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研究生:劉恩慈
研究生(外文):En-Tzu Liu
論文名稱:利用隨機增幅核酸多型性及聚合酵素連鎖反應技術
論文名稱(外文):Analysis of Fusarium oxysporum f. sp. cubense Using Random
指導教授:張碧芳張碧芳引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
中文關鍵詞:巴拿馬病聚合酵素連鎖反應隨機增幅核酸多型性分析香蕉黃葉病菌
外文關鍵詞:Fusarium oxysporum f. sp. cubensepolymerase chain reactionPCR)random amplified polymorphic DNAPanama disease
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中 文 摘 要
香蕉黃葉病 (Fusarium wlit of banana) 係由 Fusarium oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen 所引起,因於1890年在巴拿馬大發生,又稱巴拿馬病 (Panama disease) 。此病害分布極廣,而且目前除了栽培抗病品種外,並無其他有效防治方法,對世界各地的香蕉產量造成嚴重的威脅,台灣自1967年屏東縣出現疑似罹患巴拿馬病的病株之後,此病即嚴重摧毀台灣的香蕉產業。由於香蕉乃利用無性繁殖之種苗栽培,使用不帶病原菌之健康種苗為防治此土壤傳播性病害方法之一,因此建立快速篩選無病原菌香蕉苗之偵測法,為香蕉健康種苗驗證所必須。本研究利用「隨機增幅核酸多型性分析」 (random amplified polymorphic DNA, RAPD) 開發香蕉黃葉病菌之快速偵測技術,並設計專一性引子對供聚合酵素連鎖反應 (polymerase chain reaction, PCR) 檢測之用,以達到快速並準確偵測病原菌之目的。目前已自全省各地收集不同寄主的 F. oxysporum 菌株計十種15個分離株及香蕉黃葉病菌株15個分離株,經大量培養後抽取其菌絲 DNA ,並以健康香蕉組織 DNA、一種其他 Fusarium sp. 及三種非 Fusarium 屬之病原菌 DNA作為對照。利用 OPA、OPAT 及 OPAW 引子組 (Operon Technologies, Inc., Alameda, CA) 和文獻中分析香蕉黃葉病菌所使用之44個引子,針對上述各種 DNA 進行 RAPD 分析。結果顯示以 OPA-02 (5’-TGCCGAGCTG-3’) 引子進行 RAPD 增幅測試,可在香蕉黃葉病菌株 DNA 中增幅出約 400 bp 之專一性片段,經純化回收此片段作為探針,並將上述 RAPD 結果進行南方雜合分析,可知在香蕉菌株之增幅樣品中,能辨識 400 bp 專一性的條帶,此 400 bp 片段經解序分析已知和目前 GenBank 中已發表的核酸序列並無相似性,利用此序列設計二組正向及反向之專一性引子對 OPA02400A1 (5’-CAGGGGATGTATGAGGAGGCT-3’)、OPA02400A2 (5’-CGGTACTTGCTGTGCGGGGA-3’)、OPA02400S1 (5’-CAGCTATGACAAGAACACCAGA-3’) 與 OPA02400S2 (5’-GTGACAGCGTCGTCTAGTTCC -3’),利用 OPA02400A1/OPA02400S2 及 OPA02400A2/OPA02400S2 二種組合於 PCR 反應中,在合適的PCR條件下皆可分別增幅出特定之專一性片段,並可由南方雜合分析確定其專一性。

英 文 摘 要
ABSTRACT
Fusarium wilt of banana, the so- called Panama disease, caused by Fusarium oxysporum Schlechtend.:Fr. f. sp. cubense (E.F. Smith) W.C. Snyder & H.N. Hansen (Foc). In 1890, the outbreak of this disease in Panama almost completely destroyed the export trade of banana. Foc is distribute worldwide and is difficult to control in agricultural practice except planting wilt-resistant banana hybrids. Panama disease is a serious threat to banana production in the whole world. In 1967, the suspected Fusarium-wilted of banana was discovered in Pingtung, Taiwam, and this disease seriously destroyed the banana export industry there after in Taiwan. Since banana is propagated by asexual plantlets, in order to control the soilborne Panama disease, it is necessary to use health banana plantlets for production. Hence, the development of a fast detect Foc in banana plantlet is essential for rapid screening of health banana plantlets. In this study, random amplified polymorphic DNA (RAPD) analysis was first used to develop a rapid detection method for Foc. Next, specific primers for polymerase chain reaction (PCR) was designed for quick and accurate detection of Foc pathogen. The individually purified DNA from mycelia of fifteen isolates of Foc, fifteen F. oxysporum (Fo) isolates from ten species of different hosts and one other Fusarium sp. was used for RAPD and PCR analyses. The DNA sample from healthy banana tissue and three kinds of non-Fusarium pathogens pathogens were also used as control. Forty-four 8 to 15 nt of short random primers were used for analysis of the aboved DNA samples. A specific 400-bp fragment was amplified from DNA of 14 Foc and another Fo (Fo-L1) isolated from anoectochilus using OPA-02 (5’-TGCCGAGCTG-3’) primer. This 400-bp fragment (OPA02400) obtained from Foc samples was cloned and used as probe for Southern blot analysis. The OPA02400 probe could identify the specific 400-bp fragment amplified from the DNA samples of forteen Foc and Fo-L1. The sequence of OPA02400 showed no similarity to any sequence deposited in GenBank. The OPA02400 sequence was used to design two forward and reverse specific primers: OPA02400A1 (5’-CAGGGGATGTATGAGGAGGCT-3’)、OPA02400A2 (5’-CGGTACTTGCTGTGCGGGGA-3’)、OPA02400S1 (5’-CAGCTATGACAAGAACACCAGA-3’) and OPA02400S2 (5’-GTGACAGCGTCGTCTAGTTCC -3’). The results suggested that the OPA02400A1/OPA02400S2 and OPA02400A2/OPA02400S2 primer pairs could amplify specific fragments only in the 15 Foc and Fo-L1 DNA samples under proper PCR conditions, and the specificity was further confirmed by Southern blot analysis.

目 錄
壹、緒論 2
貳、材料與方法 8
供試菌株的來源與培養 8
各菌株在不同培養基上的菌落型態比較 8
菌絲總量DNA之萃取 9
RAPD 分析 10
核酸探針之製備 10
設計專一性引子及其 PCR 分析 11
南方雜合分析 11
專一性 PCR 最佳條件之測試 11
參、結果 15
香蕉黃葉病菌在不同培養基上的菌落型態比較 15
RAPD 分析 16
RAPD 之南方雜合分析 18
專一性片段 OPA02400 的序列及專一性引子對的設計 18
專一性 PCR 之結果 19
PCR 之最佳條件 19
肆、討論 21
伍、參考文獻 26
陸、中文摘要 31
柒、英文摘要 33
捌、圖表說明 35
玖、附錄 70

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