羌活药材ITS/ITS2条形码鉴定及其稳定性与准确性研究

Stability and accuracy of the identification of Notopterygii Rhizoma et Radix using the ITS/ITS2 barcodes

为验证DNA条形码鉴定的稳定性与准确性,本文选用羌活药材作为研究对象,对31份样本提取基因组DNA,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增内部转录间隔区(internal transcribed spacer,ITS)序列并进行双向测序,所得序列经CodonCode Aligner拼接后,采用MEGA5.0软件与其混伪品进行序列比对,计算种内和种间距离,构建邻接树(neighbor-joining tree,NJ Tree)。ITS2序列采用基于隐马尔可夫模型(hidden Markov model,HMMer)的注释方法获得。结果表明,羌活药材ITS序列长度为603～604 bp,ITS2序列长度均为228 bp,羌活药材ITS/ITS2序列单倍型与其基原植物叶片序列一致。两种基原植物ITS/ITS2序列种内平均kimura 2-parameter(K2P)遗传距离均远远小于其与混伪品的种间平均K2P遗传距离;NJ树结果显示羌活、宽叶羌活与其混伪品均可明显区分,表现出良好单系性。因此ITS/ITS2序列作为DNA条形码能稳定、准确鉴别羌活药材,为保障临床安全用药提供了新的技术手段。

In this study,Notopterygii Rhizoma et Radix was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time.All genomic DNAs from thirty one samples were extracted.The ITS（internal transcribed spacer） regions were amplified and sequenced bi-directionally.Obtained sequences were assembled using the CodonCode Aligner.And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter（K2P） model.The neighbor-joining（NJ） phylogenetic trees were constructed.The ITS2 regions were obtained by using the hidden Markov model（HMM）-based annotation methods from the ITS sequences.Results indicated that the lengths of ITS regions of Notopterygii Rhizoma et Radix were 603？604 bp,while the lengths of ITS2 regions were 228 bp.The haplotypes of ITS/ITS2 regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves.The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Notopterygium incisum and N.franchetii.The NJ trees showed that N.incisum,N.franchetii and its adulterants can be easily differentiated according to their monophyly.Therefore,ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.