荚蒾属植物DNA条形码鉴定
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摘要
目的:通过分析13种荚蒾属药用植物的ITS2(internal transcribed spacer 2)条形码序列,探讨荚蒾属植物属内鉴定的新方法,为荚蒾属植物的快速、准确、批量鉴定提供方法学依据。方法:该研究收集13种荚蒾属植物共32份样品,进行基因组DNA提取、PCR扩增ITS2序列并测序。所得序列经CodonCode Aligner拼接并剪切后,采用MEGA5.1软件计算遗传距离,比较种内、种间序列差异,利用邻接法(Neighbor-Joining,NJ)构建系统聚类树。结果:基于ITS2序列分析,13种药材种间存在明显差异,各个种的种内最大遗传距离均小于种间最小遗传距离;构建的系统树各个种不同样本均分别聚在一起,表现出单系性。结论:ITS2序列作为DNA条形码可较好地用于荚蒾属植物的鉴定研究,有助于为荚蒾属植物鉴定提供分子生物学方法基础,也为日后荚蒾属植物的开发打下坚实基础。
Objective:To analyze the ITS2(internal transcribed spacer 2)barcode sequence of 13 species of Viburnum medicinal plants,and to explore a new method for identification of Viburnum,and provide a method for rapid,accurate and batch identification of Viburnum plants.Methods:A total of 32 samples from 13 species of Viburnum were collected and DNA was extracted.The ITS2 of ribosomal DNA of Viburnum was amplified and sequenced by bi-directional sequencing of PCR products.Sequence assembly and consensus sequence generation were performed by using Codon Code Aligner.MEGA 5.1 was used to calculated the genetic distances and the phylogenetic tree was constructed by using the neighbor-joining(NJ)method.Results:There were significant differences in thirteen kinds of Viburnum.Their maximum intraspecific genetic distance(K2 P distance)was far lower than their minimum interspecific genetic distance with the other species.In the constructed phylogenetic tree,all species showed monophyletic.Conclusion:The ITS2 sequence can be used as a DNA barcode for the identification of Viburnum,which will help to provide molecular basis for the identification of Viburnum,and lay a solid foundation for the development of Viburnum.
Objective:To analyze the ITS2(internal transcribed spacer 2)barcode sequence of 13 species of Viburnum medicinal plants,and to explore a new method for identification of Viburnum,and provide a method for rapid,accurate and batch identification of Viburnum plants.Methods:A total of 32 samples from 13 species of Viburnum were collected and DNA was extracted.The ITS2 of ribosomal DNA of Viburnum was amplified and sequenced by bi-directional sequencing of PCR products.Sequence assembly and consensus sequence generation were performed by using Codon Code Aligner.MEGA 5.1 was used to calculated the genetic distances and the phylogenetic tree was constructed by using the neighbor-joining(NJ)method.Results:There were significant differences in thirteen kinds of Viburnum.Their maximum intraspecific genetic distance(K2 P distance)was far lower than their minimum interspecific genetic distance with the other species.In the constructed phylogenetic tree,all species showed monophyletic.Conclusion:The ITS2 sequence can be used as a DNA barcode for the identification of Viburnum,which will help to provide molecular basis for the identification of Viburnum,and lay a solid foundation for the development of Viburnum.
引文
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[10] 孙稚颖,陈士林,姚辉,等.基于ITS2序列的羌活及其混伪品的DNA条形码鉴定[J].中草药,2012,43(3):568-571.
[11] XIANG L,SONG J Y,XIN T Y,et al.DNA barcoding the commercial Chinese caterpillar fungus [J].FEMS Microb Let,2013,347(2):156-162.
[12] XIN T Y,YAO H,GAO H H,et al.Super food Lycium barbarum,(Solanaceae)traceability via an internal transcribed spacer 2 barcode [J].Food Res Inter,2013,54(2):1699-1704.
[13] CHEN S L,YAO H,HAN J P,et al.Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PLoS One,2010,5(1):e8613.
[14] 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):747-754.
[15] KIMURA M.A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.[J].J Mol Evol,1980,16(2):111-120.
