臺灣博碩士論文加值系統

爪哇擬青黴菌(Paecilomyces javanicus)屬於不完全菌亞門(Deuteromycotina)、線菌綱(Hyphomycetes)、鏈孢霉目(Moniliales)、鏈孢霉科(Moniliaceaes)、擬青黴菌屬(Paecilomyces)。爪哇擬青黴菌(P. javanicus)是一種蟲生真菌，在自然界裡主要感染雙翅目和鱗翅目昆蟲，其寄主範圍廣泛、產孢量大、在自然界中出現的頻率頗高，深具研究價值。以往傳統鑑定真菌的方法頗具爭議性，故本研究嘗試以RAPD分子標誌技術與18S rDNA基因定序法，針對由台灣北、中、南、東部各地採集到的爪哇擬青黴菌22株，分析菌株與菌株之間基因的差異性，並進一步探討在不同地理區域採集的爪哇擬青黴菌，其親緣關係是否具有相關性。利用RAPD分子標誌技術以UPGMA程序所建構的相似性樹狀圖顯示，在相似性65.2%時，產生5個群，顯示菌株與菌株間的差異很明顯，具相當高的歧異度，有效的區分出同種中菌株之差異性，但所產生的群與採集地域來源並無顯著相關性。進一步利用18S rDNA基因定序法，鑑別不同菌株在系統分類所在位置，定出每株菌株的基因序列，以分辨出菌株間之相異程度，以Neighbor-Joining (NJ)、Maximum-Likelihood (ML)及Maximum-Parsimony (MP)三種方法建構出爪哇擬青黴菌之親緣關係樹狀圖，三種樹狀圖分類結果大致相似，可將本土採集的爪哇擬青黴菌分類為3個群，所產生的群與採集地域來源間亦無顯著之相關性。而18S rDNA定序出的序列可以作為物種分類上有意義之基因序列，提供做為每一供試菌株之身分證明。

Paecilomyces javanicus belonging to Paecilomyces, Moniliaceaes, Moniliales, Hyphomycetes, Deuteromycotina, is an entomopathogenic fungus with high sporulation and specific against Diptera and Lepidoptera. It is widely dispersed among different hosts and localities. The traditional identification methodology for this fungus is rather controvertible. In this study, we tried to differentiate the genetic difference among 22 P. javanicus isolates sampled from northern, central, southern and eastern part of Taiwan with RAPD molecular makers and 18S rDNA sequencing techniques. Furthermore, the phylogeny of P. javanicus sampled from difference localities was also studied. We used UPGMA program of RAPD molecular makers techniques to establish the dendrogram. There were 5 clusters generated when similarity is 65.2%. This showed the significant difference among isolates, but it was irrelevant to the localities sampled. The 18S rDNA sequencing techniques was also applied to characterize the genetic difference among 22 P. javanicus isolates tested. The 18S rDNA of each isolates was sequenced. Those methods, namely, Neighbor-Joining (NJ)、Maximum-Likelihood (ML) and Maximum-Parsimony (MP) were employed to establish the phylogenic tree for P. javanicus. isolates. Three phylogenic trees generated from different methods established approximately the same pattern. Isolates were grouped 3 clusters according to sequence identity. It was irrelevant between the localities and classification. The 18S rDNA sequence was a useful tool for the identity description of each P. javanicus isolates.

