山茱萸药材及其混伪品的ITS／ITS2序列鉴定研究

利用DNA条形码技术准确快速鉴定山茱萸药材及其混伪品。方法：提取山莱萸及其混伪品的基因组DNA，利用PCR技术扩增它们的ITS序列。所得序列经双向测序后用CodonCode AlignerV3．5．4软件进行拼接和质量评估，用MEGAV5．0计算种内、种间的遗传距离，构建NJ（邻接）树鉴定山茱萸药材及其混伪品。结果：山茱萸药材的ITS序列长度均为659bp，种内变异较小，平均K2P遗传距离为O．005，远小于其与混伪品的种间平均K2P遗传距离0．357。不同来源的山茱萸药材ITS2序列无变异，长度均为250bp，其与混伪品的种间平均K2P遗传距离为0．571。ITS／ITS2序列的NJ系统聚类树和BLAST比对结果均可明显区分山茱萸药材及其混伪品，表现出良好的单系性。结论：ITS／ITS2序列可准确有效鉴定山茱萸药材，为临床安全用药奠定了基础，也为其它药材的分子鉴定提供了新的思路。

Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences

The DNA barcoding method was used to accurately and rapidly identify Comi Fructus and its adulterants. METHODS： Genomic DNA extracted from Comi Fructus and its adulterants were used as templates. The ITS （internal trascribed spacer） regions were amplified using polymerase chain reaction. Sequence assembly was performed using CodonCode Aligner V 3.5.4. Genetic distances were computed using MEGA V 5.0. Species identification was conducted using neighbor-joining （NJ） trees. RESULTS： The ITS sequence length of Comi Fructus was 659 bp. The average intra~specific genetic distance of Comi Fructus was 0.005, markedly lower than the inter-specific genetic distance between Corni Fructus and its adulterants （0.357）. The ITS2 sequence length of Comi Fructus was 250 bp. No variation was found among the different samples. The interspecific genetic distance of ITS2 between Corni Fructus and its adulterants was 0.571. NJ trees and BLAST results indicated that Corni Fructus and its adulterants can be easily differentiated with monophyly. CONCLUSION： ITS/ITS2 regions can accurately and efficiently distinguish Corni Fructus and its adulterants. In addition, the results not only established the foundation for the clinical safety in the utilization of Comi Fructus, but also provided reference for molecular identification of other Chinese herbal medicine and Chinese herbal pieces.