陈倩;刘义梅;刘震;陈士林;陈科力;
CHEN Qian 1,LIU Yimei 1,LIU Zhen 1,CHEN Shilin 2,CHEN Keli 1(1.Key Laboratory of Ministry of Education on Traditional Chinese Medicine Resource and Compound Prescription & Hubei University of Chinese Medicine,Wuhan 430065,China; 2.Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100193,China)

• 1:中药资源和中药复方教育部重点实验室 湖北中医药大学
• 2:中国医学科学院 北京协和医学院药用植物研究所

摘要(Abstract)：

目的:评价几条热点DNA条形码候选序列对马鞭草科药用植物的鉴别能力。方法:选择psbA-trnH,rbcL,matK,ITS2和ITS序列进行评价,使用各序列的通用引物进行扩增和测序,用扩增及测序成功率,种内种间差异,barcod-ing gap,基于BLAST 1和Nearest Distance方法的鉴定成功率等指标来评价各序列的鉴定能力。结果:对马鞭草科32个种55个样本的序列分析发现,matK的扩增成功率太低,未纳入后续分析;ITS,ITS2,psbA-trnH以及rbcL的扩增及测序成功率分别为83.6%,83.6%,96.4%,98.2%,其鉴定成功率除rbcL为77.8%,75.9%外都为100%,但ITS2序列的种间,种内差异,barcoding gap较psbA-trnH,ITS有明显优势。ITS2序列进一步纳入网上163个样品的数据后其鉴定成功率依然可以达到89.5%,87.6%。结论:考虑到psbA-trnH较高的测序成功率,ITS2和psbA-trnH是适合马鞭草科植物鉴别的一个较好DNA条形码序列组合。
Objective: To evaluate the ability of several hotspot candidate sequence of DNA barcodes for identifying medicinal plant species in Verbenaceae.Method: Using universal primers,three chloroplast sequences,psbA-trnH,rbcL,matK,two nuclear ribosomal DNA ITS2 and ITS were amplified and sequenced.PCR amplification and sequencing efficiency,intra-and inter-specific variation,barcoding gap and identification efficiency(with BLAST 1 and Nearest Distance methods) were used to evaluate these loci.Result: The rate of successful amplification using matK was too low to further analyze,and the rate of successful amplification and sequencing using psbA-trnH,ITS,and ITS2 and rbcL sequence was 83.6%,83.6%,96.4%,98.2%,respectively.The rate of successful identification using psbA-trnH,ITS,and ITS2 was 100% at the species level except that rbcL was 77.8%,75.9% for 55 samples belonging to 32 species,but ITS2 did better in intra-and inter-specific variation,barcoding gap than the other loci.The rate of successful identification of ITS2 was 89.5%,87.6% even when joining the date of 165 samples from GenBank.Conclusion: It proposes that the combination of ITS2 and psbA-trnH senquence is promising for the identification of the species in Verbenaceae.

关键词(KeyWords)： DNA条形码;ITS2;ITS;rbcL;psba-trnH;马鞭草科
DNA barcoding;ITS2;ITS;rbcL;psbA-trnH;Verbenaceae

Abstract:

Keywords:

基金项目(Foundation): 国际科技合作项目(2007DFA30990);; 卫生行业科研专项(200802043)

作者(Author): 陈倩;刘义梅;刘震;陈士林;陈科力;
CHEN Qian 1,LIU Yimei 1,LIU Zhen 1,CHEN Shilin 2,CHEN Keli 1(1.Key Laboratory of Ministry of Education on Traditional Chinese Medicine Resource and Compound Prescription & Hubei University of Chinese Medicine,Wuhan 430065,China; 2.Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100193,China)

Email:

DOI:

参考文献(References)：

• [1]裴鉴,陈守良.中国植物志.第65卷.第1分册[M].北京:科学出版社,1982.
• [2]施苏华,杜雅青,David Bufford,等.用cpDNA matK基因和nrDNA ITS区序列确定我国特有植物四棱草属的系统位置[J].科学通报,2003,48(11):1176.
• [3]Hannah E Marx,Nataly O'Leary,Yuan Y W,et al.A molecularphylogeny and classification of Verbenaceaei[J].Am J Bot,2010,97(10):1647.
• [4]Lamm R D,Slaughter D C,Giles D K.Precision weed controlsystem for cotton[J].T ASAE,2002,45(1):231.
• [5]Hebert P D N,Cywinska A,Ball S L,et al.Biological identifi-cations through DNA barcodes[J].Proc R Soc Lond B,2003,270:313.
• [6]CBOL Plant Working Group.A DNA barcode for land plants[J].Proc Natl Acad Sci USA,2009,106(31):12794.
• [7]Chen S L,Yao H,Han J P,et al.Validation of the ITS2 regionas a novel dna barcode for identifying medicinal plant species[J].PLoS ONE,2010,5(1):e8613.
• [8]Thompson J D,Higgins D G,Gibson T J.Clustal W:improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighting,position-specific gap penalties and weightmatrix choice[J].Nucleic Acids Res,1994,22(22):4673.
• [9]Sass C,Little D P,Stevenson D W,et al.DNA Barcoding in theCycadales:testing the potential of proposed barcoding markers forspecies identification of Cycads[J].PLoS ONE,2007,2(11):e1154.
• [10]罗焜,陈士林,陈科力,等.基于芸香科的植物通用DNA条形码研究[J].中国科学(C辑):生命科学,2010,40(4):342.
• [11]朱英杰,陈士林,姚辉,等.重楼属药用植物DNA条形码鉴定研究[J].药学学报,2010,45(3):376.
• [12]Lahaye R,Bank M,Bogarin D,et al.DNA barcoding the florasof biodiversity hotspots[J].Proc Natl Acad Sci USA,2008,105(8):2923.
• [13]Slabbinck B,Dawyndt P,Martens M,et al.TaxonGap:a visu-alization tool for intra-and inter-species variation among individu-al biomarkers[J].Bioinformatics,2008,24(6):866.
• [14]Thomas C.Plant bar code soon to become reality[J].Science,2009,325(5940):526.
• [15]Meyer C P,Paulay G.DNA barcoding:error rates based on com-prehensive sampling[J].PLoS Biol,2005,3(12):e422.
• [16]Song J Y,Yao H,Li Y,et al.Authentication of the family Po-lygonaceae in Chinese Pharmacopoeia by DNA barcoding tech-nique[J].J Ethnopharmacol,2009,124(3):434.
• [17]Yao H,Song J Y,Ma X Y,et al.Identification of Dendrobiumspecies by a candidate DNA barcode sequence:the chloroplast ps-bA-trnH intergenic region[J].Planta Med,2009,75(6):667.
• [18]Gregory T R.DNA barcoding does not compete with taxonomy[J].Nature,2005,434(7037):1067.
• [19]宁淑萍,颜海飞,郝刚,等.植物DNA条形码研究进展[J].生物多样性,2008,16(5):417.
• [20]Keller A,Schleicher T,Schultz J,et al.5.8S-28S rRNA inter-action and HMM-based ITS2 annotation[J].Gene,2009,430(1/2):50.
• [21]梁汉兴,李璐,彭华.广义马鞭草科花器官发生的研究[J].植物学报,2001,43(7):673.
• [22]Wagstaff S J,Olmstead R G.Phylogeny of Labiatae and Verben-aceae inferred from rbcL sequences[J].System Bot,1997,22(1):165.