蒲公英與其偽品物種5.8S rDNA序列及PCR-RFLP之分子鑑定

PCR-RFLP Marker of Ribosomal DNA Used in Detection of Adulteration Species of Taraxacum mongolicum

袁秋英(Chiou-Ing Yuan) ； 林李昌(Li-Chang Lin) ； 郭昭麟(Chao-Lin Kuo) ； 蔣慕琰(Mou-Yen Chiang)

《作物、環境與生物資訊》 4卷4期 (2007/12) Pp. 285-296

https://doi.org/10.30061/CEB.200712.0002

摘要

近年中草藥之研發與運用倍受矚目，然而藥材基原常出現代用品及偽品，使用不當易影響療效，甚至發生中毒現象。由於分子標誌普遍應用於植物物種之鑑定，其中針對基原植物18S-26S rDNA序列之比對，已漸成為鑑定中草藥真偽的有利證據之一。本研究利用ITS1-5.8S rRNA-ITS2序列之差異及PCR-RFLP技術，建立蒲公英(Taraxacum mongolicum Hand.-Mazz.)及其偽品或替代品之檢測方法。試驗結果顯示蒲公英、臺灣蒲公英(T. formosanum Kitanlura)及西洋蒲公英(T. officinale Wiggers)等3 種蒲公英，ITS1-5.8S rRNA-ITS2 序列長度皆為643 bp；而兔仔菜(Ixeris chinensis (Thunb.) Nakai)、刀傷草(I. laevigata (Bl.)Sch.) 、鵝仔草(Pterocypsela indica L.) 、苦滇菜(Sonchusoleraceus L.)、黃鵪菜(Youngia japonica L.)、地膽草(Elephantopus mollis Kunch)及紫背草(Emilia sonchifolia (L.) DC. var. javanica(Burm. F.) Mattfeld )等7 種偽品基原植物ITS1-5.8S rRNA-ITS2 序列長度界於634-645bp 之間。蒲公英屬植物之相似度為95-99%；蒲公英屬植物與7種偽品之相似度則介於80-88%之間。經由比對3種蒲公英及7種偽品基原植物5.8S rRNA-ITS 核酸片段之異同，分別利用Sph I、Mse I 及Bmr I限制酶反應，可將蒲公英屬植物5.8S rRNA-ITS核酸片段，切割為2-4 條不同長度之多型性條帶。本研究所建立之PCR-RFLP檢測技術，可有效及正確區別蒲公英藥材基原之真偽，可提供為中草藥品質控管及基原鑑定之依據。

關鍵字

蒲公英 ； 中藥 ； 分子鑑定 ； 核糖體核酸 ； 間隔區 ； 聚合酶鏈鎖反應－限制酶片段長度多型性

並列摘要

Substitutes and adulterants of traditional Chinese medicine (TCM) materials are often introduced intentionally or accidentally, thus serious interfering with their therapeutic effects, even leading to life-threatening poisoning. DNA markers have been widely used for identification of plant species in recent decade. Novel methods for molecular authentication of Taraxacum mongolicum have established in this study, based on direct sequencing of the internal transcribes spacer (ITS) region of 18S-26S ribosomal DNA (rDNA) and PCR-restriction fragment length polymorphism (RFLP). The whole length of ITS1-5.8S rRNA-ITS2 region was 643 bp in T. mongolicum, T. formosanum and T. officinale, and was 634-645 bp in the seven adulterant species. The similarity of ITS1-5.8S rRNA-ITS2 regions among Taraxacum species and between Taraxacum species and their adulterant species were 95-99% and 80-88%, respectively. The PCR product of three Taraxacum species and seven adulterants on the 5.8S rRNA-ITS were digested with the restriction endonuclease Sph I, Mse I and Bmr I. Each fragment gave unique electrophoretic profiles for three Taraxacum species. This method provides an effective and accurate identification of Taraxacum species.

並列關鍵字

Taraxacum mongolicum ； Traditional Chinese medicine TCM ； Molecular marker ； Ribosomal DNA ； Internal transcribed spacer ITS ； Polymerase chain reaction-Restriction Fragment Length Polymorphism PCR-RFLP