Screening of DNA Barcoding for Medicinal Dendrobium in Yunnan

doi:10.3969/j.issn.1009-7791.2020.06.001

Subtropical Plant Science ›› 2020, Vol. 49 ›› Issue (06): 411-419.DOI: 10.3969/j.issn.1009-7791.2020.06.001

• Plant biochemistry and molecular biology • Next Articles

Screening of DNA Barcoding for Medicinal Dendrobium in Yunnan

XU Su-su^1,2,3, GAO Jing^1,2,3, CHEN Jun-wen^2,3,4, LIU Xiang-yu^1,2,3, ZHANG Jing-li^1,2,3*

(1.College of Landscape and Horticulture, Yunnan Agricultural University, Kunming 650201, Yunnan China; 2.National & Local Joint Engineering Research Center on Germplasm Innovation & Utilization of Chinese Medicinal Materials in Southwestern China, Yunnan Agricultural University, Kunming 650201, Yunnan China; 3.The Key Laboratory of Medicinal Plant Biology of Yunnan Province, Yunnan Agricultural University, Kunming 650201, Yunnan China; 4.College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan China)

Received:2020-10-30 Revised:2020-12-12 Online:2020-12-30 Published:2020-12-30
Contact: ZHANG Jing-li

云南常见药用石斛DNA条形码筛选

徐素素^1,2,3，高静^1,2,3，陈军文^2,3,4，刘祥宇^1,2,3，张敬丽^1,2,3*

(1.云南农业大学园林园艺学院，云南昆明 650201；2.云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心，云南昆明 650201；3.云南农业大学云南省药用植物生物学重点实验室，云南昆明650201；4.云南农业大学农学与生物技术学院，云南昆明 650201)

通讯作者: 张敬丽
基金资助:
云南省重大科技专项(2017ZF003)；国家重点研发计划(2017YFC1702205)；云南省教育厅科学研究基金项目(2021Y184)

Abstract

Abstract: Twelve common medicinal Dendrobium species in Yunnan province were selected, and molecular biology identification techniques were used to screen DNA barcoding sequences suitable for Yunnan medicinal Dendrobium. A total of 36 samples of 12 kinds of medicinal Dendrobium (Dendrobium devonianum, D. chrysotoxum, D. falconeri, etc.) were used as materials to extract the total DNA of the samples, and the nuclear gene fragments ITS and ITS2, the chloroplast gene fragment psbA-trnH and matK sequences were amplified and sequenced, part of the Dendrobium sequence was downloaded in GenBank; Sequence analysis and phylogenetic analysis were used by bioinformatics software. The results showed that the success rates of amplification and sequencing of the 4 sequences were 100% and all of them had no obvious Barcoding Gap, but there was a small amount of overlap between the ITS sequence and the ITS2 sequence within and between species, which tended to be biased towards both ends. The phylogenetic tree showed that the ITS and psbA-trnH sequences could successfully distinguish the 12 medicinal Dendrobiums in this study, the ITS2 sequence failed to distinguish D. longicornu and D. bellatulum. The matK sequence only distinguished 6 species of Dendrobium. It is suggested that ITS and psbA-trnH sequences should be regarded as the identification sequence of Yunnan medicinal Dendrobium to provide theoretical basis for the provenance identification of medicinal Dendrobium.

Key words: medicinal Dendrobium, Yunnan, DNA barcoding, phylogenetic analysis, species identification, molecular identification

摘要： 选择云南省内常见的12种药用石斛，采用分子生物学鉴定技术筛选其适用的DNA条形码序列。以12种药用石斛共36个样品为材料，提取样品总DNA，对核基因片段ITS和ITS2、叶绿体基因片段psbA-trnH和matK序列进行扩增、测序，结合GenBank下载部分石斛序列；利用生物信息学软件进行序列分析和系统发育分析。结果表明，4条序列扩增和测序成功率均为100%；4条序列没有明显的Barcoding Gap，但ITS序列与ITS2序列的种内和种间重叠部分较少，有偏向两端的趋势；系统发育树显示，ITS和psbA-trnH序列能成功区分12种云南常见的药用石斛，ITS2序列未能区分长距石斛(Dendrobium longicornu)和矮石斛(D. bellatulum)，matK序列仅区分6种石斛。建议以ITS和psbA-trnH序列作为云南药用石斛鉴定序列，为药用石斛的种源鉴定提供理论依据。

关键词: 药用石斛, 云南, DNA条形码, 系统发育分析, 物种鉴定, 分子鉴定

CLC Number:

S567

XU Su-su, GAO Jing, CHEN Jun-wen, LIU Xiang-yu, ZHANG Jing-li . Screening of DNA Barcoding for Medicinal Dendrobium in Yunnan[J]. Subtropical Plant Science, 2020, 49(06): 411-419.

徐素素，高静，陈军文，刘祥宇，张敬丽. 云南常见药用石斛DNA条形码筛选[J]. 亚热带植物科学, 2020, 49(06): 411-419.

