雪胆属植物DNA条形码的鉴定分析
|
摘要
雪胆属(Hemsleya)植物属葫芦科(Cucurbitaceae)翅子瓜亚科(Zanonioudeae),主要分布于中国西南地区,该属多种植物的块茎具有良好的抗肿瘤,抗炎、抗菌、抗病毒等多种功效,是西南地区各民族常用的传统中药。本实验从常用的DNA条形码中筛选出适合鉴定雪胆属植物的核心DNA条形码,扩增并测序所需的DNA序列;分析其序列的长度、碱基组成及其变异位点;用MEGA7.0计算K-2P遗传距离,评估了各个序列的"Barcoding gap";通过构建NJ树来确定单个序列及其不同组合的物种分辨率。结果表明ITS+psb A-trn H+trn L具有最大的分辨率,为75%,推荐将ITS+psb A-trn H+trn L作为雪胆属植物鉴定的核心DNA条形码。
Hemsleya, a genus of Cucurbitaceae Zanonioudeae, is mainly distributed in the southwest of China. Many plants of this genus has good antitumor, anti-inflammatory, antibacterial, antiviral and other effects, so Hemsleya is used as a traditional Chinese medicine for the nationalities in the southwest of China. In this study, the core DNA barcode suitable for the identification of Hemsleya was screened out from the common DNA barcode, and these DNA sequences were amplified and sequenced. The length of the sequence, the composition of the base and its variation sites were analyzed. The K-2 P genetic distance was calculated by MEGA7.0, and the "Barcoding gap" of each sequence was evaluated. In addition, the species resolution of a single sequence and its combinations were determined by constructing a NJ tree. The results showed that the combination fragment ITS+psb A-trn H+trn L had the maximum resolution, which was 75%. We recommend ITS+psb A-trn H+trn L as the core DNA barcode for the identification of Hemsleya plants.
Hemsleya, a genus of Cucurbitaceae Zanonioudeae, is mainly distributed in the southwest of China. Many plants of this genus has good antitumor, anti-inflammatory, antibacterial, antiviral and other effects, so Hemsleya is used as a traditional Chinese medicine for the nationalities in the southwest of China. In this study, the core DNA barcode suitable for the identification of Hemsleya was screened out from the common DNA barcode, and these DNA sequences were amplified and sequenced. The length of the sequence, the composition of the base and its variation sites were analyzed. The K-2 P genetic distance was calculated by MEGA7.0, and the "Barcoding gap" of each sequence was evaluated. In addition, the species resolution of a single sequence and its combinations were determined by constructing a NJ tree. The results showed that the combination fragment ITS+psb A-trn H+trn L had the maximum resolution, which was 75%. We recommend ITS+psb A-trn H+trn L as the core DNA barcode for the identification of Hemsleya plants.
引文
CBOL Plant Working Group,2009,A DNA barcode for land plants,Proc.Natl.Acad.Sci.USA,106(31):12794-12797
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.,and Leon C.,2010,Validation of the ITS2region as a novel DNA barcode for identifying medicinal plant species,PLoS One,5(1):e8613