第一章 緒論.......................................................................................................1
1.1. 前言.......................................................................................................1
1.2. 擬青黴菌屬(Paecilomyces)之特性及分類研究..................................4
1.2.1. 擬青黴菌屬之特性......................................................................4
1.2.2. 擬青黴菌屬之分類沿革..............................................................4
1.3. 聚合酵素長鏈連鎖反應(PCR)...........................................................12
1.3.1. PCR原理..................................................................... ...............12
1.3.2. 影響PCR反應因素................................................................ ...13
1.3.3. RAPD分子標誌技術.................................................................13
1.3.4. RAPD親緣關係之分析..............................................................15
1.4. 利用核醣體基因探討真菌之分類.....................................................16
1.4.1. 真菌分類技術之發展................................................................16
1.4.2. 真菌核醣體基因結構................................................................17
1.4.3. 核醣體基因在真菌分類之運用................................................19
1.5. 實驗目的及研究範圍.........................................................................21
第二章 實驗方法與步驟.................................................................................22
2.1. 儀器設備.............................................................................................22
2.2. 實驗藥品.............................................................................................23
2.3. 利用RAPD分子標誌鑑定擬青黴菌.................................................24
2.3.1. 菌株來源............................................................. ..................... 24
2.3.2. 擬青黴菌菌株的培養方法................................................. ..... 28
2.3.3. 擬青黴菌DNA之萃取與定量.................................................28
2.3.4. RAPD供試引子. ..................................................................... 29
2.3.5. RAPD反應條件..........................................................................30
2.3.6. RAPD增幅圖譜之資料分析......................................................32
2.4. 利用18S rDNA基因序列進行擬青黴菌分類研究...........................34
2.4.1. 引子的設計.......................................................................... .....34
2.4.2. .PCR反應條件....................................................................... ....35
2.4.3. PCR產物定序....................................................................... .....39
2.4.4. 18S rDNA基因序列處理...........................................................39
2.4.5. 不同種間的擬青黴菌進行18S rDNA序列比對......................39
2.4.6. 資料整理與分析....................................................................... 43
第三章 結果.....................................................................................................44
3.1. RAPD分子標誌鑑定擬青黴菌...........................................................44
3.1.1. 圖譜分析....................................................................... ..... ......44
3.1.2. 相似性矩陣(similarity matrix) ....................................... ..... ....55
3.1.3. 相似性樹狀圖(dendrogram) ....................................... ..... ...... 55
3.2. 18S rDNA定序進行爪哇擬青黴菌分類研究....................................60
3.2.1. 引子的設計....................................................................... ..... ..60
3.2.2. PCR反應結果....................................................................... .....60
3.2.3. 18S rDNA基因序列比對的差異...............................................60
3.2.4. 爪哇擬青黴菌18S rDNA和基因庫中相近序列的比對..........64
3.2.5. 擬青黴菌18S rDNA序列之親緣關係樹狀圖..........................64
第四章 討論.....................................................................................................73
4.1. RAPD分子標誌鑑定擬青黴菌..........................................................73
4.1.1. 引子的篩選................................................................................73
4.1.2. 相似性矩陣................................................................................73
4.1.3. 相似性樹狀圖............................................................................74
4.2. 18S rDNA定序進行擬青黴菌分類研究............................................76
4.2.1. 引子的設計................................................................................76
4.2.2. .PCR反應結果............................................................................76
4.2.3. .18S rDNA基因序列比對的差異..............................................76
4.2.4. 擬青黴菌18S rDNA序列之親緣關係樹狀圖..........................77
4.3. 以RAPD分子標誌與18S rDNA定序探討爪哇擬青黴菌...............79
第五章 參考文獻.............................................................................................83
附錄................................................................................ ................................... 90
作者簡歷................ ............................................................................................92
表 1.1. 擬青黴菌之分類沿革.............................................................................8
表 2.1. 供試擬青黴菌之物種及來源...............................................................25
表 2.2. 爪哇擬青黴菌各族群採集地點一覽表...............................................26
表 2.3. 本研究所使用之RAPD引子序列與GC%..........................................31
表 2.4. 18S序列分析所使用的引子一覽表.....................................................41
表 2.5. NCBI Genbank中擬青黴菌18S核醣體基因序列資料表...................42
表 3.1. 利用10個RAPD引子合併分析擬青黴菌，經計算所得之相
似性矩陣..............................................................................................56
表 3.2. 爪哇擬青黴菌18S rDNA序列長度表................................................ 62
表 3.3. 爪哇擬青黴菌和NCBI Genbank Accession number AB099944
、AY526463之18S rDNA序列相同度分析...........................................63
表 3.4. 爪哇擬青黴菌E1 18S rDNA序列相同度最高的40段序列............. 66
圖 1.1. 爪哇擬青黴菌感染不同蟲體之情形.....................................................5
圖 1.2. 爪哇擬青黴菌在PDA培養基上的菌落外觀........................................6
圖 1.3. 爪哇擬青黴菌之顯微構造............................................................. .......7
圖 1.4. 真核生物核醣體組成圖.......................................................................18
圖 1.5. 蟲生真菌核醣體基因大小示意圖.......................................................20
圖 2.1. 爪哇擬青黴菌採樣地點分佈圖...........................................................27
圖 2.2. Accession number AY526463的18S rDNA基因序列..........................36
圖 2.3. Accession number AB099944的18S rDNA, ITS1, 5.8S rDNA,
ITS2, 28S rDNA基因序列................................................................37
圖 2.4. 擴增18S rDNA序列之引子位置圖.....................................................40
圖 3.1. 以引子OPM-1分析擬青黴菌之RAPD圖譜......................................45
圖 3.2. 以引子OPM-2分析擬青黴菌之RAPD圖譜......................................46
圖 3.3. 以引子OPM-4分析擬青黴菌之RAPD圖譜......................................47
圖 3.4. 以引子OPM-6分析擬青黴菌之RAPD圖譜......................................48
圖 3.5. 以引子OPM-9分析擬青黴菌之RAPD圖譜......................................49
圖 3.6. 以引子OPM-12分析擬青黴菌之RAPD圖譜....................................50
圖 3.7. 以引子OPM-14分析擬青黴菌之RAPD圖譜....................................51
圖 3.8. 以引子OPM-16分析擬青黴菌之RAPD圖譜....................................52
圖 3.9. 以引子OPM-18分析擬青黴菌之RAPD圖譜....................................53
圖 3.10. 以引子OPM-19分析擬青黴菌之RAPD圖譜..................................54
圖 3.11. 10個RAPD引子合併分析擬青黴菌，UPGMA建構之樹狀圖
............................................................................ ................................. 57
圖 3.12. 聚合酵素長鏈連鎖反應電泳圖.........................................................61
圖 3.13. 爪哇擬青黴菌E1 18S rDNA序列和資料庫比對的結果............... .65
圖 3.14. 擬青黴菌的18S rDNA部分序列比對圖...........................................67
圖 3.15. 以Neighbor-Joining (NJ)方法建構的擬青黴菌18S rDNA序列
之親緣關係圖...................................................................................68
圖 3.16. 以Maximum-Likelihood (ML)方法建構的擬青黴菌18S rDNA
序列之親緣關係圖...........................................................................69
圖3.17. 以Maximum-Parsimony (MP)方法建構的擬青黴菌18S rDNA
序列之親緣關係圖.............................................................................70

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