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研究生:王正如
論文名稱:利用AFLP指紋分析尋找尖鐮胞菌分化型特異性DNA片段
論文名稱(外文):Characterizing formae speciales-specific DNA fragments by the AFLP fingerprinting in Fusarium oxysporum
指導教授:藍清隆
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:88
中文關鍵詞:Fusarium oxysporumAFLP
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尖鐮胞菌(Fusarium oxysporum)的形態、生理與病原性都具有高度變異性,造成檢測及鑑定上極大的困難,本研究旨在利用分子生物學技術,尋找輔助快速檢測連胞菌的分子標記。利用AFLP (Amplified fragment length polymorphism )指紋分析法,配合銀染PAGE (polyacrylamide gel electrophoresis)膠體,以七種常見之尖鐮胞菌分化型為主,找尋分化型間特異性DNA片段。AFLP分析步驟中,以MseI限制酵素切割尖鐮胞菌基因組DNA,其後接合相對應之轉接子,限制酵素切割與轉接子接合分別為37℃作用兩小時,20℃作用過夜為最佳。研究中選用的二個延伸鹼基選擇性放大引子M-AC、M-AG、M-CA、M-CG、M-CT、M-GA、M-GG、M-TC、M-TG能有效簡化分子複雜度。9組選擇性引子組合放大出的條帶數大約在21條至53條間,對於7個參試樣本總共得到2075個AFLP譜帶,平均一組引子對於一個樣本可產生33個AFLP譜帶。9組選擇性延伸引子組合共得到108個特異性DNA片段,平均一組選擇性延伸引子組合能得到12個特異性DNA片段。隨機選出63個特異性DNA片段自銀染之PAGE中回收,其中46個DNA片段成功回收並選殖入yT&A載體。將上述46個特異性DNA片段定序,分析DNA與氨基酸序列後發現其中有5個序列可能與跳躍子(transposon)有關,4個是已知基因的序列,17個是待確認蛋白質的序列,20個與Genbank中的序列相似度極低。而其中有4個特異性DNA片段可能為尖鐮胞菌跳躍子Fot3、Folyt 1及轉位酵素(transposase)序列;其餘尚有6個DNA序列中具有鋅結合模組(zinc binding domain) “ZnF_NFX domain”及訊號胜肽(signal peptide),因此推測這些DNA片段上下游可能有基因存在。另有一個片段氨基酸序列具有訊號胜肽與4段穿透細胞膜的片段(transmembrane segment),故推測上下游可能有表現外泌性蛋白質的基因存在。
目錄
中文摘要 ---------------------- I
英文摘要 --------------------- II
前言 ---------------------- 1
材料與方法 ---------------------- 8
菌株來源與培養方 ---------------------- 8
基因組DNA萃取 ---------------------- 8
瓊脂膠體電泳 ---------------------- 9
AFLP分析 ---------------------- 10
自聚丙烯醯胺膠體回收特異性DNA片段 ---------------------- 12
自瓊脂膠體回收DNA片段 ---------------------- 13
載體與DNA片段的接合反應 ---------------------- 14
大腸桿菌勝任細胞之製備 ---------------------- 14
熱休克轉形作用 ---------------------- 15
質體DNA萃取 ---------------------- 15
序列分析 ---------------------- 17
結果 ---------------------- 19
AFLP條件測試 ---------------------- 19
AFLP再現性測試 ---------------------- 20
AFLP分析結果 ---------------------- 21
AFLP特異性片段回收與選殖 ---------------------- 22
特異性DNA片段選殖與DNA及氨基酸序列比對分析 ----------------- 23
討論 ---------------------- 29
參考文獻 ---------------------- 42
圖 ---------------------- 49
表 ---------------------- 71
附錄 ---------------------- 88
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