基于ITS2序列鉴别道地药材岷县当归及其混伪品

Identification of Angelica sinensis and its adulterants based on ITS2 sequence

目的利用核糖体r RNA基因内转录间隔区(ITS2)序列,对甘肃道地药材岷县当归(岷归)及其混伪品和近缘属药材进行鉴定分析。方法对供试材料ITS2序列进行PCR并双向测序,所得序列经Codon Code Aligner校对拼接后,利用MEGA 6.0对序列进行分析比对,计算种内、种间遗传距离,利用邻接法(NJ)构建系统聚类树,并应用ITS2数据库网站预测其ITS2二级结构。结果经PCR扩增测序,15份甘肃栽培当归与标准样品岷归1号序列比对无差异,序列长度230 bp,GC含量为55.46%,37份当归混伪品与近缘属药材ITS2序列长度为228～233 bp,GC含量为51.53%～65.65%。岷归与混伪品、近缘属药材共53条序列中共检测到变异位点220个,保守位点10个,信息位点213个。根据K2P遗传距离计算,岷县当归种内遗传距离明显小于种间遗传距离,基于ITS2序列构建的NJ树和网站预测的ITS2二级结构,均能将岷归与其混伪品和近缘属药材区分。结论 ITS2序列可作为当归鉴定的DNA条形码,为当归市场的健康可持续发展提供有效的技术手段。

Objective In this study,Angelica sinensis and its adulterants,close relatives were analyzed by ITS2 sequences to investigate the genetic structure from Min County,Gansu Province.Methods In this study,the internal transcribed spacer 2（ITS2） regions of samples were amplified by PCR and sequenced bi-directionally.Obtained sequences were assembled using Codon Code Aligner.The genetic distances were computed by MEGA 6.0 in accordance with the kimura 2-parameter（K2 P） model and the phylogenetic tree was constructed by Neighbor-joining（NJ） method.Moreover,the secondary structure of ITS2 was predicted using ITS2 database websites.Results The ITS2 region was 230 bp and base composition was GC content of 55.46% of A.sinensis in Min County.In its adulterants and close relatives of A.sinensis,the ITS2 region ranged in size from 228—233 bp and base composition was with G ＋ C content ranged from 51.53%—65.65%.A total of 220 polymorphic sires were detected from 53 sequences,in which parsimony informative sires were up to 213.By K2 P model,the intra-specific genetic distances of A.sinensis in Min County were smaller than inter-specific ones of A.sinensis and its adulterants in ITS2 regions.NJ tree and secondary structure results could distinctly differentiate quality product and adulterants.Conclusion ITS2 sequence can accurately identify the authenticity of A.sinensis,which provide a effective technology for the healthy and sustainable development in A.sinensis market.