基于高通量测序技术对五种白头翁药材混合样品的DNA分子鉴定研究

Study on DNA molecular identification of mix samples of five species of Baitouweng medicinal materials based on high-throughput sequencing technology

中药白头翁应用历史悠久,药典收载为毛莨科植物白头翁Pulsatilla chinensis (Bge.) Regel的干燥根。市场流通中易混淆种有朝鲜白头翁、兴安白头翁、细叶白头翁、金县白头翁等,伪品众多,准确鉴定十分迫切。本研究采用宏基因组学的方法,基于高通量测序技术对5种白头翁药材混合样品的ITS2序列进行测序。首先对药材混合粉末中的总DNA进行提取,并对总DNA中ITS2片段进行PCR扩增,再利用Illumina MiSeq平台对DNA片段进行双端测序,最后采用FLASH、QIIME以及GraPhlAn等软件对序列进行整理分析并与本课题组前期上传至GenBank中的序列进行聚类分析。结果显示,混合样品中共得到53 024条ITS2序列,有效序列52 295条,白头翁属5种药材总计49 079条,其代表序列与本课题组前期上传至GenBank中的序列进行聚类分析后,5种白头翁分别聚为一支,呈单系性。结果表明,利用高通量测序技术,以ITS2序列作为DNA条形码,可以对5种白头翁药材的混合粉末进行有效鉴定,为混合中药材的基原鉴定提供新的方法和思路。

Traditional Chinese medicine Baitouweng have a long history of application.The pharmacopoeia included dry roots of Pulsatilla chinensis（Bge.） Regel of Ranunculaceae.There are easily confused species in the market circulation,such as P.cernua（Thunb.） Bercht.et Opiz.,P.dahurica（Fisch.） Spreng.,P.turczaninoviiKryl.et Serg.,and P.chinensis（Bge.） Regel var.kissii（Mandl） S.H.Li et Y.H.Huang,etc.In this study,using the method of metagenomics,based on high-throughput sequencing technology,the ITS2 sequence of mixed samples of five species of Baitouweng medicinal materials was sequenced.First,the total DNA extraction of medicinal materials mixing powder,and the ITS2 fragment of total DNA was amplified by PCR.Second,the Illumina MiSeq platform was used to carry out Paired-end sequencing for DNA fragments.Last,using FLASH,QIIME and GraPhlAn software to arrange and analyze,and clustering analysis with the sequences of uploaded to GenBank by our group in the early stage.The results showed that a total of 53 024 sequences of ITS2 were obtained from the mixed samples,there are 52 295 effective sequences,there are a total of 49 079 of five species of medicinal materials of P.Miller.After the representative sequences and the sequence of uploaded to GenBank by our group in the early stage were clustering analysis,5 species of Baitouweng medicinal materials were clustered into one branch separately,presenting monophyletic.The results showed that using the high-throughput sequencing technology,using ITS2 sequence as DNA barcode,the mix powder of 5 species of Baitouweng medicinal materials could be effectively identified.It provides a new method and thought for the origin identification of mixed Chinese medicinal materials.