摘要
目的：本文选用金樱子作为研究对象验证DNA条形码鉴定中药材的稳定性及准确性。方法：通过改良的CTAB法提取10份样品的基因组DNA,扩增得到ITS2片段。将得到的片段进行双向测序,运用软件CodonCode Aligner进行拼接,其它6份样品来源于GenBank。将金樱子与混伪品ITS2序列应用MEGA 5.0软件进行序列比对,计算种内、种间距离,构建邻接树（neighbor-joining tree,NJTree）。结果：金樱子药材ITS2序列长度为219~221 bp,存在两个Poly C结构。金樱子种内平均Kimura 2-parameter（K-2-P）遗传距离为0.005,远小于与混伪品的种间平均K-2-P遗传距离0.574。NJ树结果表明金樱子与混伪品分为两枝,Bootstrap支持率为99%,表现出良好的单系性。结论：ITS2序列作为DNA条形码能准确稳定的鉴别金樱子药材,本研究为保证金樱子临床用药安全提供了技术保障。
In order to verify the sstability and accuracy of DNA barcode technique,we chose Rosa laevigata Michx as study object.Genomic DNAs of 10 samples were extracted by modified CTAB method.ITS2 sequences were obtained by direct PCR sequencing;the other 6 sequences were obtained from GenBank.The sequences were assembled using the CodonCode Aligner.All of the 16 ITS2 sequences were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the Kimura 2-parameter（K-2-P） model.Results indicated that the lengths of ITS2 regions of R.laevigata ranged from 219 to 221 bp with two Poly C structure in it.The intra-specific genetic distances were smaller than inter-specific ones in ITS2 regions of R.laevigata.The NJ tree showed that R.laevigata and adulterants were divided into two clades,with 99% bootstrap value,showing good monophyly.So,ITS2 was considered a good marker to identify R.laevigata and its adulterants.
出处
《世界科学技术-中医药现代化》
北大核心
2013年第3期435-440,共6页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委青年科学基金项目(81001608):基于全叶绿体基因组分析筛选乌头属药用植物DNA条形码序列,负责人:韩建萍
关键词
金樱子
DNA条形码
ITS2
物种鉴定
Rosa laevigata Michx
DNA bardcoding
ITS2
species identification
