基于DNA条形码技术对23种未知黎药进行物种鉴定
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摘要
该研究利用DNA条形码技术对23种未知黎药进行物种鉴定。采用试剂盒法提取了23种药材的总DNA,并利用PCR技术扩增其ITS2,psbA-trnH序列并双向测序,利用Codon Code Aligner v7. 0. 1软件对测序峰图进行正反向序列拼接,去除引物区及低质量序列获得ITS2,psbA-trnH序列,并将其输入到中药材DNA条形码数据库和NCBI Gen Bank数据库中进行检索,尝试对这23种药材进行物种鉴定。并以鉴定结果的相似度≥97%即确定到物种水平;介于97%和90%即确定为属级水平;低于90%即确定到科级水平这一原则作为鉴定标准,有17种药材可以鉴定到物种水平,5种药材可以鉴定到属级水平,1种可以确定到科级水平。由此可见DNA条形码技术可以快速、准确的鉴别无背景信息的未知药材。
DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.
DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.
引文
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[8]陈士林,姚辉,韩建萍,等.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.
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[12]汤欢,向丽,李西文,等.濒危兰科药用植物DNA条形码鉴定[J].中国中药杂志,2017,42(11):2058.
[13]陈小露,柏宁宁,向丽,等.基于DNA条形码鉴定龙葵及其近缘种[J].中药材,2017,40(2):290.
[14]郭梦月,任莉,陈新连,等.基于ITS2条形码鉴别半枝莲及其混伪品[J].世界中医药,2016(5):796.
[15] Xin T,Yao H,Gao H. Super food Lycium barbarum Solanaceae traceability via an internal transcribed spacer 2 barcode[J].Food Res Int,2013,2(54):1699.
[16] Chen X,Liao B,Song J,et al. A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding[J]. Gene,2013,530(1):39.
[17] Di Pinto A,Di Pinto P,Terio V,et al. DNA barcoding for detecting market substitution in salted cod fillets and battered cod chunks[J]. Food Chem,2013,141(3):1757.
[18] Santhosh Kumar J U,Krishna V,Seethapathy G S,et al. DNA barcoding to assess species adulteration in raw drug trade of"Bala"(genus:Sida L.)herbal products in South India[J]. Biochem Syst Ecol,2015,61:501.
[19] Duan B Z,Fang H L,Li X W. Survey of traditional Dai medicine reveals species confusion and potential safety concerns:a case study on Radix Clerodendri Japonicum[J].中国天然药物:英文版,2017,15(6):417.
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[29] Qiu S X,Nguyen V H,Le T X,et al. A pregnane steroid from Aglaia lawii and structure confirmation of cabraleadiol monoacetate by X-ray crystallography[J]. Phytochemistry,2001,56(7):775.
[30] Long C L,Li R. Ethnobotanical studies on medicinal plants used by the Red-headed Yao People in Jinping,Yunnan province,China[J]. J Ethnopharmacol,2004,90:389.
[31] Li S,Long C,Liu F,et al. Herbs for medicinal baths among the traditional Yao communities of China[J]. J Ethnopharmacol,2006,108(1):59.
[32]戴好富,梅文莉.黎族药志.第1册[M].北京:中国科学技术出版社,2008.
[33]胡明勋,马逾英,蒋运斌,等.鸡矢藤的研究进展[J].中国药房,2017(16):2277.
[34]汪海斌,王燕燕.豆腐柴有效成分及药理作用[J].医药导报,2015(4):492.
[35]王园园,宋晓虹,李成仁,等.八种山茶属植物种子油脂的脂肪酸分析[J].中国油脂,2007,32(9):78.
[36] Sripisut T,Wong P,Thunwadee R. Alkaloids from Glycosmis cochinchinensis twigs[J]. Phytochem Lett,2013,6:337.
[37] Shapcott A,Forster P I,Guymer G P,et al. Mapping biodiversity and setting conservation priorities for SE Queensland's rainforests using DNA barcoding[J]. PLoS ONE,2015,10(3):e122164.
[38]龙丽,戴云,胡琳,等.密花豆属植物化学成分研究进展[J].云南中医中药杂志,2013(4):56.
[39]徐艳琴,刘小丽,黄小方,等.草珊瑚的研究现状与展望[J].中草药,2011,42(12):2552.
[40]舒永志,成亮,杨培明.黑老虎的化学成分及药理作用研究进展[J].中草药,2011,42(4):805.
[41]董玉琼.榼藤化学成分与药理活性研究[D].上海:上海交通大学,2011.
