目的:基于ITS2序列对中药材王不留行种子及其混伪品进行鉴定研究,为王不留行种子及其混伪品的鉴定提供新方法.方法:对王不留行种子及其混伪品样品提取基因组DNA、PCR扩增、双向测序获得ITS2序列.应用MEGA 6.0软件计算种内、种间遗传距离,构建邻接树,基于自行编写的代码和开源代码PHP QR Code的编码方式,将王不留行及其混伪品ITS2序列转换为条形码图片,获得各物种二维DNA条形码图片,并在中药材DNA条形码鉴定系统(www.tcmbarcode.cn)对所获得ITS2序列进行分析鉴定.结果:王不留行药材ITS2序列长度为219-221 bp,种内最大K2P遗传距离为0.009,小于其与混伪品的种间K2P遗传距离,王不留行及其混伪品ITS2序列间存在的变异位点较多.NJ树结果表明王不留行与其混伪品分别聚为一支,可明显区分.结论:ITS2序列可以准确鉴别王不留行种子,为其种质资源鉴定和中药材种子质量标准的建立提供了新方法,将获得的ITS2序列转换为二维DNA条形码可为王不留行药材流通管理提供便利,为保障王不留行临床用药安全提供了新的技术手段.
Identification of Seeds of Vaccaria segetalis (Neck.) Garcke Based on ITS2 Sequence
This study was aimed to explore a new method to identify the seeds of Vaccaria segetalis (Neck.) Garcke from its adulterants by the internal transcribed spacer 2 (ITS2) regions.In this study,all genomic DNAs of samples of V.segetalis and its adulterants were extracted.And then,the ITS2 sequences were PCR amplified and sequenced bidirectionally.The intraspecific and interspecific genetic distances were calculated by the MEGA 6.0 software for the construction of neighbor-joining (N J) tree.Based on self written code and open source PHP QR Code coding way,the ITS2 sequences of V.segetalis and its adulterants were transferred into the two-dimensional barcoding images,which can be used in the analysis and identification in the Traditional Chinese Medicine (TCM) DNA barcoding species identification system (www.tcmbarcode.cn).The results showed that the sequence lengths of ITS2 regions of V.segetalis were ranged from 219 to 221 bp.The largest intraspecific K2P genetic distance was 0.009,which was smaller than the interspecific K2P genetic distance compared to its adulterants.There were many variation sites of ITS2 sequences between V.segetalis and its adulterants.The NJ tree showed that V.segetalis and its adulterants can be easily differentiated according to their monophyly.It was concluded that the ITS2 sequences could be used to identify V.segetalis and its adulterants,which not only provided a new method for the identification of germplasm resources of TCM,but also supplied some guidelines for the establishment of the quality standards of Chinese herbal seeds and seedlings.To transfer the two-dimensional DNA barcoding image of the obtained ITS2 sequences of V.segetalis were convenient for its circulation management.It provided new technical means for the clinical medication safety of V.segetalis.
NETL
NSTL
万方数据
维普