Chen X.B.,Chen G.Y.,Liu J.H.,Lei M.,Meng Y.H.,Guo D.A.,Liu X.,and Hu L.H.,2014,Cytotoxic cucurbitane triterpenoids isolated from the rhizomes of Hemsleya amabilis,Fitoterapia,94(2):88-93
China Plant BOL Group.,Li D.Z.,Gao L.M.,Li H.T.,Wang H.,Ge X.J.,Liu J.Q.,Chen Z.D.,Zhou S.L.,Chen S.L.,Yang J.B.,Fu C.X.,Zeng C.X.,Yan H.F.,Zhu Y.J.,Sun Y.S.,Chen S.Y.,Zhao L.,Wang K.,Yang T.,and Duan G.W.,2011,Comparative analysis of a large dataset indicates that internal transcribed spacer(ITS)should be incorporated into the core barcode for seed plants,Proc.Natl.Acad.Sci.USA,108(49):19641-19646
Cuenoud P.,Savolainen V.,Chatrou L.W.,Powell M.,Grayer R.J.,and Chase M.W.,2002,Molecular phylogenetics of Caryophyllales based on nuclear 18S r DNA and plastid rbc L,atp B,and mat K DNA sequences,Am.J.Bot.,89(1):132-144
Editorial Committee of Chinese Botany of the Chinese Academy of Sciences,ed.,1986,Flora of China,Science Press,Beijing,China,pp.102-129(中国科学院中国植物志编辑委员会,编著,1986,中国植物志,科学出版社,中国,北京,pp.102-129)
Gao L.M.,Liu J.,Cai J.,Yang J.B.,Zhang T.,and Li D.Z.,2012,A synopsis of technical notes on the standards for plant DNA barcoding,Zhiwu Fenlei Yu Ziyuan Xuebao(Plant D-iversity and Resources),34(6):592-606(高连明,刘杰,蔡杰,杨俊波,张挺,李德铢,2012,关于植物DNA条形码研究技术规范,植物分类与资源学报,34(6):592-606)
Gao T.,Yao H.,Song J.Y.,Zhu Y.J.,Liu C.,and Chen S.L.,2010,Evaluating the feasibility of using candidate DNAbarcodes in discriminating species of the large Asteraceae family,BMC Evol.Biol.,10(1):324
Guo W.W.,2016,DNA barcoding identification of Hemsleya macrosperma and its adulterants,Thesis for M.S.,Yunan University of TCM,Supervisor:Yang Y.W.,pp.47-48(郭蔚文,2016,罗锅底及其混伪品的DNA条形码鉴定研究,硕士学位论文,云南中医学院,导师:杨耀文,pp.47-48)
Hebert P.D.N.,Cywinska A.,Ball S.L.,and Dewaard J.,2003,Biological identifications through DNA barcode,Proceedings of the Royal Society B.,270(1512):313-321
Huang W.J.,2014,Identification of buckwheat and its wild relatives by DNA barcoding technique,Thesis for M.S.,Minzu University of China,Supervisor:Long C.L.,pp.24-29(黄卫娟,2014,荞麦及其野生近缘种的DNA条形码鉴定,硕士学位论文,中央民族大学,导师:龙春林,pp.24-29)
Huang Y.,Sun B.,and He P.M.,2018,Application of DNA barcoding technology in molecular identification of red macroalgae,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(3):1321-1333(黄艳,孙彬,何培民,2018,DNA条形码技术在大型红藻分子鉴定中的应用,基因组学与应用生物学,37(3):1321-1333)
Keller A.,Schleicher T.,Schultz J.,Müller T.,Dandekar T.,and Wolf M.,2009,5.8S~28S r RNA interaction and HMM-based ITS2 annotation,Gene,430(1):50-57
Kress W.J.,and Erickson D.L.,2007,A two-locus global DNAbarcode for land plants:the coding rbc L gene complements the non-coding trn H-psb A spacer region,PLoS One,2(6):e508
Li D.Z.,1990 Systematics and evolution of Hemsleya(Cucurbitaceae),Dissertation for Ph.D.,Kunming Institute of Botany,Academia Sinica,Supervisor:Wu Z.Y.,pp.12-21(李德铢,1990,雪胆属植物的系统与进化,博士学位论文,中国科学院昆明植物研究所,导师:吴征镒,pp.12-21)
