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研究生:陳巧宜
研究生(外文):Chiao-Yi Chen
論文名稱:黑殭菌(Metarhizium anisopliae)變種間核糖體DNA內轉錄間隔區序列及二次代謝物之分析
論文名稱(外文):Analysis of Ribosomal DNA Internal Transcribed Spacer Sequence and Secondary Metabolites of Metarhizium anisopliae Varieties
指導教授:郭章信古國隆
指導教授(外文):Chang-Hsin KuoKuo-Lung Ku
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物資源學系研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:80
中文關鍵詞:黑殭菌親緣關係二次代謝物化學多樣性
外文關鍵詞:Metarhizium anisopliaephylogenysecondary metaboliteschemodiversity
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黑殭菌 (Metarhizium anisopliae (Metsch.) Sorokin ) 是一種昆蟲病原真菌,寄主範圍極廣,可感染 200 種以上的昆蟲,深具經濟及研究的價值。本研究收集國內外共計 32 株供試黑殭菌菌株,配合使用分類上常利用之核糖體核酸內轉錄間隔區 (rDNA-ITS) 定序法直接比對各菌株間核酸序列的差異度,並以鄰近接連法 (Neighbor-Joining , NJ) 及最大簡約法 (Maximum-Parsimony, MP) 建構出親緣關係樹 (phylogenetic tree)。同時本研究更進一步分析黑殭菌各菌株的二次代謝物 (secondary metabolites),檢測目標以黑殭菌素 (destruxins, DTX) 和細胞鬆弛素 (cytochalasins) 為主,而此二類化合物皆各有其生物活性及醫學上之利用價值。本研究室在以往曾自南投採集所得之 Ma0407 菌株發現其可產生類細胞鬆弛素之新化合物,而購自食品工業發展研究所之菌株 BCRC35507 可產生黑殭菌素,因此由 Ma0407 及 BCRC35507 兩菌株產生之已知代謝物作為對照組,將供試菌株代謝萃取物經由高效液相層析儀 (high-performance liquid chromatography; HPLC) 分析後結果顯示 ML 13 可產生和 Ma0407 相同的類細胞鬆弛素,而 Ma0319及 BCRC 35520 和 ARSEF 488、7488、5369 則可比對出和 BCRC 35507 相同的黑殭菌素,但各菌株間所產之黑疆菌素種類及含量均有差異,而黑疆菌素A、B、B2/E2 和 C 則普遍存在於個菌株間。經由 rDNA-ITS 定序法探討 Metarhizium 種間及變種間之親緣關係及了解變種間二次代謝物之化學多樣性 (chemodiversity),發現縱使 ITS 序列完全相同、親緣關係相近者也未必會產生完全相同之代謝物。
Metarhizium anisopliae (Metsch.) Sorokin, a fungal entomopathogen, can infect more than 200 kinds of insect; the it is a value fungus for extensive studies both in industry and medicine. In the present study, 32 isolates of M. anisopliae collected from domestic and abroad were analyzed for ribosomal DNA internal transcribed spacer (rDNA-ITS) and secondary metabolites. The results of the difference between varieties were subjected the collected species. The difference of DNA sequence were analyzed by method to construct Neighbor-Joining (NJ) and Maximum-Parsimony (MP) phylogeny tree. The differences of secondary metabolites, mainly destruxins and cytochalasins of M. anisopliae varieties were also to be subjected to statistically analysis further. Ourrently. We use Ma0407, which collected from Nanto with analogs of cytochalasin, and BCRC 35507 purchased from Food Industry Research and Development Institute with destruxins. I employ Ma0407 and BCRC 35507 as reference group for chemodiversity study. After high-performance liquid chromatography (HPLC) analysis, the data reveals that ML13 and Ma0407 could produce the identical analogs of cytochalasins, in addition, BCRC 35507, Ma0319, BCRC 35520, and ARSEF 488, 7488, 5369 have the same destruxins; however, the classification and that quantity of destruxins in M. anisopliae strains are generally different, but destruxins A, B, B2/E2 and C exist commonly in every M. anisopliae isolate. In the present study, we explore the phylogeny of Metarhizium species and of Metarhizium varieties through rDNA-ITS sequencing method, and then investigate the chemodiversity of secondary metabolites between varieties. Results show that even if different M. anisopliae strains that possess identical rDNA-ITS sequence, meaning they have close phylogenetic relationship, cannot produce completely identical metabolites.
摘要..........................................II
Abstract ....................................III
第一章、 前言 .............................1
第二章、 文獻回顧 .............................3
一、 黑殭菌 (M. anisoplia) 之介紹..........3
二、 黑殭菌屬的分類沿革及特性 ..........4
三、 黑殭菌系統分類的研究進展 ..........6
1. 同功酵素分析 ...................6
2. RFLP 和 RAPD 技術的研究 ..........7
3. rDNA 序列分析 ...................8
四、 黑殭菌所產之代謝物 ..................10
1. 黑殭菌素 ...........................11
2. 細胞鬆弛素 ..................13
第三章、 材料與方法 ..................15
一、 供試菌株的來源、保存與製備 .........15
二、 黑殭菌親緣關係之建立 .........15
(一)、 DNA 萃取 ...........................15
(二)、 PCR 與 ITS 定序 ..................16
(三)、 序列排列及親緣圖建立 .........18
三、 黑殭菌代謝物質分析 ..................18
(一)、 樣品萃取 ...........................18
(二)、 HPLC分析 ...........................18
第四章、 結果 ...........................20
一、 黑殭菌親緣關係之建立 .........20
二、 黑殭菌代謝產物之分析 .........22
第五章、 討論 ...........................23
第六章、 參考文獻 ...........................28
附錄、 ...........................65
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