[42]刘遥.小叶买麻藤茎的化学成分研究[D].广州:南方医科大学,2015.
[43] Zhou S,Wang Y H,Lin M. Gnetuhainin S,a new resveratrol dimer from Gnetum hainanense[J]. Chin Chem Lett,2002,13:549.
[44] He Z,Ma C,Tan G T,et al. Rourinoside and rouremin,antimalarial constituents from Rourea minor[J]. Phytochemistry,2006,67(13):1378.
[45] Kulkarni P,Patel V,Shukla S T.Antidiabetic potential of Rourea minor(Gaertn.)root in streptozotocin—induced diabetic rats[J]. Orient Pharm Exp Med,2014,14(1):69.
[46] Zheng X L,Wei J H,Sun W. Ethnobotanical study on medicinal plants around Limu Mountains of Hainan Island,China[J].J Ethnopharmacol,2013,148(3):964.
[47]郑希龙,饶显龙,李榕涛,等.海南黎族治疗肝病的药用植物资源[J].时珍国医国药,2013,24(8):1999.
[48]郑希龙,陈红锋,李榕涛,等.海南润方言黎族药用民族植物学研究[J].云南植物研究,2008(2):195.
[49]李栎,肖憬,苏振宇,等. ITS2条形码序列对茜草科黎药植物的鉴定[J].中草药,2013,44(13):1814.
[50] Li X,Yang Y,Henry R J,et al. Plant DNA barcoding:from gene to genome[J]. Biol Rev Camb Philos Soc,2015,90(1):157.
[2]矢弓.做大做强海南南药黎药[J].今日海南,2017(3):36.
[3]郑希龙,戴好富,刘寿柏,等.海南黎族药用植物资源调查研究——以万宁市黎族为例[J].中国民族医药杂志,2013(4):20.
[4] Zheng X L,Xing F W. Ethnobotanical study on medicinal plants around Mt. Yinggeling,Hainan Island,China[J]. J Ethnopharmacol,2009,124(2):197.
[5]陈士林,郭宝林,张贵君,等.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.
[6] Chen S,Yao H,Han J,et al. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PLoS ONE,2010,5(1):e8613.
[7] Yao H,Song J,Liu C,et al. Use of ITS2 region as the universal DNA barcode for plants and animals[J]. PLoS ONE,2010,5(10):e13102.
[8]陈士林,姚辉,韩建萍,等.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.
[9] Chen S,Pang X,Song J,et al. A renaissance in herbal medicine identification:from morphology to DNA[J]. Biotechnol Adv,2014,32(7):1237.
[10]陈士林,姚辉,宋经元,等.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,2007(3):7.
[11]陈士林,庞晓慧,姚辉,等.中药DNA条形码鉴定体系及研究方向[J].世界科学技术——中医药现代化,2011,13,(5):747.
[12]汤欢,向丽,李西文,等.濒危兰科药用植物DNA条形码鉴定[J].中国中药杂志,2017,42(11):2058.
[13]陈小露,柏宁宁,向丽,等.基于DNA条形码鉴定龙葵及其近缘种[J].中药材,2017,40(2):290.
[14]郭梦月,任莉,陈新连,等.基于ITS2条形码鉴别半枝莲及其混伪品[J].世界中医药,2016(5):796.
[15] Xin T,Yao H,Gao H. Super food Lycium barbarum Solanaceae traceability via an internal transcribed spacer 2 barcode[J].Food Res Int,2013,2(54):1699.
[16] Chen X,Liao B,Song J,et al. A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding[J]. Gene,2013,530(1):39.
[17] Di Pinto A,Di Pinto P,Terio V,et al. DNA barcoding for detecting market substitution in salted cod fillets and battered cod chunks[J]. Food Chem,2013,141(3):1757.
[18] Santhosh Kumar J U,Krishna V,Seethapathy G S,et al. DNA barcoding to assess species adulteration in raw drug trade of"Bala"(genus:Sida L.)herbal products in South India[J]. Biochem Syst Ecol,2015,61:501.
[19] Duan B Z,Fang H L,Li X W. Survey of traditional Dai medicine reveals species confusion and potential safety concerns:a case study on Radix Clerodendri Japonicum[J].中国天然药物:英文版,2017,15(6):417.
[20]顾选.基于DNA条形码—产地—形态分析的药材鉴定方法及巴西草药品种整理研究[D].北京:北京中医药大学,2015.
