Rhizoma et Radix Heraclei,Radix Angelicae Sinensis,Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii by integrating ITS2 and its secondary structure

本文利用ITS2序列及其二级结构对传统中药材牛尾独活、当归、独活和羌活进行分类鉴定。通过GenBank数据库中下载牛尾独活等药材共7个物种26条ITS序列,采用隐马尔可夫模型注释获得ITS2序列。采用MEGA软件计算种内、种间K2P遗传距离,构建NJ系统发育树。同时利用ITS2 Database预测各样本ITS2二级结构,并利用4Sale软件比对一级结构和二级结构的联合矩阵,最终采用ProfDistS软件基于距离法构建PNJ进化树。结果表明,各样本种间平均遗传距离均远大于种内平均遗传距离,各药材间存在明显的Barcoding Gap;系统发育树显示各样本均独立分支;样本间的二级结构均有一定的差异;PNJ进化树比NJ树显示出更高的分辨率。因此,本研究建议将ITS2作为鉴别药材牛尾独活、当归、独活和羌活及其基源植物的DNA条形码,为4种中药材的分类与准确鉴定提供一定的科学依据。

This study is to identify Chinese medicinal materials Rhizoma et Radix Heraclei,Angelicae Sinensis,Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii based on ITS2 and its secondary structure.Total 26 ITS sequences of 7 species were downloaded from GenBank,the ITS2 sequences were annotated by HMMer method.The NJ phylogenetic tree was built by MEGA software,the intraspecific and interspecific K2P genetic distance were analyzed by MEGA as well.The ITS2 secondary structures of all taxa were predicted by ITS2 database.Sequence matrix of primary structure and secondary structure was aligned by 4Sale software.And the profile neighbor joining(PNJ)phylogenetic tree was constructed via the the ProfDistS software based on the distance method.The results show that,the average interspecific genetic distance was far greater than the average intraspecific genetic distance,an obvious barcoding gap was noted among all taxa;NJ tree showed that all species were clustered into seperate branches;each species had different secondary structures;the PNJ tree showed higher resolution than NJ tree.Therefore,ITS2 is suggested to be used as a barcode for distinguishing the original plants of Rhizoma et Radix Heraclei,Radix Angelicae Sinensis,Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii in this study,this provides some scientific basis for classification and accurate identification of these Chinese medicinal materials.