References

[1] 宋广青,刘新民,王琼,刘靖. 石斛药理作用研究进展[J]. 中草药, 2014,45(17): 2576–2580. [2] He L, Su Q, Bai L, Li M F, Guo L. Recent research progress on natural small molecule bibenzyls and its derivatives in Dendrobium species[J]. European Journal of Medicinal Chemistry, 2020,204: 112530. [3] Nie X Q, Chen Y, Li W, Lu Y L. Anti-aging properties of Dendrobium nobile Lindl.: From molecular mechanisms to potential treatments[J]. Journal of Ethnopharmacology, 2020,257: 112839. [4] Yoo S R, Jeong S J, Lee N R, Shin H K, Seo C S. Simultaneous determination and anti-inflammatory effects of four phenolic compounds in Dendrobii Herba[J]. Natural Product Research, 2017,31(24): 2923–2926. [5] 贾元,胡利娟,冯群,田忠静,余雨,翁庆北,朱斌. 基于ISSR标记的西南地区石斛资源亲缘关系分析[J]. 贵州农业科学, 2019,47(12): 8–12. [6] 中国科学院中国植物志编辑委员会. 中国植物志(第19卷)[M]. 北京: 科学出版社, 1999: 67–68. [7] 中国科学院昆明植物研究所. 云南植物志(第14卷)[M]. 北京: 科学出版社, 2003. [8] 白音,包英华,金家兴,阎玉凝,王文全. 我国药用石斛资源调查研究[J] .中草药, 2006,37 (9): 4–6. [9] 姚能昌. 浅述云南石斛资源现状及开发利用技术[J]. 林业调查规划, 2004,29 (4): 80–82. [10] 云南省药材公司. 云南中药资源名录[M]. 北京: 科学出版社, 1993: 669–672. [11] 贾敏如,李星炜. 中国民族药志要[M]. 北京: 中国医药科技出版社, 2005: 217–218. [12] 黄海,李劲松,符岸军,严海. 石斛属植物DNA条形码序列的筛选[J]. 热带作物学报, 2010,31(10): 1769–1777. [13] 陈士林,郭宝林,张贵君,严铸云,罗光明,孙素琴,吴和珍,黄林芳,庞晓慧,陈建波. 中药鉴定学新技术新方法研究进展[J]. 中国中药杂志, 2012,37(8): 1043–1055. [14] Li D Z, Liu J Q, Chen Z D, Wang H, Ge X J, Zhou S L, Gao L M, Fu C X, Chen S L. Plant DNA barcoding in China[J]. Journal of Systematics and Evolution.2011,49(3): 165–168. [15] 程芳婷,李忠虎,刘春艳,原超,李雪童,刘占林. 地黄属植物的DNA条形码研究[J]. 植物科学学报, 2015,33 (1): 25–32. [16] Kress W J, Wurdack K J, Zimmer E A, Weigt L A, Janzen D H. Use of DNA barcodes to identify flowering plants[J]. Proceedings of the National Academy of Sciencesof the United States of America, 2005,102: 869–8374. [17] Chen S L, Yao H, Han J P, Liu C, Song J Y, Shi L C, Zhu Y J, Ma X Y, Gao T, Pang X H, Luo K, Li Y, Li X W, Jia X C, Lin Y L, Leon C. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J]. PLoS ONE, 2010,5: e8613. [18] Hollingsworth P M. Refining the DNA barcode for land plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2011,108(49): 19451–2. [19] 陈士林,姚辉,韩建萍,辛天怡,庞晓慧,石林春,罗焜,宋经元,侯典云,石上梅,钱忠直. 中药材DNA条形码分子鉴定指导原则[J]. 中国中药杂志, 2013,38(2): 141–148. [20] 国家药典委员会. 中国药典(一部)[M]. 北京: 中国医药科技出版社, 2015. [21] Shi L C, Zhang J, Han J P, Song J Y, Yao H, Zhu YJ, Li J C, Wang Z Z, Xiao W, Lin Y L, Xie C X, Qian Z Z, Chen S L. Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae[J]. Journal of Systematics and Evolution, 2011,49(3): 261–266. [22] Ran J H, Wang P P, Zhao H J, Xiao Q W. A test of seven candidate barcode regions from the plastome in Picea (Pinaceae)[J]. Journal of Integrative Plant Biology, 2010,52(12): 1109–1126. [23] Zhang J, Chen M, Dong X Y, Lin R Z, Fan J H, Chen Z D. Evaluation of four commonly used DNA barcoding loci for Chinese medicinal plants of the family Schisandraceae[J]. PLoS One, 2015,10(5): e125574. [24] Zhang C Y, Wang F Y, Yan H F, Hao G, Hu C M, Ge X J. Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae) [J]. Molecular EcologyResources., 2012,12: 98–108. [25] 刘枫,赵群,戴军,陈存武,陈乃富,韩邦兴. DNA分子标记技术在石斛属鉴别中的应用进展[J]. 皖西学院学报, 2017(2): 14–18,36. [26] 叶子,卢叶,王峥涛,徐红,胡之璧. 基于ITS序列鉴别石斛类药材[J]. 中国中药杂志, 2014,39(20): 3928–3935. [27] Lau D T, Shaw P C, Wang J, Bu P. Authentication of medicinal Dendrobium Species by the internal transcribed spacer of ribosomal DNA [J]. Planta Med, 2001,67(5): 456–460. [28] Yao H, Song J Y, Ma X Y, Liu C, Li Y, Xu H X, Han J P, Duan L S, Chen S L. Identification of Dendrobium Species by a candidate DNA barcode sequence: The chloroplast psbA-trnH intergenic region [J]. Planta Med, 2009,75(6): 667–669. [29] 李金璐,王硕,于婧,王玲,周世良. 一种改良的植物DNA提取方法[J]. 植物学报, 2013,48(1): 72–78. [30] Keller A, Schleicher T, Schultz J, Muller T, Dandekar T, Wolf M. 5.8S-28S rRNA interaction and HMM-based ITS2 annotation[J].Gene, 2009,430(1–2): 0–57. [31] Meyer P, Paulay G. DNA barcoding: Error rates based on comprehensive sampling[J]. PLoS Biology, 2005,3(12): e422 [32] 刘依丽,冯尚国,何仁锋,赵妙玉,吴慧,崔艺庆,陈倩,王慧中. 基于ITS2条形码对铁皮石斛及其混伪品分子鉴定的初步研究[J]. 杭州师范大学学报(自然科学版), 2014(1): 35–41. [33] 邵世光,韩丽,马艳红,沈洁,张伟超,丁小余. 枫斗类石斛cpDNA psbA–trnH的序列分析与鉴别[J]. 药学学报, 2009(10): 1173–1178. [34] 滕艳芬,吴晓俊,徐红,王峥涛,余国奠,徐珞珊. 石斛及其常见混淆品的matK基因序列比较[J]. 中国药科大学学报, 2002,33(4): 280–283. [35] 张爱丽,高亚芳,黎太米,谭文红,钱子刚. DNA条形码在主要滇产石斛鉴别中的应用[J]. 中国民族民间医药, 2017,26(2): 11–16. [36] Li D Z, Gao L M, Li H T, Wang H, Ge X J, Liu J Q, Chen D Z, Zhou S L, Chen S L, Yang J B. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants[J]. Proceedings of the National Academy of Sciences, 2011,108(49): 19641–19646. [37] Kress W J, Wurdack K J, Zimmer E A, Weigt L A, Janzen D H. Use of DNA barcodes to identify flowering plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005,102(23): 8369.

