基于ITS2序列片段的药用植物栝楼及混淆品分子鉴定研究
Molecular Identification of Trichosanthes kirilowii and Its Adulterants Using ITS2 Sequence Fragments

应用ITS2条形码序列片段对我国市场药用植物栝楼与其混淆品共8份材料进行分子鉴定.对所有材料根部进行DNA提取纯化、PCR扩增并双向测序得到ITS2序列，将所得8条序列与Genbank中7条序列用Clustal X软件进行比对，BioEdit软件人工校正；利用MEGA5.0软件计算种内、种间遗传距离，并采用K2P距离法构建NJ和ML树，评价序列的鉴定效果.中药材植物各物种内遗传距离变异区间为0~0.077 9，平均为0.019，物种间遗传距离变异区间为0.004~0.594，平均0.436，种间距离明显大于种内距离；构建药用植物栝楼各物种NJ和ML系统进化树，二者结果一致，各物种均聚为一支，形成单系类群，支持率均在50%以上.ITS2条形码序列能够快速准确地从种的水平鉴定药用植物栝楼真伪.
Molecular identification based on ITS2 sequence fragments was conducted of Trichosanthes kirilowii, a Chinese herbal medicine plant, and its adulterants on the medicine market.We treated the roots of 8 experimental samples for DNA extraction and purification, PCR amplification and bidirectional sequencing to get the ITS2 sequences.Then the 8 ITS2 sequences obtained by experiment and 7 downloaded ITS2 sequences from GenBank database were aligned using ClustalX, with manual adjustments assisted by BioEdit. MEGA program version 5.0 was employed to estimate the intraspecific and interspecific genetic distance. NJ and ML trees were constructed using K-2P distance method in order to evaluate the identification results.The range of intraspecific difference space of all Chinese herbal plants was 0-0.077 9, with a mean value of 0.019, while the interspecific distance ranged from 0.004 to 0.594, with a mean value of 0.436.The interspecific value was significantly greater than the intraspecific value. NJ and ML phylogenetic trees of different species of T. kirilowii and its adulterants were constructed and the two phylogenetic trees were consistent with each other.Each species was clustered into one branch, thus forming a monophyletic group, with a support rate of > 50%.The results showed that the ITS2 barcoding is a fast and accurate method for the identification of T. kirilowii and its adulterants