基于DNA条形码-产地-形态联用的药材溯源新方法研究——以黑果枸杞1种伪品为例

A new herbs traceability method based on DNA barcoding-origin-morphology analysis——an example from an adulterant of 'Heiguogouqi'

利用NCBI核酸数据库中ITS(internal transcribed spacer)序列,结合产地分析和形态分析对新发现的一种黑果枸杞混伪品进行溯源研究。通过提取样品DNA,PCR扩增及双向测序,获得样品的ITS序列。所得序列用Conting Express软件进行拼接,截取ITS序列全长;利用NCBI核酸数据库中已登录的ITS序列进行相似度分析对其进行DNA条码溯源;根据其产地分析DNA条形码溯源结果;再根据形态特征进一步验证结果,3种方法联用获得溯源结果。新发现的黑果枸杞混伪品来源于小檗科小檗属植物喀什小檗Berberis kaschgarica。与黑果枸杞的主要区别:喀什小檗果实表面平滑,质地轻脆,黑果枸杞表面皱缩,质地坚实而硬;喀什小檗外果皮细胞壁连珠状增厚,石细胞壁平直,层纹不明显,黑果枸杞外果皮细胞壁不增厚,石细胞壁波浪状,层纹明显;喀什小檗的ITS序列长度为606 bp,黑果枸杞的长度为654 bp,二者序列对齐后的相似度为53.32%。

The fruit of Lycium ruthenicum is a common folk medicine in China. Now it is popular for its antioxidative effect and other medical functions. The adulterants of the herb confuse consumers. In order to identify a new adulterant of L. ruthenicum, a research was performed based on NCBI Nucleotide Database ITS Sequence, combined analysis of the origin and morphology of the adul- terant to traceable varieties. Total genomic DNA was isolated from the materials, and nuclear DNA ITS sequences were amplified and sequenced;DNA fragments were collated and matched by using ContingExpress. Similarity identification of BLAST analysis was performed. Besides, the distribution of plant origin and morphology were considered to further identification and verification. Families and genera were identified by molecular identification method. The adulterant was identified as plant belonging to Berberis. Origin analysis narrowed the range of sample identification. Seven different kinds of plants in Berberis were potential sources of the sample. Adulterants variety was traced by morphological analysis. The united molecular identification-origin-morphology research proves to be a preceding way to medical herbs traceability with time-saving and economic advantages and the results showed the new adulterant of L. ruthenicum was B. kaschgarica. The main differences between B. kaschgarica and L. ruthenicum are as follows： in terms of the traits, the surface of B. kaschgarica is smooth and crispy, and that of L. ruthenicum is shrinkage, solid and hard. In microscopic characteristics, epicarp ceils of B. aschgarica thickening like a string of beads, stone cells as the rectangle, and the stone cell walls of L. ruthenicum is wavy, obvious grain layer. In molecular sequences, the length of ITS sequence of B. kaschgarica is 606 bp, L. ruthenicum is 654 bp, the similarity of the two sequences is 53.32%.