Metrics

Copyright © Editorial office of Subtropical Plant Science
Tel: 0592-5654157　E-mail: yrdzwkx@126.com
Supported by Beijing Magtech Co., Ltd.

Screening of DNA Barcoding for Medicinal Dendrobium in Yunnan

云南常见药用石斛DNA条形码筛选

PDF

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 8

Recommended Articles

Metrics

[1]	LONG Bo, ZHANG Ze, ZHANG Yu, XU Ru-hua, ZHAO Da-ke. Tainia epiphytica, a New Record of Orchidaceae from China [J]. Subtropical Plant Science, 2023, 52(4): 343-346.
[2]	YAN Sheng-ze, LUO Qing-qing, YE Kai-wun, WANG Ai-qin, ZHAN Mei-rong. New Distribution of Wild Thelephora ganbajun M. Zang in China [J]. Subtropical Plant Science, 2021, 50(04): 313-317.
[3]	FEI Xi-tong,JU Miao-miao,LIN Yuan,LI Bin,LI De-long,XIN Pei-yao. Application and Research Progress of ITS2 Sequence in Plant Identification Using DNA Barcoding [J]. , 2014, 43(04): 339-342.
[4]	YANG Hong-pei,YU Dong-li,YANG Zheng-bin,LIU Qiang. New Records of Orchids from Yunnan, China [J]. Subtropical Plant Science, 2013, 42(04): 350-352.
[5]	GUO Li,GUAN Wen-ling,DU Yan-yan. The Resources of Ornamental Fruit Plants in Yunnan Province and the Landscape Application in Kunming City [J]. Subtropical Plant Science, 2013, 42(03): 255-260.
[6]	LI Chun-fang,CHEN Zhi-ying,YANG Jun-bo,XIONG Zhi. Rapid Propagation and In vitro Conservation of Dendrocalamus yunnanicus [J]. Subtropical Plant Science, 2012, 41(02): 27-31.
[7]	HE Lu, DUAN Yue-tang, SHA Yu-cang, LUO Jing-ping, QU Wen-lin, TAN Hong. Introduction and Adaptability of Several Taiwanese Fruits in Dry-hot Valley of Yuanmou,Yunnan Province [J]. Subtropical Plant Science, 2006, 35(02): 24-26,23.
[8]	GUAN Wen-ling, LI Shi-feng. Ornamental germplasm resources of genus Delphinium in Yunnan [J]. Subtropical Plant Science, 2002, 31(S1): 61-64.