Li H.T.,Yang J.B.,Li D.Z.,Michael M.,and Amin S.,2010,Amolecular phylogenetic study of Hemsleya(Cucurbitaceae)based on ITS,rpl16,trn H-psb A,and trn L DNA sequences,Plant Syst.Evol.,285(1-2):23-32
Li N.,Chen S.L.,Liu Y.M.,and Chen K.L.,2011,Screening of universal DNA barcodes for plants in Cucurbitaceae,Zhongcaoyao(Chinese Traditional and Herbal Drugs),42(7):1396-1401(李妮,陈士林,刘义梅,陈科力,2011,葫芦科植物通用DNA条形码的筛选,中草药,42(7):1396-1401)
Ma X.Y.,Xie C.X.,Liu C.,Song J.Y.,Yao H.,Luo K.,Zhu Y.J.,Gao T.,Pang X.H.,Qian J.,and Chen S.L.,2010,Species identification of medicinal pteridophytes by a DNA barcode marker,the chloroplast psb A-trn H intergenic region,Biol.Pharm.Bull.,33(11):1919-1924
Meyer C.P.,and Paulay G.,2005,DNA barcoding:error rates based on comprehensive sampling,PLoS Biol.,3(12):e422
Nie R.L.,and Chen Z.L.,1986,The research history and present status on the chemical components of genus Hemsleya(Cucurbitaceae),Yunnan Zhiwu Yanjiu(Acta Botanica Yunnanica),8(1):115-124(聂瑞麟,陈宗莲,1986,雪胆属植物化学研究历史和现状,云南植物研究,8(1):115-124)
Pang X.,Song J.Y.,Zhu Y.J.,Xu H.X.,Huang L.F.,and Chen S.L.,2011,Applying plant DNA barcodes for Rosaceae species identification,Cladistics,27(2):165-170
Selvaraj D.,Sarma R.K.,and Sathishkumar R.,2008,Phylogenetic analysis of chloroplast mat K gene from Zingiberaceae for plant DNA barcoding,Bioinformation,3(1):24-27
Song J.Y.,Yao H.,Li Y.,Li X.W.,Lin Y.L.,Liu C.,Han J.P.,Xie C.X.,and Chen S.L.,2009,Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique,J.Ethnopharmacol.,124(3):434-439
Stoeckle M.Y.,Gamble C.C.,Kirpekar R.,Young G.,Ahmed S.,and Little D.P.,2011,Commercial teas highlight plant DNAbarcode identification successes and obstacles,Sci.Rep.,1:42
Tang Y.L.,Liu Y.,Yao H.,Chen Z.D.,Wu Y.S.,Wu P.,and Jiang D.,2012,Evaluation on the application value of identification in cucurbitaceae by ITS2,Zhiwu Kexue Xuebao(Plant Science Journal),30(6):631-638(唐银琳,刘镛,姚辉,陈振东,吴耀生,吴鹏,蒋东,2012,DNA条形码(ITS2)在葫芦科鉴定中应用价值的评估,植物科学学报,30(6):631-638)
Wu Z.Y.,and Chen Z.L.,1985,Materia ad floram cucurbitacearum sinensium-Hemsleya Cogn.,Zhiwu Fenlei Xuebao(Acta Phytotaxonomica Sinicao,23(2):121-143(吴征镒,陈宗莲,1985,中国葫芦科植物志资料-雪胆属,植物分类学报,23(2):121-143)
Zheng Y.H.,Alverson A.J.,Wang Q.F.,and Palmer J.D.,2013,Chloroplast phylogeny of Cucurbita:evolution of the domesticated and wild species,J.Syst.Evol.,51(3):326-334
Zhou J.B.,Wang Q.,Li L.L.,and Liu J.Q.,2013,The combina tion of chloroplast and nuclear DNA fragments could improve the discrimination power for barcoding Spruce(Picea)species,Zhiwu Fenlei Yu Ziyuan Xuebao(Plant Diversity and Resources),35(3):375-385(邹嘉宾,王茜,李丽丽,刘建全,2013,结合叶绿体和核DNA序列变异可提高云杉属物种的分子鉴别率,植物分类与资源学报,35(3):375-385)
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.,and Leon C.,2010,Validation of the ITS2region as a novel DNA barcode for identifying medicinal plant species,PLoS One,5(1):e8613