[16] KELLER A,SCHLEICHER T,SCHULTZ J,et al.5.8 S-28S rRNA interaction and HMM-based ITS2 annotation[J].Gene,2009,430(1/2):50-57.
[17] 杜灵洁,杨晨娜,李尔敏,等.荚蒾属植物药用资源研究进展[J].台州学院学报,2017,39(6):6-9.
[18] IWAI K,KIM M Y,ONODERA A,et al.α-Glucosidase Inhibitory and Antihyperglycemic Effects of Polyphenols in the Fruit of Viburnum dilatatum Thunb[J].J Agr Food Chem,2006,54(13):4588-4592.
[19] 郑慧,张璐,全丽秋,等.水红木的化学成分及抗氧化活性研究[J].昆明理工大学学报(自然科学版),2018,43(2):79-87.
[20] 杨军.水红木及威灵仙的化学成分研究[D].郑州:河南大学,2010.
[21] 刘震,陈科力,陈士林,等.忍冬科药用植物DNA条形码通用序列的筛选[J].中国中药杂志,2010,35(19):2527-2532.
[2] 祝之友.荚迷属药用植物资源调查及开发利用[J].基层中药杂志,1999(1):24-25.
[3] 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):1043-1055.
[4] 辛天怡,雷美艳,宋经元.中药材DNA条形码鉴定研究进展[J].中国现代中药,2015,17(2):170-176.
[5] 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):141-148.
[6] 潘绿昌,李园园,刘红昌,等.基于ITS2和psbA-trnH序列的忍冬属5种中药材DNA条形码鉴定研究[J/OL].基因组学与应用生物学,2018[2018-11-15].http://kns.cnki.net/kcms/detail/45.1369.Q.20181115.0924.002.html.
[7] NEWMASTER S G,GRGURIC M,SHANMUGHANANDHAN D,et al.DNA barcoding detects contamination and substitution in North American herbal products [J].BMC Med,2013,11(1):222.
[8] 陈芙蓉,汪涛,郭巧生,等.基于DNA条形码ITS2序列的野菊及其易混品鉴定研究[J].中国中药杂志,2019,44(4):654-659.
[9] 王晖,时玲玲,朱国萍,等.基于DNA条形码分析的霍山石斛及其常见混伪品的初步研究[J].中国中药杂志,2018,43(20):4055-4061.
[10] 孙稚颖,陈士林,姚辉,等.基于ITS2序列的羌活及其混伪品的DNA条形码鉴定[J].中草药,2012,43(3):568-571.
[11] XIANG L,SONG J Y,XIN T Y,et al.DNA barcoding the commercial Chinese caterpillar fungus [J].FEMS Microb Let,2013,347(2):156-162.
[12] XIN T Y,YAO H,GAO H H,et al.Super food Lycium barbarum,(Solanaceae)traceability via an internal transcribed spacer 2 barcode [J].Food Res Inter,2013,54(2):1699-1704.
[13] CHEN S L,YAO H,HAN J P,et al.Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PLoS One,2010,5(1):e8613.
[14] 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):747-754.
[15] KIMURA M.A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.[J].J Mol Evol,1980,16(2):111-120.
[16] KELLER A,SCHLEICHER T,SCHULTZ J,et al.5.8 S-28S rRNA interaction and HMM-based ITS2 annotation[J].Gene,2009,430(1/2):50-57.
[17] 杜灵洁,杨晨娜,李尔敏,等.荚蒾属植物药用资源研究进展[J].台州学院学报,2017,39(6):6-9.
[18] IWAI K,KIM M Y,ONODERA A,et al.α-Glucosidase Inhibitory and Antihyperglycemic Effects of Polyphenols in the Fruit of Viburnum dilatatum Thunb[J].J Agr Food Chem,2006,54(13):4588-4592.
[19] 郑慧,张璐,全丽秋,等.水红木的化学成分及抗氧化活性研究[J].昆明理工大学学报(自然科学版),2018,43(2):79-87.
[20] 杨军.水红木及威灵仙的化学成分研究[D].郑州:河南大学,2010.
[21] 刘震,陈科力,陈士林,等.忍冬科药用植物DNA条形码通用序列的筛选[J].中国中药杂志,2010,35(19):2527-2532.