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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15062

Title: DNA鑑定技術在金絲桃屬種別辨識上應用之探討
Studies on the Application of DNA Techniques for Spceies Identification on Hypericum spp.
Authors: Cheng-Li Fang
方成立
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 金絲桃
Hypericum;ITS
Date: 2006
Issue Date: 2011-06-30T07:58:50Z
Abstract: 文獻記載金絲桃屬植物具有抗發炎、抗肝炎及抗病毒之功能,因此廣被栽培採集使用,但由於同科屬植物種類相當多,外觀及成分類似,以植物形態與化學法往往不易區分其基原。為確保藥材基原之正確性,本研究利用DNA分子標記技術來鑑定13種不同來源的金絲桃屬植物種間的差異及親緣關係,分別使用核醣體rDNA基因間(Internal Transcribed Spacer,ITS)序列、ITS限制片段長度多型性分析(Restriction Fragment Length Polymorphism,RFLP) 、逢機擴增多型性核酸分析(Random Amplified Polymorphic DNA,RAPD)等三種方法進行分析比對。 結果顯示,使用特定ITS引子對,擴增出金絲桃屬植物ITS片段後將片段定序,由序列分析比對結果:可區分同科不同屬之蘭嶼福木(Garcinia linii)及同屬不同種貫葉連翹(Hypericum perforatum)、地耳草(Hypericum japonicum)、紅旱蓮(Hypericum aseyron)與大陸金絲桃屬No.13的樣品。然而,清水金絲桃(Hypericum nakamurai)與雙花金絲桃(Hypericum geminiflorum)之間的ITS序列沒有差異;大陸金絲桃屬樣品No.11與No.12 的ITS序列相同無法區分;臺灣金絲桃(Hypericum formosanum)與方莖金絲桃(Hypericum subalatum)的ITS序列沒有差異。由於RFLP是利用ITS片段之DNA的限制酶片段進行分析,不需要定序即可快速分析出金絲桃屬植物之間的區別。因此利用金絲桃屬ITS片段之DNA再進行限制酶切位分析片段長度的多型性,來區別同科不同屬之蘭嶼福木及同屬不同種貫葉連翹、地耳草、紅旱蓮與大陸金絲桃屬樣品No.13的樣品之間的親緣關係,但是也無法區別清水金絲桃與雙花金絲桃、臺灣金絲桃與方莖金絲桃及大陸金絲桃屬No.11與No.12的樣品之間的差異。 當ITS序列與ITS-RFLP無法區分時,可使用RAPD的方法進一步分析。以S61、S62、S130等3種不同引子進行RAPD分析結果,S130引子可以同時區別清水金絲桃和雙花金絲桃,以及臺灣金絲桃和方莖金絲桃的差異。 DNA鑑定藥材之技術應用在本研究中13種樣品的基原鑑定,以ITS定序、ITS-RFLP及RAPD可以漸進式的區分同科屬內親緣接近的植物,以於快速、準確的鑑別中藥材。
Hypericum plants have been used as Chinese medicinal herbs with functions of anti-infection, anti-liver dysfunction and anti-virus, whereas materials of similar morphology and chemical fingerprints are difficult to be differentiated. This study attempts to clarify the relationship among 13 Hypericum species using DNA technology, in which analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS), ITS- restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) were investigated to identify Hypericum species. The results demonstrated that sequence analyses of ITS successfully differentiated Hypericum plants of the same family, such as Garcinia linii, Hypericum perforatum, Hypericum japonicum, Hypericum aseyron, and an unknown sample No.13 collected from mainland China, whereas no sequence variations were observed between Hypericum nakamurai and Hypericum geminiflorum, between Hypericum formosanum and Hypericum subalatum, and between unknown samples No.11 and No.12 Moreover, Garcinia linii, Hypericum perforatum, Hypericum japonicum, Hypericum aseyron, and No.13 could be quickly differentiated using ITS-RFLP without knowing the sequence information, but ITS-RFLP fails to distinguish Hypericum nakamurai from Hypericum geminiflorum, Hypericum formosanum from Hypericum subalatum, and samples No.11 from No.12. RAPD is an alternative to authenticate closely related herbal medicines instead of ITS sequence analyses. In this study, random primers S61, S62 and S130 were examined, but only S130 could distinguish Hypericum geminiflorum, from Hypericum formosanum, Hypericum subalatum, from Hypericum perforatum, Hypericum japonicum and unidentified samples No.11, No.12, and No.13 simultaneously. In conclusion, sequence comparisons of ITS are currently widely used as an improved method for the identification of herbal medicines. This study explored a step-by-step approach to clarify 13 closely related Hypericum species, thus providing a rapid and reliable method to authenticate herbal medicines.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0T94420026
http://ntour.ntou.edu.tw/ir/handle/987654321/15062
Appears in Collections:[食品科學系] 博碩士論文

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