Chen X.B.,Chen G.Y.,Liu J.H.,Lei M.,Meng Y.H.,Guo D.A.,Liu X.,and Hu L.H.,2014,Cytotoxic cucurbitane triterpenoids isolated from the rhizomes of Hemsleya amabilis,Fitoterapia,94(2):88-93
China Plant BOL Group.,Li D.Z.,Gao L.M.,Li H.T.,Wang H.,Ge X.J.,Liu J.Q.,Chen Z.D.,Zhou S.L.,Chen S.L.,Yang J.B.,Fu C.X.,Zeng C.X.,Yan H.F.,Zhu Y.J.,Sun Y.S.,Chen S.Y.,Zhao L.,Wang K.,Yang T.,and Duan G.W.,2011,Comparative analysis of a large dataset indicates that internal transcribed spacer(ITS)should be incorporated into the core barcode for seed plants,Proc.Natl.Acad.Sci.USA,108(49):19641-19646
Cuenoud P.,Savolainen V.,Chatrou L.W.,Powell M.,Grayer R.J.,and Chase M.W.,2002,Molecular phylogenetics of Caryophyllales based on nuclear 18S r DNA and plastid rbc L,atp B,and mat K DNA sequences,Am.J.Bot.,89(1):132-144
Editorial Committee of Chinese Botany of the Chinese Academy of Sciences,ed.,1986,Flora of China,Science Press,Beijing,China,pp.102-129(中国科学院中国植物志编辑委员会,编著,1986,中国植物志,科学出版社,中国,北京,pp.102-129)
Gao L.M.,Liu J.,Cai J.,Yang J.B.,Zhang T.,and Li D.Z.,2012,A synopsis of technical notes on the standards for plant DNA barcoding,Zhiwu Fenlei Yu Ziyuan Xuebao(Plant D-iversity and Resources),34(6):592-606(高连明,刘杰,蔡杰,杨俊波,张挺,李德铢,2012,关于植物DNA条形码研究技术规范,植物分类与资源学报,34(6):592-606)
Gao T.,Yao H.,Song J.Y.,Zhu Y.J.,Liu C.,and Chen S.L.,2010,Evaluating the feasibility of using candidate DNAbarcodes in discriminating species of the large Asteraceae family,BMC Evol.Biol.,10(1):324
Guo W.W.,2016,DNA barcoding identification of Hemsleya macrosperma and its adulterants,Thesis for M.S.,Yunan University of TCM,Supervisor:Yang Y.W.,pp.47-48(郭蔚文,2016,罗锅底及其混伪品的DNA条形码鉴定研究,硕士学位论文,云南中医学院,导师:杨耀文,pp.47-48)
Hebert P.D.N.,Cywinska A.,Ball S.L.,and Dewaard J.,2003,Biological identifications through DNA barcode,Proceedings of the Royal Society B.,270(1512):313-321
Huang W.J.,2014,Identification of buckwheat and its wild relatives by DNA barcoding technique,Thesis for M.S.,Minzu University of China,Supervisor:Long C.L.,pp.24-29(黄卫娟,2014,荞麦及其野生近缘种的DNA条形码鉴定,硕士学位论文,中央民族大学,导师:龙春林,pp.24-29)
Huang Y.,Sun B.,and He P.M.,2018,Application of DNA barcoding technology in molecular identification of red macroalgae,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(3):1321-1333(黄艳,孙彬,何培民,2018,DNA条形码技术在大型红藻分子鉴定中的应用,基因组学与应用生物学,37(3):1321-1333)
Keller A.,Schleicher T.,Schultz J.,Müller T.,Dandekar T.,and Wolf M.,2009,5.8S~28S r RNA interaction and HMM-based ITS2 annotation,Gene,430(1):50-57
Kress W.J.,and Erickson D.L.,2007,A two-locus global DNAbarcode for land plants:the coding rbc L gene complements the non-coding trn H-psb A spacer region,PLoS One,2(6):e508