[21]《海南植物志》电子版[EB/OL].[2018-03-26]. http://gjk.scib.ac.cn/hnzwz/result.asp? keyword=.
[22]郑希龙、邢福武、周劲松.海南民族植物学研究[M].武汉:华中科技大学出版社,2012.
[23]戴好富.黎族药志.第2册[M].北京:中国科学技术出版社,2010:6.
[24]中医药百科.中华医典官网[EB/OL].[2018-03-26]. http://www.tcmbook.cn/.
[25] FRPS.《中国植物志》全文电子版网站[EB/OL].[2018-01-27]. http://frps.eflora.cn/.
[26]戴好富.黎族药志.第3册[M].北京:中国科学技术出版社,2014.
[27]张培成,毋艳,于德泉.海南黄檀化学成分研究[J].中国中药杂志,2003,28(6):527.
[28] FOC.Flora of China《中国植物志》英文修订版官方网站[EB/OL].[2018-01-27]. http://foc.eflora.cn/.
[29] Qiu S X,Nguyen V H,Le T X,et al. A pregnane steroid from Aglaia lawii and structure confirmation of cabraleadiol monoacetate by X-ray crystallography[J]. Phytochemistry,2001,56(7):775.
[30] Long C L,Li R. Ethnobotanical studies on medicinal plants used by the Red-headed Yao People in Jinping,Yunnan province,China[J]. J Ethnopharmacol,2004,90:389.
[31] Li S,Long C,Liu F,et al. Herbs for medicinal baths among the traditional Yao communities of China[J]. J Ethnopharmacol,2006,108(1):59.
[32]戴好富,梅文莉.黎族药志.第1册[M].北京:中国科学技术出版社,2008.
[33]胡明勋,马逾英,蒋运斌,等.鸡矢藤的研究进展[J].中国药房,2017(16):2277.
[34]汪海斌,王燕燕.豆腐柴有效成分及药理作用[J].医药导报,2015(4):492.
[35]王园园,宋晓虹,李成仁,等.八种山茶属植物种子油脂的脂肪酸分析[J].中国油脂,2007,32(9):78.
[36] Sripisut T,Wong P,Thunwadee R. Alkaloids from Glycosmis cochinchinensis twigs[J]. Phytochem Lett,2013,6:337.
[37] Shapcott A,Forster P I,Guymer G P,et al. Mapping biodiversity and setting conservation priorities for SE Queensland's rainforests using DNA barcoding[J]. PLoS ONE,2015,10(3):e122164.
[38]龙丽,戴云,胡琳,等.密花豆属植物化学成分研究进展[J].云南中医中药杂志,2013(4):56.
[39]徐艳琴,刘小丽,黄小方,等.草珊瑚的研究现状与展望[J].中草药,2011,42(12):2552.
[40]舒永志,成亮,杨培明.黑老虎的化学成分及药理作用研究进展[J].中草药,2011,42(4):805.
[41]董玉琼.榼藤化学成分与药理活性研究[D].上海:上海交通大学,2011.
[42]刘遥.小叶买麻藤茎的化学成分研究[D].广州:南方医科大学,2015.
[43] Zhou S,Wang Y H,Lin M. Gnetuhainin S,a new resveratrol dimer from Gnetum hainanense[J]. Chin Chem Lett,2002,13:549.
[44] He Z,Ma C,Tan G T,et al. Rourinoside and rouremin,antimalarial constituents from Rourea minor[J]. Phytochemistry,2006,67(13):1378.
[45] Kulkarni P,Patel V,Shukla S T.Antidiabetic potential of Rourea minor(Gaertn.)root in streptozotocin—induced diabetic rats[J]. Orient Pharm Exp Med,2014,14(1):69.
[46] Zheng X L,Wei J H,Sun W. Ethnobotanical study on medicinal plants around Limu Mountains of Hainan Island,China[J].J Ethnopharmacol,2013,148(3):964.
[47]郑希龙,饶显龙,李榕涛,等.海南黎族治疗肝病的药用植物资源[J].时珍国医国药,2013,24(8):1999.
[48]郑希龙,陈红锋,李榕涛,等.海南润方言黎族药用民族植物学研究[J].云南植物研究,2008(2):195.
[49]李栎,肖憬,苏振宇,等. ITS2条形码序列对茜草科黎药植物的鉴定[J].中草药,2013,44(13):1814.
[50] Li X,Yang Y,Henry R J,et al. Plant DNA barcoding:from gene to genome[J]. Biol Rev Camb Philos Soc,2015,90(1):157.