基于ITS2序列的紫草PCR-RFLP鉴别研究

目的：通过对市面大量流通的非中国药典收载紫草基原的所谓紫草进行分析，建立紫草正品与非中国药典品的鉴别方法。方法：通过聚合酶链式反应（PCR）对紫草的ITS2序列进行扩增，通过与GenBank数据库进行Blast比对，确定非中国药典品的科属；利用距离法和建树法比较非中国药典品与软紫草属、滇紫草属、紫草属的亲缘关系；利用限制性内切酶AluⅠ酶切PCR产物，将酶切产物经琼脂糖凝胶电泳检测后紫外成像。结果：非中国药典品经Blast比对为软紫草属；非中国药典品与软紫草属新疆紫草平均遗传距离为0.071，均小于与紫草属和滇紫草属的平均遗传距离，通过建立Neighbor-Joining（N-J）树聚类分析，非中国药典品与软紫草属新疆紫草和黄花软紫草明显区分，并与滇紫草属和紫草属均具有良好的单系性。正品紫草能被限制性内切酶AluⅠ酶切为两条条带，非中国药典品不能被酶切。结论：市场流通大量紫草非中国药典收载的新疆紫草（Arnebia euchroma（Royle）Johnst.）和内蒙紫草（Arnebia guttata Bunge），利用聚合酶链式反应限制性长度片段多态性法可鉴别紫草正品与非中国药典品。

Objective: To analyze a large number of Arnebiae Radix which are circulating on market but not the origin record in ChP,and to establish an identification method for Arnebiae Radix and other non-ChP samples.Methods: ITS2 sequences of samples were amplified with polymerase chain reaction(PCR),and then blasted with GenBank database to determine family and genus.The genetic relationship of non-ChP with Arnebia Forssk,Lithospermum L.,and Onosma L. was evaluated by the methods of nearest distance and N-J tree.The PCR products were incubated with the restrictive endonuclease AluⅠ,followed by the agarose gel electrophoresis and image obtaining under UV light.Results: non-ChP belonged to Arnebia Forssk.The mean inter-specific genetic distance between non-ChP and Arnebia euchroma (Royle)Johnst.(0.071),were much lower than that between non-ChP and Lithospermum L. and between non-ChP and Onosma L. N-J trees indicated that non-ChP,Arnebia euchroma (Royle)Johnst. and Arnebia guttata Bunge could be easily identified,and revealed a better monophyly with Lithospermum L. and Onosma L.The PCR products amplified from the ITS2 region of Arnebia euchroma (Royle)Johnst. and Arnebia guttata Bunge could be cleaved into two fragments after incubation with AluⅠ,while not observed in the PCR products from non-ChP.Conclusion: Most of the Arnebiae Radix circulating on market are not the one recorded in ChP.The established PCR-RFLP method can identify Arnebia euchroma (Royle)Johnst. and Arnebia guttata Bunge with non-ChP.