基于ITS2序列的中药材苍术种苗DNA条形码鉴定  

Identification of DNA Barcoding of Atractylodis Rhizoma Seedlings by ITS2 Sequence

作　　者：刘金欣[1,2] 李耿 陈彩霞[2] 魏妙洁[1] 王燕 赵春颖[1] 石林春[2] 

机构地区：[1]承德医学院河北省中药研究与开发重点实验室,河北承德067000 [2]中国医学科学院,北京协和医学院药用植物研究所,北京100193 [3]中日友好医院,北京100029

出　　处：《中国实验方剂学杂志》2018年第2期34-38,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81303158,81703659);河北省教育厅青年拔尖人才项目(BJ201602);河北省自然科学基金项目(H2017406031)

摘　　要：目的：建立基于DNA条形码技术的中药材苍术种苗鉴定方法。方法：收集中药材苍术及其易混品原植物样品28份，构建苍术种苗鉴定参考核糖体内部转录间隔区2（ITS2）条形码数据库。从河北、山东、山西、内蒙古、辽宁及湖北等苍术主产地收集苍术种苗52份。通过提取基因组DNA，聚合酶链式反应（PCR）扩增，双向测序获得其ITS2条形码序列。应用PUAP4．0软件计算种内、种间遗传距离，利用MEGA7．0软件构建邻接树，对中药材苍术种苗进行DNA条形码鉴定。结果：中药材苍术及其易混品原植物ITS2条形码序列具有稳定的序列差异，邻接树呈现单系性，可以明显区分苍术及其易混品；基于标准参考数据库，42份苍术种苗为朝鲜苍术（80．8％），7份苍术种苗为苍术（13．5％），3份苍术种苗为白术（5．7％）。结论：基于ITS2序列可以准确区分中药材苍术及其混伪品种苗。苍术种苗基原物种鉴定结果以朝鲜苍术为主，提示苍术临床用药存在潜在安全风险。Objective： In order to guarantee the species correction of Atractylodis Rhizoma seedlings, a molecular identification method with internal transcribed spacer 2 （ITS2） as DNA barcode has been tested. Method： Here, 28 original plant specimens of Atractylodis Rhizoma and their common adulterants have been collected to construct its standard reference ITS2 sequence database. Fifty-two Atractylodis Rhizoma seedlings have been collected from Hebei province, Shandong province, Shanxi province, Inner Mongolia autonomous region, Liaoning province and Hubei province to verify their original species. Their ITS2 sequences have been obtained after DNA extraction, polymerase chain reaction （PCR） and bi-directional sequencing. The intra-species and inter-species genetic distances were calculated by PUAP 4.0 software. The neighbor-joining （N J） trees were constructed by MEGA 7.0 software. Result： The stable sequence divergence has been found between ITS2 barcode sequences of Atractylodis Rhizoma and their common aduherants, their NJ trees showed monophyly. With the help of standard reference ITS2 sequence koreana, 7 Atractylodis Rhizoma see database, we found 42 Atractylodis Rhizoma seedlings were from Atractylodes dlings were from A. lancea, 3 Atractylodis Rhizoma seedlings were from A macrocephala. Conclusion： Atractylodis Rhizoma seedlings are mainly come from A. koreana. This result is consistent with market survey of medicinal resources, suggesting that Atractylodis Rhizoma has safety risk. The DNA barcoding method based on the ITS2 sequence can accurately distinguish between Atractylodis Rhizoma seedlings and their adulterants seedlings

关 键 词：苍术 种苗 DNA条形码 核糖体内部转录间隔区2 朝鲜苍术 

分 类 号：R282[医药卫生—中药学] R931[医药卫生—中医学]