长白山地区药用植物龙胆DNA条形码鉴定  

DNA Barcoding Identification of Gentiana scabra in Changbai Mountains

作　　者：张强[1,2] 张维维 丁勇 王佳雪 方春秋 齐伟辰 ZHANG Qiang;ZHANG Wei-wei;DING Yong;WANG Jia-xue;FANG Chun-qiu;QI Wei-chen(School of Pharmaceutical Sciences,Changchun University of Chinese Medicine,Changchun 130117,China;Jilin Engineering Center for Traditional Chinese Medicine Resources in Changbai Mountain,Changchun 130117,China)

机构地区：[1]长春中医药大学药学院,吉林长春130117 [2]吉林省长白山中药资源工程研究中心,吉林长春130117

出　　处：《中国现代中药》2020年第11期1817-1821,共5页Modern Chinese Medicine

基　　金：中医药行业科研专项(201207002);吉林省代表区域中药资源保护利用(201207002-05);2017年吉林省中医药科技项目(2017148);2020年吉林省中医药科技项目(2020052);中央本级重大增减支项目(2060302)。

摘　　要：目的:基于内转录间隔区2(internal transcribed spacer 2,ITS2)碱基序列,运用DNA条形码鉴定技术对长白山地区药用植物龙胆进行鉴别研究,为龙胆药材的基原植物鉴定和药材的真伪鉴别提供参考。方法:采用十六烷基三甲基溴化铵(CTAB)提取法提取龙胆叶片及药材的DNA,对其中特定ITS2碱基序列进行聚合酶链式反应(PCR)扩增,检测后测序,并从GenBank下载同属植物序列,运用SeqMan软件对碱基序列拼接后,采用MEGA 7.0软件对数据进行分析处理及对比,计算Kimura 2-parameter(K2P)遗传距离,建立邻接(NJ)法,进行对比分析。结果:根据NJ聚类树可知,不同种龙胆属植物笔龙胆、三花龙胆、高山龙胆、坚龙胆都自为一支,区分明显,龙胆和条叶龙胆聚在一支未能区分开,同时结合变异位点及K2P遗传距离发现,条叶龙胆和龙胆应用ITS2碱基序列区分时种间差异十分小。吉林省长白山10个不同地区的龙胆种内几乎无差异,经Blast比对确定碱基序列的确为所需要鉴定的品种。结论:运用ITS2碱基序列的DNA分子条形码鉴别方法对龙胆植物及药材进行鉴别有效可行且成功率较高,可以用于龙胆属种间及种内鉴别。Objective:Based on base sequence of the internal transcribed spacer 2(ITS2),DNA barcoding was used to identify Gentiana scabra in Changbai Mountain area,to provide scientific basis for the identification of G.scabra.Methods:CTAB extraction method was used to extract DNA of gentian leaf and medicinal herbs of DNA,some specific ITS2 base sequences were amplified by PCR and sequenced after detection.And the sequence of plants of the same species was downloaded from GenBank.After the base sequence was splited with SeqMan software,MEGA 7.0 software was used to analyze,process and compare the data,calculate the Kimura 2-parameter(K2P)genetic distance,and establish neighbor-joining(NJ)tree for comparative analysis.Results:According to the NJ clustering tree,G.zollingeri,G.triflora,G.rigescens and G.algida are all of their own species,which are clearly distinguished from each other.G.scabra and G.manshurica were clustered in one branch and could not be distinguished.At the same time,it was found that G.scabra and G.manshurica were distinguished by ITS2 base sequence by combining mutation site and K2P genetic distance,and the difference between species was very small.There was almost no difference among gentiana species in ten different areas of Changbai Mountain,Jilin Province.Blast confirmed the base sequence as the species that needed to be identified.Conclusion:ITS2 base sequence is an effective and feasible method for the identification of G.scabra.DNA molecular barcode identification method can be used for the identification of Dentiana between species and within species.

关 键 词：长白山地区 龙胆 内转录间隔区2 DNA条形码 

分 类 号：R282.5[医药卫生—中药学]