药学学报, 2019, 54(5): 937-943
引用本文:
李军, 张燃, 于淑萍, 王晓敏, 冷晓红, 石林春. 中药材银柴胡及其混伪品的DNA条形码鉴定研究[J]. 药学学报, 2019, 54(5): 937-943.
LI Jun, ZHANG Ran, YU Shu-ping, WANG Xiao-min, LENG Xiao-hong, SHI Lin-chun. Identification of Chinese medicinal materials Stellariae Radix and its adulterants using DNA barcoding[J]. Acta Pharmaceutica Sinica, 2019, 54(5): 937-943.

中药材银柴胡及其混伪品的DNA条形码鉴定研究
李军1, 张燃1, 于淑萍2, 王晓敏3, 冷晓红1, 石林春4
1. 宁夏职业技术学院, 宁夏中药材开发与利用工程技术研究中心, 宁夏 银川 750021;
2. 承德医学院附属医院, 河北 承德 067000;
3. 宁夏大学农学院, 宁夏 银川 750021;
4. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193
摘要:
筛选合适的DNA条形码序列,以建立基于DNA条形码技术的银柴胡及其混伪品鉴定方法。本文收集并提取60份银柴胡及其混伪品的DNA,对4种DNA条形码序列ITS、psbA-trnH、rbcL和matK进行PCR扩增和测序,计算各序列PCR扩增和测序成功率;利用MEGA7.0分析序列特征、计算遗传距离,并构建Neighbour-joining(NJ)系统进化树;利用种间种内变异分析做Barcoding gap图;运用BLAST方法计算各序列鉴定成功率,通过以上指标综合评价不同序列的鉴别能力。结果显示,ITS序列的PCR扩增及测序成功率最高,为95.2%,matK最低,为75%;4种序列的长度范围为211~797 bp,ITS的GC含量在各序列中最高,为54.35%;matK和ITS的鉴定成功率较高,分别为92%和90%;Barcoding gap图显示ITS有明显的间隔区,rbcL、matK和psbA-trnH序列间隔区不明显或种间种内变异有部分重叠;NJ系统进化树显示,ITS可将银柴胡聚为一支,同属混伪品二柱繁缕与银柴胡明显分为不同支,rbcL和matK不能把混伪品二柱繁缕分开。因此,ITS序列可作为银柴胡及其混伪品鉴定的优选条形码序列,为银柴胡的真伪鉴定提供科学依据。
关键词:    银柴胡      混伪品      DNA条形码      ITS      分子鉴定     
Identification of Chinese medicinal materials Stellariae Radix and its adulterants using DNA barcoding
LI Jun1, ZHANG Ran1, YU Shu-ping2, WANG Xiao-min3, LENG Xiao-hong1, SHI Lin-chun4
1. College of Ningxia Vocational and Technical, Ningxia Research Center for the Development and Utilization of Chinese Herbal, Yinchuan 750021, China;
2. Affiliated Hospital of Chengde Medical College, Chengde 067000, China;
3. School of Agriculture, Ningxia University, Yinchuan 750021, China;
4. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
Abstract:
To accurately discriminate Stellariae Radix from its adulterants, four leading candidate DNA barcoding markers were evaluated. Sixty samples including Stellariae Radix and its adulterants have been newly collected and their total genomic DNA was extracted. Four DNA barcoding markers ITS, rbcL, psbA-trnH and matK were amplified and sequenced. Their sequence characteristic analyses, Kimura-2-parameter (K2P) distance calculation and Neighbor-joining (NJ) phylogenetic tree constructions were accomplished using the MEGA 7.0 software. DNA Barcoding gaps of the four DNA barcoding markers were estimated by the distributions of interand intra-sequence specific variations. Species identification efficiency was calculated using the BLAST method. The results showed that ITS had the highest (95.2%) while matK demonstrated the lowest (75%) PCR and sequencing efficiency. The length range of the four markers were in the ranger of 211-797 bp, and the G+C content of ITS was highest (54.35%). The identification efficiency of matK and ITS was 92% and 90% respectively. Barcoding gap could be found in ITS sequences. The NJ phylogenetic tree constructed using ITS sequences showed that samples of Stellariae Radix were separately formed into one clade, and samples of adulterants like Stellaria bistyla were clearly belong to different branches from Stellariae Radix, whereas NJ trees constructed using psbA-trnH, rbcL and matK could not differentiate Stellariae Radix from its adulterants. Therefore, ITS regions as DNA barcodes can stably and accurately distinguished Stellariae Radix from its adulterants, and provide a new technique for modern identification of Stellariae Radix.
Key words:    Stellariae Radix    adulterants    DNA barcoding    ITS    molecular identification   
收稿日期: 2019-01-07
DOI: 10.16438/j.0513-4870.2019-0012
基金项目: 2017年宁夏青年科技人才托举工程项目;宁夏回族自治区教育厅服务地方经济发展项目(NGY2016259);承德市科学技术局项目(201601A049);四川省科技计划项目重点研发项目(2019YFS0017).
通讯作者: 石林春,Tel:86-10-57833194,E-mail:lcshi@implad.ac.cn
Email: lcshi@implad.ac.cn
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