藏药加哇及其易混品的ITS2序列鉴定研究  

Identification of Tibetan Medicine Jiawa and Easily Mixed Species Based on ITS2 Barcode

作　　者：杨吉玉 高必兴 齐景梁 王艺娱 李倩 连艳[3] YANG Jiyu;GAO Bixing;QI Jingliang;WANG Yiyu;LI Qian;LIAN Yan(Key Laboratory of Quality Evaluation of Chinese Patent Medicines,Sichuan Institute for Drug Control,NMPA,Chengdu 611731,China;Luzhou College of Medical Devices,Luzhou 646000,China;College of Pharmacy,Chengdu University of Traditional Chinese Medicine,State Key Laboratory of Traditional Chinese Medicine Resources of Southwestern China,Key Laboratory of Standardization of Chinese Herbal Medicine,Chengdu 611137,China)

机构地区：[1]四川省药品检验研究院,国家药品监督管理局中成药质量评价重点实验室,成都611731 [2]泸州医疗器械职业学院,四川泸州646000 [3]成都中医药大学药学院,西南特色中药资源国家重点实验室,中药材标准化教育部重点实验室,四川成都611137

出　　处：《中国药学杂志》2024年第8期687-693,共7页Chinese Pharmaceutical Journal

基　　金：国家药品监督管理局药品监管科学体系建设重点项目资助(RS2024Z006)。

摘　　要：目的 利用DNA条形码核基因内部转录间隔区(ITS2)序列,建立加哇及其易混品的有效鉴定方法。方法 采用试剂盒提取72批加哇及其易混品的DNA,针对其ITS2片段进行聚合酶链式反应(PCR)扩增并双向测序,获取ITS2序列信息,同时从GenBank下载相关序列30条,应用MEGA6.0软件对102条ITS2序列进行序列比对分析,计算种内和种间Kimura双参数遗传距离(K2-P),构建邻接法(neighbor-joining, N-J)系统进化树,并预测ITS2二级结构。结果 K2-P遗传距离分析显示,除松潘棱子芹与瘤果棱子芹外,西藏棱子芹、迷果芹、西藏凹乳芹、葛缕子、裂叶独活、羽苞藁本及刺果峨参具有明显的条形码间距。N-J树聚类分析显示,各物种均能以>50%的自展支持率各自聚为1支,单系性良好。ITS2二级结构分析显示,加哇与其易混品在4个螺旋区的茎环数目、大小、位置有所差异。结论 ITS2序列可用来鉴别加哇及其易混品,有利于加哇的质量控制及资源利用。OBJECTIVE To establish an effective identification method for Jiawa and easily mixed species by using DNA barcoding ITS2 sequence.METHODS The total DNA of 72 Jiawa and its similar species samples were extracted by using the plant genomic DNA Extraction kit,PCR amplification and bidirectional sequencing were carried out for the ITS2 fragments,another 30 items of the genus were downloaded from GenBank.MEGA6.0 software was applied to analyze 102 ITS2 sequences,calculate the intraspecific and interspecific K2-P distance,construct neighber-jioning(N-J)phylogenetic tree,and predict the secondary structure of ITS2.RESULTS K2-P genetic distance analysis showed that,in addition to Pleurospermum franchetianum Hemsl.and Pleurospermum wrightianum de Boiss.,Pleurospermum hookeri var.thomsonii C.B.Clarke.,Sphallerocarpus gracilis(Bess.)K.-Pol.,Vicatia thibetica de Boiss.,Carum carvi L.,Heracleum millefolium Diels.,Ligusticum daucoides(Franch.)Franch.and Anthriscus sylvestris subsp.Nemorosa have obvious barcode spacing.N-J tree cluster analysis showed that each species could be clustered into one branch with a bootstrap support rate of>50%,and the monophyleticity was good.The secondary structure analysis of ITS2 showed that the number,size and position of stem-loops in the four helical regions of Jiawa and its miscible were different.CONCLUSION The ITS2 sequence can be used to identify Jiawa and its easily mixed species,which is beneficial to the rational development and utilization of Jiawa resources.

关 键 词：加哇 核基因内部转录间隔区序列 易混品 藏药 

分 类 号：R282[医药卫生—中药学]