透骨草药材的ITS2条形码鉴定研究

Identification on ITS2 Barcode of Tougucao of genus Vicia L.

目的:基于ITS2序列对透骨草药材进行分子鉴别研究,为透骨草准确鉴别提供理论基础。方法:利用试剂盒提取透骨草DNA,对透骨草药材的ITS2序列进行PCR扩增和测序,并从GenBank中下载同科属植物ITS2序列,利用CodonCode Aligner软件拼接,采用MEGA 5.10进行数据分析,基于K2P模型构建聚类树(NJ树),应用BLAST方法进行鉴定分析。结果:ITS2序列可将87个样本互相区分开并聚为3类,山野豌豆、毛山野豌豆、狭山野豌豆、蚕豆、大花野豌豆聚为一类,其中蚕豆和大花野豌豆各自单聚小类;广布野豌豆单聚一类;其他聚为一大类,区分明显。透骨草药材的二级结构构型存在一定的特征。结论:基于ITS2序列的DNA条形码能够有效鉴定透骨草药材。

Objective:To perform molecular identification research of Tougucao based on ITS2 sequence,in order to provide a theoretical basis for the accurate identification of Tougucao.Methods:The DNA of Tougucao was extracted by the kit extraction method,the ITS2 sequences of Tougucao were amplified and sequenced by PCR and ITS2 sequences of the same family were downloaded from GenBank.CodonCode Aligner software was used for sequence splicing and data Analysis was performed with MEGA 5.10.Clustering tree(NJ tree)was constructed based on K2 P model,BLAST method was used for identification and analysis.Results:Eighty-seven samples could be divided into 3 groups by ITS2 sequence.Vicia amoena,Vicia amoena var.sericea,Vicia amoena var.angusta,Vicia faba,Vicia bungei were clustered into one group,in which Vicia faba and Vicia bungei were respectively grouped into small groups,Vicia cracca were singly clustered into one group,and the others were grouped into a large category,which could be clearly distinguished.There were some characteristics in the secondary structure and configuration of Tougucao.Conclusion:The DNA barcode based on ITS2 sequence can effectively indentify Tougucao.