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中药材锁阳的ITS2条形码分子鉴定研究
引用本文:侯典云,宋经元,石林春,杨培,陈士林,姚辉.中药材锁阳的ITS2条形码分子鉴定研究[J].中国中药杂志,2013,38(23):4028-4032.
作者姓名:侯典云  宋经元  石林春  杨培  陈士林  姚辉
作者单位:河南科技大学 农学院, 河南 洛阳 471003;中国医学科学院 北京协和医学院 药用植物研究所 濒危药材繁育国家工程实验室, 北京100193;中国医学科学院 北京协和医学院 药用植物研究所 濒危药材繁育国家工程实验室, 北京100193;中国医学科学院 北京协和医学院 药用植物研究所 濒危药材繁育国家工程实验室, 北京100193;中国医学科学院 北京协和医学院 药用植物研究所 濒危药材繁育国家工程实验室, 北京100193;中国医学科学院 北京协和医学院 药用植物研究所 濒危药材繁育国家工程实验室, 北京100193;中国中医科学院 中药研究所, 北京 100700;中国医学科学院 北京协和医学院 药用植物研究所 濒危药材繁育国家工程实验室, 北京100193
基金项目:国家自然科学基金项目(81130069);国家高技术研究发展计划(863 )项目(2012AA021602);教育部长江学者和创新团队发展计划项目(IRT1150)
摘    要:目的:应用DNA条形码技术准确、快速鉴定中药材锁阳及其混伪品。方法:采用试剂盒法提取药材的基因组DNA,PCR扩增其核糖体DNA的内转录间隔区(internal transcribed spacer 2,ITS2)并进行双向测序,应用CodonCode Aligner V 3.7.1对测序峰图进行校对拼接,比较ITS2序列的GC含量,采用MEGA 5.0计算种内、种间 Kimura 2-parameter(K2P)遗传距离,通过中药材DNA条形码鉴定系统比对和构建NJ 系统聚类树进行鉴定分析。结果:锁阳药材的ITS2序列长度为229 bp,种内变异较小,平均K2P遗传距离为0.003,锁阳药材及其混伪品的种间平均K2P遗传距离为0.760,种间最小K2P遗传距离明显大于锁阳种内的最大K2P遗传距离。中药材DNA条形码鉴定系统比对和NJ树鉴定结果均表明ITS2 序列可区分锁阳及其混伪品。结论:ITS2条形码可有效鉴定中药材锁阳及其混伪品,且具有较好的稳定性和准确性,为保障临床安全用药提供了新的技术手段。

关 键 词:锁阳  DNA条形码  ITS2  分子鉴定
收稿时间:2013/5/13 0:00:00

Molecular identification of Cynomorii Herba using ITS2 DNA barcoding
HOU Dian-yun,SONG Jing-yuan,SHI Lin-chun,YANG Pei,CHEN Shi-lin and YAO Hui.Molecular identification of Cynomorii Herba using ITS2 DNA barcoding[J].China Journal of Chinese Materia Medica,2013,38(23):4028-4032.
Authors:HOU Dian-yun  SONG Jing-yuan  SHI Lin-chun  YANG Pei  CHEN Shi-lin and YAO Hui
Institution:Agricultural College, Henan University of Science and Technology, Luoyang 471003, China;The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China;The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China;The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China;The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China;The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China;Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
Abstract:Objective: To identify the Cynomorii Herba and its analogues species using DNA barcoding technique. Method: Total genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2(ITS2) regions were amplified using polymerase chain reaction (PCR), and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter (K2P) distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) trees. Result: The ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification. Conclusion: The ITS2 region is an efficient barcode for identification of Cynomorii Herba,which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.
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