名贵中药材血竭的DNA条形码鉴定研究

DNA Barcodes Identification of One Rare Traditional Chinese Medicine Draconis Sanguis

目的筛选适合树脂类药材血竭鉴定的DNA条形码序列,探索血竭药材鉴定的新方法。方法提取41份药材和基原植物样本的基因组DNA,对药材样本基因组DNA的7条候选条形码序列[ITS、ITS2、psb A-trn H、rbc L、mat K、trn L(UAA)内含子、trn L(UAA)内含子的P6环]进行PCR扩增及测序。采用软件Codon Code Aligner V4.2对测序峰图进行校对拼接,应用MEGA5.0软件计算种内种间遗传距离(Kimura 2-parameter),构建邻接树(Neighbor-joing tree,NJ Tree)。结果血竭药材的trn L(UAA)内含子的P6环序列可以成功扩增并得到高质量序列,而其他6个候选序列[ITS、ITS2、psb A-trn H、rbc L、mat K、trn L(UAA)内含子]均不能成功扩增,难以用于鉴别。血竭的trn L(UAA)内含子的P6环序列长度为45 bp,麒麟竭的种内最大K2P遗传距离远小于麒麟竭与品剑叶龙血树的种间最小K2P遗传距离,NJ树显示血竭与其混伪品各自聚为一支,可明显区分。结论 trn L(UAA)内含子的P6环序列可作为血竭药材的DNA条形码鉴定序列,成为血竭药材真伪的鉴定依据。

OBJECTIVE To choose suitable barcodes for Draconis Sanguis and find the new method to identify Draconis Sanguis. METHODS The genomic DNAs from 41 samples were extracted. Draconis Sangnis samples were amplified and sequenced based on seven candidate DNA barcodes [ ITS , ITS2, psbA-trnH, rbcL, marK, trnL （UAA）intron, P6 loop of trnL （UAA） intron ]. The obtained sequences were assembled using the CodonCode Aligner V 4.2. The Kimura 2-Parameter （K2P） distances were calculated using MEGA 5.0. The neighbor-joining （ N J） phylogenetie trees were constructed. RESULTS Six out of the seven candidates [ ITS, ITS2, psbA-trnH, rbcL, matK, trnL （UAA） intron] were not able to be amplified, while P6 loop of trnL （UAA） intron could be used for identification. The length of the P6 loop of trnL（UAA） intron sequence of Daemonorops draco was 45 bp. The maximum K2P intra-spe- cific genetic distances of Daemonorops draco were much smaller than the minimum inter-specific genetic distances between Daemonorops draco and its adulterants. The NJ tree showed that Draconis Sanguis differed from its adulterants obviously. CONCLUSION The P6 loop of trnL （UAA） intron can be used as DNA barcode for the identification of Draconis Sanguis.