基于ITS条形码的维药克孜力乔古鲁克药材的分子鉴定研究

摘要

目的 对维药克孜力乔古鲁克药材(Paeoniae radix hybridae,PRH)的品种进行精准鉴别,同时将其原植物块根芍药Paeonia hybrida Pall.与窄叶芍药P.anomala L.和新疆芍药P.sinjiangensis K.Y.Pan进行区分,为保证药材来源的准确性提供科学依据. 方法 提取块根芍药的DNA,基于内部转录间隔区的核糖体DNA序列(ITS)进行PCR引物扩增并测序,并从GenBank数据库下载块根芍药和其原变种窄叶芍药和近缘种新疆芍药的ITS序列,采用DNAMAN、Editseq、MeGA5等软件,计算块根芍药种间、种内的Kimura 2-parameter(K 2-P)遗传距离,寻找块根芍药的特异位点,并构建Neighbor-joining(N-J)系统聚类树. 结果 块根芍药G+C含量范围为55.82%~56.45%,与另外两个种G+C含量未重叠;所有样本ITS序列的K 2-P遗传距离范围为0~0.026,其中种间遗传距离分布为0.014~0.026,大于块根芍药种内遗传距离0.000~0.012;通过序列比对分析,发现块根芍药与另外2个种有6个特异位点;N-J系统聚类树也将19个样品聚为两个大支系,所有块根芍药聚为一支,有84%支持率,其中中国地区分布的块根芍药聚为一小支,俄罗斯地区分布的块根芍药亲缘关系较远. 结论 经过遗传距离、系统聚类、特异位点分析等系统研究发现ITS序列能区分块根芍药与其原变种窄叶芍药和近缘种新疆芍药,可以应用于维药PRH的精准鉴别,并且在变种与原变种的区分方面有很大潜力.%Objective To accurately identify the variety of Paeoniae radix hybridae and distinguish Paeonia hybrida Pall.with Paeonia anomala L.and Paeonia sinjiangensis K.Y.Pan to provide a scientific basis for ensuring the accuracy of material sources.Methods Total genomic DNA was extracted from P.hybrida and internal transcribed spacer (ITS) was amplified by PCR and the product sent to sequence.ITS sequences of P.hybrida, P.anomala and P.sinjiangensis were downloaded from GenBank database.Interspecific and intraspecific Kimura 2-parameter(K 2-P) genetic distance, specific site and Neighbor-joining(N-J)system cluster tree were acquired based on softwares of ContigExpress、DNAMAN、Editseq、MeGA5.Results The content of G+C in Paeonia hybrid was ranged from 55.82% to 56.45%, which not overlapped with the other two species;The K 2-P genetic distance of all sequences was ranged from 0 to 0.026.The K 2-P genetic distance of interspecific and intraspecific samples was respectly ranged from 0.014 to 0.026 and 0.000 ~ 0.012;Compared with the other two categories, P.hybrid has 6 specific sites;Total samples were classified into 2 branches which containing one branch of P.hybrida with 84% in favor.Among of those, P.hybrid distributed in region of China clustered into one small branch, while the P.hybrid distributed in Russia was far away from those in genetic relationship.Conclusion ITS can distinguish P.hybrid from P.anomala and P.sinjiangensis by genetic distance, systematic cluster tree, site-specific analysis research and could be used for precise discrimination of Paeoniae radix hybridae and have great potential to make a distinction between the varieties and the original variety.