中药材银柴胡及其混伪品的DNA条形码鉴定研究

Identification of Chinese medicinal materials Stellariae Radix and its adulterants using DNA barcoding

筛选合适的DNA条形码序列,以建立基于DNA条形码技术的银柴胡及其混伪品鉴定方法。本文收集并提取60份银柴胡及其混伪品的DNA,对4种DNA条形码序列ITS、psb A-trn H、rbc L和mat K进行PCR扩增和测序,计算各序列PCR扩增和测序成功率;利用MEGA7.0分析序列特征、计算遗传距离,并构建Neighbour-joining (NJ)系统进化树;利用种间种内变异分析做Barcoding gap图;运用BLAST方法计算各序列鉴定成功率,通过以上指标综合评价不同序列的鉴别能力。结果显示, ITS序列的PCR扩增及测序成功率最高,为95.2%, matK最低,为75%; 4种序列的长度范围为211～797 bp, ITS的GC含量在各序列中最高,为54.35%; matK和ITS的鉴定成功率较高,分别为92%和90%; Barcoding gap图显示ITS有明显的间隔区, rbc L、mat K和psb A-trn H序列间隔区不明显或种间种内变异有部分重叠; NJ系统进化树显示, ITS可将银柴胡聚为一支,同属混伪品二柱繁缕与银柴胡明显分为不同支, rbc L和mat K不能把混伪品二柱繁缕分开。因此, ITS序列可作为银柴胡及其混伪品鉴定的优选条形码序列,为银柴胡的真伪鉴定提供科学依据。

To accurately discriminate Stellariae Radix from its adulterants,four leading candidate DNA barcoding markers were evaluated.Sixty samples including Stellariae Radix and its adulterants have been newly collected and their total genomic DNA was extracted.Four DNA barcoding markers ITS,rbc L,psb A-trn H and mat K were amplified and sequenced.Their sequence characteristic analyses,Kimura-2-parameter(K2P)distance calculation and Neighbor-joining(NJ)phylogenetic tree constructions were accomplished using the MEGA 7.0 software.DNA Barcoding gaps of the four DNA barcoding markers were estimated by the distributions of interand intra-sequence specific variations.Species identification efficiency was calculated using the BLAST method.The results showed that ITS had the highest(95.2%)while mat K demonstrated the lowest(75%)PCR and sequencing efficiency.The length range of the four markers were in the ranger of 211-797 bp,and the G+C content of ITS was highest(54.35%).The identification efficiency of mat K and ITS was 92%and 90%respectively.Barcoding gap could be found in ITS sequences.The NJ phylogenetic tree constructed using ITS sequences showed that samples of Stellariae Radix were separately formed into one clade,and samples of adulterants like Stellaria bistyla were clearly belong to different branches from Stellariae Radix,whereas NJ trees constructed using psb A-trn H,rbc L and mat K could not differentiate Stellariae Radix from its adulterants.Therefore,ITS regions as DNA barcodes can stably and accurately distinguished Stellariae Radix from its adulterants,and provide a new technique for modern identification of Stellariae Radix.