Molecular Identification of Ilicis Chinensis Folium from Its Closely Related Species and Adulterants Based on Ribosomal Internal Transcribed Spacer 2 (ITS2) Sequence
Abstract Ilicis Chinensis Folium is a traditional Chinese herbal medicine, which derived from one kind of ornamental plants named Ilex chinensis. In order to identify adulterants emerged in the market and closely related species in Ilex rapidly and reliably by DNA barcoding, ribosomal internal transcribed spacer 2 (ITS2) from 37 materials of I. chinensis, Ilex rotunda, Ilex asprella, Ilex cornuta and Ligustrum lucium was amplified and sequenced, respectively. Assembled by CodonCode Aligner 4.2.7, all of the 37 ITS2 sequences ampilified and 28 GenBank sequences of Ilex, L. lucium, Symplocos setchuensis were annotated based on hidden Markov model (HMM) to cut off 5.8S and 28S so that complete ITS2 region could be gained. Kimura 2-parameter (K2P) genetic distance analysis of intraspecific and interspecific accompanied by neighbor-joining tree (NJ tree) construction was performed by molecular evolutionary genetics analysis (MEGA6.0). Results indicated that unique bands were obtained from gel electrophoresis demonstrating the successful amplification of ITS2 sequences by the universal primer. Sequencing results showed that the aligned sequence length of I. chinensis was 239 bp and the interspecific one was 269 bp. Four variable sites of intraspecies were discovered, which divided the I. chinensis into 6 haplotypes. In contrast, there were 143 interspecific variable sites which were much more than those of I. chinensis. Sequence characteristics analysis of intraspecific and interspecific by MEGA6.0 showed the maximum K2P genetic distance of intraspecies was 0.013, which was much shorter than the minimum K2P genetic distance of interspecies of 0.094. L. lucium and S. setchuensis clustered into separate clade with bootstrap scores both of 100%. I. chinensis distributed to a single cluster that belonged to the cluster of the Ilex. Species in the same subgenus of Ilex could also cluster together. Therefore, ITS2, as one of the universal DNA barcodes, could differentiate I. chinensis from its adulterants and closely related species effectively. As a molecular biological method for discrimination, DNA barcoding possessed huge potential to authenticate the Ilex, meanwhile, it provides scientific foundation for clinical medication safety, exploration and application.
YU Li-Li,QIAO Wen-Jing,CHEN Xiao-Chen, et al. Molecular Identification of Ilicis Chinensis Folium from Its Closely Related Species and Adulterants Based on Ribosomal Internal Transcribed Spacer 2 (ITS2) Sequence[J]. , 2015, 23(5): 598-605.
Chen S L, Pang X H, Song J Y, et al. 2014. A renaissance in herbal medicine identification: From morphology to DNA[J]. Biotechnology Advances, 32(7):1237-1244. Chen S L, Yao H, Han J P, et al. 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J]. PloS ONE,5(1):e8613. Hebert P D, Cywinska A, Ball S L, et al. 2003. Biological identifications through DNA barcodes[J]. Proceedings of the Royal Society of London. Series B: Biological Sciences,270(1512):313-321. Keller A, Schleicher T, Schultz J, et al. 2009. 