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研究生: 李肇晉
Gou-Gin Lee
論文名稱: 台灣地區栓菌屬親緣關係之研究
Phylogenetic analysis of Trametes Fr. in Taiwan
指導教授: 葉增勇
Yeh, Zeng-Yung
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2005
畢業學年度: 93
語文別: 中文
論文頁數: 61
中文關鍵詞: 栓菌親緣關係
英文關鍵詞: Trametes, Phylogeny
論文種類: 學術論文
相關次數: 點閱:609下載:33
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  • 栓菌屬(Trametes),是一種廣泛分佈於全世界的多孔菌,雲芝Trametes versicolor(L.:Fr.)Pilt又稱彩絨栓菌,一種廣為人知的藥用真菌,分類地位隸屬於擔子菌門(Basidiomycota),帽菌綱(Hymenomycetes),無褶菌目(Aphyllophorales),多孔菌科(Polyporaceae)。本實驗由台灣 新竹食品工業發展研究所之「生物資源保存及研究中心」購買Trametes elegans, T. feei, T. gibbosa, T. heteromorpha, T. hirsuta, T. lactinea, T. orientalis, T. pubescens, T. suaveolens 9株台灣產栓菌屬純菌株,及國立自然科學博物館 吳聲華研究員提供台灣產雲芝(T. versicolor)種內不同採集地14株及毛栓菌1株(T. hirsuta)之純培養菌株,併以野外採集所得之毛栓菌2株,於實驗室純培養並抽取基因體DNA(genomic DNA),針對核醣體DNA內轉錄區(ITS1-5.8S-ITS2 rDNA)及部分粒線體次單位核醣DNA(partial mt SSU rDNA),經由聚合連鎖反應(PCR)及核酸定序(sequencing)將目標序列解序,再加上美國國家衛生院的NCBI網站下載之5株多孔菌科真菌,所得之序列以生物資訊常用相關軟體Bioedit, Clustal, DnaSP等作序列排序(alignment)及族群分析,藉此探討本屬真菌種內遺傳分化及種間之親緣關係,並計算遺傳距離(genetic distance),再以統計軟體PAUP4.0之Maximum parsimony及Neighbor-Joining兩種分析法進行演算並建構演化樹,結果發現本實驗中分析的雲芝(T. versicolor)種內發生些微變異,但不足以產生種的分化現象;T. orientalis與T. lactinea成一共系群並自成一格;T. heteromorpha, T. feei兩株栓菌在早期便與其他栓菌分歧演化,其遺傳距離也比較遠,甚至超過不同屬的多孔菌,因此這兩株栓菌的歸類有再檢討之必要;朱紅栓菌(Pycnoporus cinnabarinus)則是與栓菌群有著非常密切的親緣關係,對於其過去在形態分類的變動上,提供了分子分類訊息供參考。本實驗所建立的親緣關係樹,期望能對於日後在相關產品開發或相近物種之藥用特性比較提供發展基礎。

    Trametes versicolor(L.:Fr.)Pilt, is a common polypore fungus used for medicinal purposes. It belongs to Basidiomycetes, Hymenomycetes, Aphyllophorales, Polyporaceae, and is widely distributed in the world. Nine pure cultures of Trametes purchased from BCRC of FIRDI in Hsinchu, Taiwan, 14 cultures of Trametes versicolor and 1 culture of Trametes hirsuta from Dr. Wu, and 2 collections of Trametes hirsuta are totally used in this study. Genomic DNA were extracted from the above cultures, then the internal transcribed spacer rDNA(ITS1-5.8S-ITS2 rDNA)and partial mitochondrial small subunit rDNA(partial mt SSU rDNA)were further amplified by polymerase chain reaction(PCR)with primers. The DNA sequences of the 26 tested cultures and 5 polypores sequnces from NCBI were used in analysis. Bioinformation software like BioEdit, Clustal, and DnaSP were used to align the sequences and to analyze the genetic distance and genetic differentiation. Maximum parsimony tree and Neighbor-joining tree were constructed with PAUP 4.0(Phylogeny Analysis Using Parsimony). The results revealed that there are minor variations in T. versicolor. Trametes orientalis and T. lactinea were located in the same clade. Another two Trametes species, namely, T. heteromorpha and T. feei have long genetic distance from other Trametes species, and may be divergent from other species early in the past. The phylogenetic relationship of Pycnoporus cinnabarinus is closely related to Trametes group, and this result provides the classification information between Pycnoporus cinnabarinus and Trametes genus. The phylogenetic research can provide a base for further medicinal development of Trametes species and other related polypores.

    圖表目錄……………………………………………………………ii 中文摘要…………………………………………………………iii Abstract …………………………………………………………iv 前 言…………………………………………………………1 材料與方法……………………………………………………10 一. 形態觀察與整理……………………………………10 二. 菌株來源…………………………………………………10 三. 菌絲體之培養及保存……………………………………10 四. 液態培養………………………………………………12 五. 染色體DNA之製備………………………………………12 六. 染色體DNA純度與定量分析……………………………13 七. 聚合連鎖反應………………………………………13 八. 聚合連鎖反應產物之檢測…………………………14 九. DNA定序………………………………………………14 十. 序列分析……………………………………………14 結 果……………………………………………………19 一. 聚合連鎖反應產物之檢測…………………………20 二. 序列比對與網路序列搜尋……………………………22 三. 遺傳距離………………………………………………24 四. 中性假說檢測…………………………………………38 五. 親緣關係樹之建構………………………………………42 討 論…………………………………………51 結 論………………………………………………………55 參考文獻…………………………………………………………56

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