臺灣博碩士論文加值系統

尖鐮胞菌(Fusarium oxysporum)在世界各地引起許多植物病害，鐮胞菌菌落形態與生理上的變異極大，鑑定困難。本研究運用RAPD方法尋找尖鐮胞菌各分化型的特殊DNA片段。實驗中使用200支OPERON的RAPD引子，以R-1程式進行PCR，利用不同濃度組合的Mg2+或瓊脂膠體進行分析；結果顯示以1.5 mM Mg2+進行PCR後，再搭配1.4％瓊脂膠體分離PCR產物之解析度效果最佳。經多次重複試驗後，挑選出21支能在尖鐮胞菌基因組中放大出特異性片段的引子，各引子均具有CCA/TC/G、CC或GG dimer，其中有20支引子的3'-端第一個鹽基是G或C，且GC含量比都在60％∼70％之間。DNA多形性分析研究發現在具有致病性尖鐮胞菌之基因組較為一致，甚至只要使用單一個引子，就可明顯的將不同致病性分化型的尖鐮胞菌區分。將15個特異性片段經選殖定序，分析其DNA及氨基酸序列後發現：有兩個片段（685 bps與522 bps）與F. oxysporum f. sp. lycopersici的插入（insertion sequence）Foxy序列( FOX250814)相似，具有與SINE相似的RNA polymerase III結合點序列Box A與Box B，兩Box之間相隔都是33 bps DNA片段，而其中一個片段具有和Foxy相同的(TATG)5重複序列。由BA5引子所放大出約1800 bps片段的胺基酸序列經序列比對後和多種生物的酒精脫氫脢基因相似，相同度為34∼40 %，相似度為50∼55%；且具有高度保留的Motif Cytochrome c family heme-binding site。另外由引子AY11所放大出429 bps片段的DNA序列中，具有高度保留的Motif Basic - leucine zipper domain及zinc-binding region 2；這兩個序列是常見的轉錄因子(transcriptional factors)，所以猜測在此DNA片段的上下游，可能有基因存在，其餘的11個片段中有6個片段與資料庫中的序列有>40%的序列相似性。為了進一步確認實驗中所挑出的特異性片段之專一性，利用南方墨點法進行分析，結果中顯示Dig探針和致病性尖鐮胞菌各分化型基因組DNA都有雜合訊號。

Fusarium oxysporum causes many plant diseases worldwide. Fusarium spp., with a high level of morphological and physiological diversity, are well-known to be difficult to identify. The RAPD (random amplified polymorphic DNAs ) method were employed to find out the DNA fragments specific to given F. oxysporum formae speciale. In this study, 200 OPERON RAPD primers were screened, R-1 program and 1.5 mM Mg2+ was used in the PCR amplification step, and the PCR products were then separated electrophoretically in the 1.4％ agarose gels. Finally, 21 primers were found to amplify specific-DNA fragments from the F. oxysporum genomes. These primers all comprised CCA/TC/G、CC or GG dimer. Among these primers, the 3’- terminal nucleotide of 20 primers was a G or a C, and the GC content ranged from 60％∼70％. The DNA pattern of pathogenic F. oxysporum isolates was more similar to each other than the non-pathogenic isolates. Using the DNA amplification pattern from only one primer could discriminate difference among F. oxysporum formae speciales. Fifteen specific DNA fragments amplified from F. oxysporum genomic DNAs were cloned and sequenced. Two fragments of 685 bps and 522 bps, respectively aligned to the Foxy ( FOX250814) insertion sequence of F. oxysporum lycopersici. Both two sequences comprised Box A and Box B which are the SINE consensus sequence of RNA polymerase III binding site, a 33 bps spacer was also identified between two Box sequences. One fragment had a (TATG)5 repeat sequence typical of the Foxy sequence. The 1800 bps fragment amplified from the BA5 primer was aligned to alcohol dehydrogenase gene of several species, with a sequence identity of 34∼40 %, and a sequence similarity of 50∼55%. In addition, a consensus sequence of motif cytochrome c family heme-binding site was identified. A 429 bps fragment amplified from the AY11 primer comprised of consensus sequence of motif basic - leucine zipper domain and zinc-binding region 2. These motifs are transcriptional factors commonly found in different genomes, and it was tempted to suggest that gene was likely to exit in its up-stream or down-stream. Six fragments, among the rests of 11 DNA fragments, were aligned, with a sequence similarity greater than 40%, of sequences in the databases. To confirm specificity of the DNA fragment, the Southern dot blotting method was employed to probe the DNA templates of 33 different genomes. A positive hybridization signal was examined in most of the different formae speciales of F. oxysporum.

中文摘要------------------------------------------------------I
英文摘要------------------------------------------------------II
前言----------------------------------------------------------1
材料與方法----------------------------------------------------8
菌株來源------------------------------------------------------8
尖鐮胞菌菌株的培養方法----------------------------------------8
真菌基因組DNA的萃取-------------------------------------------9
植物基因組DNA的抽取-------------------------------------------10
RAPD供試引子--------------------------------------------------11
RAPD測試條件及分析--------------------------------------------11
基礎分生技術--------------------------------------------------11
序列分析------------------------------------------------------19
實驗結果------------------------------------------------------20
尖鐮胞菌菌株之RAPD 初步篩選-----------------------------------21
RAPD之PCR及電泳條件測試---------------------------------------21
尖鐮胞菌菌株之RAPD多形性--------------------------------------22
尋找尋找尖鐮胞菌特異性引子------------------------------------23
尖鐮胞菌特異性片段之選殖與DNA及氨基酸序列比對分析-------------24
特殊DNA片段的偵測---------------------------------------------27
討論----------------------------------------------------------28
參考文獻------------------------------------------------------39
圖表----------------------------------------------------------48
附錄----------------------------------------------------------69

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