5.8S-28S rRNA interaction and HMM-based ITS2 annotation [J]. Gene,430(1-2):50-57. Chen X C, Liao B S, Song J Y, et al. 2013. A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding [J]. Gene,530(1):39-43. Yao H, Song J Y, Liu C, et al. 2010. Use of ITS2 region as the universal DNA barcode for plants and animals [J]. PLoS ONE,5(10):e13102. 陈景艳, 姜运力, 潘德权. 2006. 贵州野生冬青属树木观赏生物学特性研究[J]. 贵州林业科技,34(3):25-28. (Chen JY, Jiang YL, Pan DQ.2006. Study on the ornamental characteristics of wild Ilex in Guizhou[J]. Guizhou Forestry Science and Technology,34(3): 25-28.) 陈士林, 郭宝林, 张贵君,等. 2012. 中药鉴定学新技术新方法研究进展[J]. 中国中药杂志,37(8):1043-1055.(Chen S L, Guo B L, Zhang G J, et al. 2012. Advances of studies on new technology and method for identifying traditional Chinese medicinal materials[J]. China J ournal of Chinese Materia Medica, 37(8): 1043-1055.) 陈士林, 庞晓慧, 罗焜,等. 2013. 生物资源的DNA条形码技术[J]. 生命科学,25(5):458-466.(Chen S L, Pang X H, Luo K, et al. 2013. DNA barcoding of biological resources[J]. Chinese Bulletin of Life Sciences, 25(5):458-466.) 陈士林, 姚辉, 韩建萍,等. 2012. 中药DNA条形码分子鉴定 [M]. 人民卫生出版社，中国，北京，pp. 1-53.(Chen S L, Yao H, Han J P, et al. 2012. Molecular identification of traditional Chinese materia medica using DNA barcoding[M]. People’s Medical Publishing House, Beijing, China, pp. 1-53.). 陈士林, 姚辉, 韩建萍,等. 2013. 中药材DNA条形码分子鉴定指导原则[J]. 中国中药杂志,38(2):141-148.(Chen S L, Yao H, Han J P, et al. 2013. Principles for molecular identification of traditional Chinese materia medica using DNA barcoding[J]. China J ournal of Chinese Materia Medica,38(2):141-148.) 陈士林, 姚辉, 宋经元,等. 2007. 基于DNA barcoding(条形码)技术的中药材鉴定[J]. 世界科学技术-中医药现代化,9(3):7-12. (Chen S L, Yao H, Song J Y, et al. 2007. Use of DNA barcoding to identify Chinese medicinal materials[J]. Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology, 9(3):7-12.) 谷婧. 2011. 苦丁茶植物资源调查整理与DNA分子鉴定及系统亲缘关系探讨[D].博士学位论文，北京协和医学院，导师：肖培根，pp.129-142. (Gu J.2011. Investigation, molecular identification and systematic relationship of Ku Ding Cha plant resources [D]. Thesis for Ph.D.,Peking Union Medical College,Supervisor: Xiao P G, pp.129-142.) 国家药典委员会. 2010. 中华人民共和国药典（一部） [M]. 中国医药科技出版社，中国，北京，pp.92-93.( National Pharmacopoeia Committee. 2010. Pharmacopoeia of People’s Republic of China[M]. The medicine science and technology press of China, Beijing, China, pp.92-93.) 惠梓航, 姜卫兵, 魏家星,等. 2011. “冬青”类树种的园林特性及其应用[J]. 江西农业学报(02):46-49..(Hui ZH,Jiang WB,Wei JX,et al.2011.Landscape Characeristics of Holly and Its Application[J].Acta Agriculturae Jiangxi,(2):46-49.) 谢军波，李萍.2002.四季青酚酸类化学成分研究[J]. 中国药科大学学报, 33(1): 76-77.(Xie J B, Li P. 2002. Stuies on phenolic acids from Ilex pururea Hassk.[J]. Journal of China Pharmaceutical University, 33(1): 76-77.) 廖保生, 韩建萍, 陈晓辰,等; 2013. 三七快速鉴定方法. 中国专利， CN102888456A 廖立平, 毕志明, 李萍,等. 2005. 四季青叶中的三萜类化学成分[J]. 中国天然药物, 3(6): 344-347.(Liao L P, Bi Z M, Li P, et al. 2005. Triterpenoids from Leaves of Ilex purpurea[J]. Chinese Journal of Natural Medicines, 3(6): 344-347.) 唐祝兴, 张东瑾, 王巧玲. 2010. 毛细管电泳-安培检测法测定中药四季青中的6种活性成分[J]. 辽宁化工, 39(3): 332-335. (Tang Z X, Zhang D J, Wang Q L. 2010. Determination of Six Kinds of Active Ingredients of Si Ji Qing by Capillary Electrohoresis With Amperom etric Detection[J]. Liaoning Chemical Industry, 39(3): 332-335.)