Li D.Z.,1990 Systematics and evolution of Hemsleya(Cucurbitaceae),Dissertation for Ph.D.,Kunming Institute of Botany,Academia Sinica,Supervisor:Wu Z.Y.,pp.12-21(李德铢,1990,雪胆属植物的系统与进化,博士学位论文,中国科学院昆明植物研究所,导师:吴征镒,pp.12-21)
Li H.T.,Yang J.B.,Li D.Z.,Michael M.,and Amin S.,2010,Amolecular phylogenetic study of Hemsleya(Cucurbitaceae)based on ITS,rpl16,trn H-psb A,and trn L DNA sequences,Plant Syst.Evol.,285(1-2):23-32
Li N.,Chen S.L.,Liu Y.M.,and Chen K.L.,2011,Screening of universal DNA barcodes for plants in Cucurbitaceae,Zhongcaoyao(Chinese Traditional and Herbal Drugs),42(7):1396-1401(李妮,陈士林,刘义梅,陈科力,2011,葫芦科植物通用DNA条形码的筛选,中草药,42(7):1396-1401)
Ma X.Y.,Xie C.X.,Liu C.,Song J.Y.,Yao H.,Luo K.,Zhu Y.J.,Gao T.,Pang X.H.,Qian J.,and Chen S.L.,2010,Species identification of medicinal pteridophytes by a DNA barcode marker,the chloroplast psb A-trn H intergenic region,Biol.Pharm.Bull.,33(11):1919-1924
Meyer C.P.,and Paulay G.,2005,DNA barcoding:error rates based on comprehensive sampling,PLoS Biol.,3(12):e422
Nie R.L.,and Chen Z.L.,1986,The research history and present status on the chemical components of genus Hemsleya(Cucurbitaceae),Yunnan Zhiwu Yanjiu(Acta Botanica Yunnanica),8(1):115-124(聂瑞麟,陈宗莲,1986,雪胆属植物化学研究历史和现状,云南植物研究,8(1):115-124)
Pang X.,Song J.Y.,Zhu Y.J.,Xu H.X.,Huang L.F.,and Chen S.L.,2011,Applying plant DNA barcodes for Rosaceae species identification,Cladistics,27(2):165-170
Selvaraj D.,Sarma R.K.,and Sathishkumar R.,2008,Phylogenetic analysis of chloroplast mat K gene from Zingiberaceae for plant DNA barcoding,Bioinformation,3(1):24-27
Song J.Y.,Yao H.,Li Y.,Li X.W.,Lin Y.L.,Liu C.,Han J.P.,Xie C.X.,and Chen S.L.,2009,Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique,J.Ethnopharmacol.,124(3):434-439
Stoeckle M.Y.,Gamble C.C.,Kirpekar R.,Young G.,Ahmed S.,and Little D.P.,2011,Commercial teas highlight plant DNAbarcode identification successes and obstacles,Sci.Rep.,1:42
Tang Y.L.,Liu Y.,Yao H.,Chen Z.D.,Wu Y.S.,Wu P.,and Jiang D.,2012,Evaluation on the application value of identification in cucurbitaceae by ITS2,Zhiwu Kexue Xuebao(Plant Science Journal),30(6):631-638(唐银琳,刘镛,姚辉,陈振东,吴耀生,吴鹏,蒋东,2012,DNA条形码(ITS2)在葫芦科鉴定中应用价值的评估,植物科学学报,30(6):631-638)
Wu Z.Y.,and Chen Z.L.,1985,Materia ad floram cucurbitacearum sinensium-Hemsleya Cogn.,Zhiwu Fenlei Xuebao(Acta Phytotaxonomica Sinicao,23(2):121-143(吴征镒,陈宗莲,1985,中国葫芦科植物志资料-雪胆属,植物分类学报,23(2):121-143)
Zheng Y.H.,Alverson A.J.,Wang Q.F.,and Palmer J.D.,2013,Chloroplast phylogeny of Cucurbita:evolution of the domesticated and wild species,J.Syst.Evol.,51(3):326-334
Zhou J.B.,Wang Q.,Li L.L.,and Liu J.Q.,2013,The combina tion of chloroplast and nuclear DNA fragments could improve the discrimination power for barcoding Spruce(Picea)species,Zhiwu Fenlei Yu Ziyuan Xuebao(Plant Diversity and Resources),35(3):375-385(邹嘉宾,王茜,李丽丽,刘建全,2013,结合叶绿体和核DNA序列变异可提高云杉属物种的分子鉴别率,植物分类与资源学报,35(3):375-385)