17种毛茛科藏药材的分子鉴别  

Molecular Identification of 17 Tibetan Medicinal Materials of Ranunculaceae Based on ITS2 Sequence

作　　者：慕泽泾 吴晓鑫 游茜茜 杜小浪 钟国跃 王晓云 Mu Zejing;Wu Xiaoxin;You Xixi;Du Xiaolang;Zhong Guoyue;Wang Xiaoyun(Research Center for Traditional Chinese Medicine Resources and Ethnic Minority Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西中医药大学中药资源与民族药研究中心,南昌330004

出　　处：《世界科学技术-中医药现代化》2022年第7期2594-2601,共8页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家科学技术部国家重点研发计划(2019YFC1712302):50种藏药品种整理与质量评价研究,负责人:冯育林

摘　　要：目的毛茛科的多种植物是民族药的基原,本研究以ITS2序列作为DNA条形码对17种毛茛科藏药材进行鉴定,为药材的安全使用提供可靠的检测方法。方法从四个省份采集或购买17种毛茛科藏药材样本,分布于8个属。采用试剂盒法提取基因组DNA,PCR扩增ITS2序列后,将扩增产物进行双向测序。利用CodonCode Aligner(v.9.0.1)软件进行序列拼接,将拼接后的序列在NCBI网站进行在线比对。在GenBank数据库中下载相关物种的rDNA序列(包含完整的ITS2区域)。上述序列经过CodonCode Aligner(v.9.0.1)软件剪切后,采用BLAST方法进行比对,然后再采用两种方法评估ITS2序列的鉴定能力。第一种是系统聚类树法(Tree-based method),用MEGA(v.5.0)软件构建NJ系统聚类树(Neighbour-Joining tree);第二种是遗传距离法(Distance-based method),用TaxonGap(v.2.4.1)软件分析物种的种内与种间变异关系。结果29份试验样本的ITS2序列测序成功率为100%,与93条数据库中下载的ITS2序列合并后,长度为213-230 bp,比对长度为244 bp,变异位点数72.1%。BLAST结果表明,29条ITS2序列可以将全部物种鉴定到属。系统聚类分析结果表明,ITS2序列能够成功鉴别8种药材;遗传距离分析结果表明,ITS2序列能够成功鉴别7种毛茛科药材。两种方法总共可以鉴别9种药材,它们是露蕊乌头(Aconitum gymnandrum)、草玉梅(Anemone rivularis)、多小叶升麻(Cimicifuga foetida var.foliolosa)、短尾铁线莲(Clematis brevicaudata)、长花铁线莲(Clematis rehderiana)、甘青铁线莲(Clematis tangutica)、腺毛黑种草(Nigella glandulifera)、拟耧斗菜(Paraquilegia microphylla)和芸香叶唐松草(Thalictrum rutifolium),鉴定效率为52.9%。结论ITS2序列可以快速、准确地识别和鉴定部分毛茛科藏药材,为原植物和药材的分子鉴定提供依据。Objective To provide a reliable method for the safe use of medicinal materials,i.e.used the ITS2 sequence as a DNA barcode to identify 17 Tibetan medicinal plants of the Ranunculaceae family,because that many species of the Ranunculaceae family are the original plants of ethnic medicine.Methods A total of 29 Tibetan medicinal plant samples belong to 17 species distributed in 8 genera were collected from Sichuan Province and other places.The genomic DNA of the sample was extracted by the kit method.The ITS2 sequence was obtained by PCR and bidirectional sequencing,then spliced and blasted online.The rDNA sequence containing the complete ITS2 region of related species was downloaded from GenBank database.After the above sequences were spliced by the CodonCode Aligner(v.9.0.1)software,they were blasted firstly,and then two methods were used to evaluate the identification ability of ITS2.The first was the tree-based method,i.e.building Neighbour-Joining tree by MEGA(v.5.0)software;the second was the distance-based method,i.e.analyzing intra-species and inter-species variation of species by TaxonGap(v.2.4.1)software.Results The success rate of ITS2 sequence sequencing of 29 samples was 100%.After merging with 93 ITS2sequences downloaded from the database,the length was predicted as 213-230,the alignment length was 244bp,and the number of mutation sites was 72.1%.BLAST results showed that 29 ITS2 sequences can identify all species to genera.The result of the tree-based method showed that the ITS2 sequence could successfully identify 8 Tibetan medicinal plant species;the result of the distance-based method showed that the ITS2 sequence could successfully identify 7Tibetan medicinal plant species.The combined results of two methods have proved that the ITS2 region can be used to distinguish nine Tibetan medicinal species,i.e.Aconitum gymnandrum,Anemone rivularis,Cimicifuga foetida var.foliolosa,Clematis brevicaudata,Clematis rehderiana,Clematis tangutica,Nigella glandulifera,Paraquilegia microphylla and Thalictrum rutifo

关 键 词：毛茛科 ITS2序列 DNA条形码 分子鉴定 

分 类 号：R282.5[医药卫生—中药学]