茅莓根及其混伪品的DNA条形码鉴定研究

Identification of Rubi Parvifolii Radix and Its Adulterants Using DNA Barcoding

目的茅莓根与同科属其他物种存在混用现象,为保证安全用药,本实验利用ITS2序列对茅莓根及其混伪品进行鉴定研究。方法提取茅莓及其混伪品的DNA,经PCR扩增后测序,通过Codon Code Aligner V3.7.1对测序序列进行质量分析和拼接,基于MEGA5.1中的K2P模型计算茅莓根及其混伪品的种内、种间遗传距离并构建系统聚类树。结果茅莓ITS2序列比对后长度为212 bp,种内最大K2P遗传距离为0.014,存在3个变异位点,平均GC含量为57.42%。基于ITS2序列构建NJ树,茅莓及其混伪品能够表现出良好的单系性,均能相互明显区分。从药材市场购买的23份茅莓根样品,DNA提取和扩增成功率为100%,采用NJ树分析,其中13份药材样品与茅莓聚为一支,其余10份属于其他物种。结论茅莓及其混伪品鉴定难题可用ITS2条形码技术解决。

OBJECTIVE To identify Rubi Parvifolii Radix from its adulterants using ITS2 sequence. METHODS All the DNA of Rubi Parvifolii Radix and its adulterants were extracted. All the sequences were assembled using the CondonCode Aligner V3.7. 1. The Kimura 2-parameter （K2P） genetic distances and the neighbor joining （NJ） phylogenetic tree were calculated by using MEGAS. 1. RESULTS The ITS2 sequences were succesful]y amplified and sequenced. The length of ITS2 sequences of Rubus parvifolius was 212 bp, and the average GC content was 57.42%. Among 20 ITS2 sequences of R. parvifolius, three transversions were detected at site 66,118 and 177. The maximum intra-specific K2P distance of R. parvifolius was 0. 014, lower than the minimum interspecific K2P distances of adulterants, except for R. coreanus. Additionally, the ITS2 sequences of all the polytypic species were separated into pairs of divergent clusters in the NJ tree and R. parvifolius can be distinguished clearly from its adulterants. The ITS2 sequences of 23 samples of Rubi Parvifolii Radix collected from different herb markets, were successfully amplified. The NJ tree analysis indicated that 13 samples clustered with R. parvifolius, while the other 10 samples were clustered into other divergent clusters. CONCLUSION ITS2 Sequence can be used as DNA barcode to correctly identify Rubi Parvifolii Radix from its adulterant.