摘要
目的:建立基于核糖体DNA内转录间隔区(ITS)序列的北柴胡种子分子鉴定方法,保障北柴胡栽培种子的物种准确。方法:收集北柴胡及主要栽培柴胡属物种种子、市售北柴胡种子样品共59份,探究不同取样量和不同水浴条件对种子DNA提取质量的影响,建立柴胡属种子DNA提取方法;通过聚合酶链式反应(PCR)和双向测序得ITS条形码序列。从GenBank下载34条北柴胡、红柴胡、黑柴胡等主要栽培柴胡属物种ITS序列,丰富北柴胡种子鉴定数据库;利用MEGA-X10.0.5进行变异位点分析并构建邻接(NJ)系统发育树,考察ITS序列对北柴胡种子的物种鉴定能力;基于BLAST方法和NJ系统发育树法对市售北柴胡种子进行DNA条形码鉴定。结果:共获得59条北柴胡、红柴胡、三岛柴胡及市售北柴胡种子的ITS序列,北柴胡ITS序列可分为6个单倍型,包含7个变异位点,NJ系统发育树表明北柴胡所有单倍型可形成独立的枝,与所收集样品中其他柴胡属栽培物种可相互区分,具备北柴胡种子物种鉴定能力。基于ITS条形码序列鉴定发现19份市售北柴胡种子中有3份为三岛柴胡,伪品率15.8%。结论:基于ITS序列的DNA条形码技术可以准确可靠地鉴定北柴胡种子及其易混品,可为我国中药材种子规范化建设提供参考。
Objective:To establish a molecular identification method for Bupleurum chinense seeds based on ribosomal DNA internal transcribed spacer(ITS)sequence,ensuring the species authenticity of the cultivated seeds of B.chinense.Method:A total of 59 seeds samples of B.chinense and its main cultivated species,marketed B.chinense were collected.The effect of different sampling amounts and different water bath conditions on DNA extraction quality of the seeds was investigated,a DNA extraction method for seeds of Bupleurum was established.Their ITS sequences were obtained by polymerase chain reaction(PCR)and bidirectional sequencing.In addition,34 ITS sequences of main cultivated Bupleurum species,such as B.chinense,B.scorzonerifolium,B.falcatum and B.smithii,were downloaded from GenBank to enrich identification database of B.chinense seeds.The neighbor-joining(NJ)dendrogram were constructed by MEGA-X 10.0.5 software to investigate the the species identification ability of ITS sequences for B.chinense seeds.And DNA barcoding identification of marketed B.chinense seeds was conducted based on BLAST method and NJ dendrogram method.Result:In total,59 ITS sequences were obtained.ITS sequences of B.chinense could be divided into six haplotypes,including seven variable sites.The NJ dendrogram indicated that all the haplotypes of B.chinense could form independent branches,which could be distinguished from other cultivated species of Bupleurum in the collected samples,and possessed the ability to identify species of B.chinense seeds.Based on ITS sequence barcoding identification,3 of the 19 marketed B.chinense seeds were B.falcatum with a counterfeit rate of 15.8%.Conclusion:DNA barcoding technology based on ITS sequence can accurately and reliably identify B.chinense seeds and its adulterants,providing reference for the standardization construction of Chinese medicinal materials seeds.
作者
赵晴
谢红波
央拉
魏妙洁
石林春
刘金欣
赵春颖
ZHAO Qing;XIE Hong-bo;YANG La;WEI Miao-jie;SHI Lin-chun;LIU Jin-xin;ZHAO Chun-ying(Hebei Key Laboratory of Research and Development of Chinese Medicine,Chengde Medical University,Chengde 067000,China;School of Medicine,Tibet University,Lhasa 850000,China;Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第14期182-189,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81703659)
河北省自然科学基金项目(H2017406031)
河北省教育厅青年拔尖人才项目(BJ201602)
河北省“三三三人才工程”项目(A2017002090)
河北省高校重点学科建设项目(冀教高[2013]4号)
河北省教育厅在读研究生创新能力培养项目(CXZZSS2019132)。
