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研究生:呂昀陞
研究生(外文):Yun-Seng Lue
論文名稱:鑑定及偵測茄科植物細菌性斑點病菌Xanthomonasvesicatoria之聚合酵素連鎖反應技術
論文名稱(外文):The polymerase chain reaction Technique for Identification and Detection of
指導教授:曾國欽曾國欽引用關係
指導教授(外文):Kuo-Ching Tzeng
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
論文頁數:65
中文關鍵詞:茄科植物細菌性斑點病菌隨機增幅核酸多型性分析無毒力基因
外文關鍵詞:Xanthomonas axonopodis pv. vesicatoria and X. vesicatoriarandom amplified polymorphic DNARAPDavirulence gene
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茄科植物細菌性斑點病為世界各國番茄及甜椒產區之重要病害,其病原菌為Xanthomonas axonopodis pv. vesicatoria(Xav)與X. vesicatoria(Xv)。在國外已研發出對Xav具專一性之引子對RST13/14,本研究利用隨機增幅核酸多型性分析 (random amplified polymorphic DNA, RAPD)之技術,針對X. vesicatoria篩選出具有專一性之核酸片段(約500 bp),將此專一性DNA片段進行選殖及核酸序列解序,並依其序列,設計出C-2-2L/2R兩組引子對,將此組引子對以聚合酵素連鎖反應進行分析,可對X. vesicatoria之菌株產生一407bp之DNA片段,但對其他種之Xanthomonas病菌則無法產生此一核酸片段。利用此引子對偵測X. vesicatoria之靈敏度可達1 pg之基因體核酸及1.5個細菌數。此外利用引子對C-2-2L/2R偵測人工帶菌種子之檢測效率可達1000個種子中含0.1%之帶菌率。將混合接種Xav與Xv之罹病葉片以引子對C-2-2L/2R與RST13/14進行偵測,結果顯示此兩組引子對可分別對Xav與Xv增幅出預期之條帶。此外根據無毒力基因序列設計出之引子對bsta1/a2與Rxv1/2以PCR反應測試結果顯示, bsta1/a2可對番茄菌群之菌株增幅出1.9 kb之專一性片段;而Rxv1/2可針對tomato race 1之菌株增幅出1.8 kb之專一性片段。由結果顯示引子對C-2-2L/2R可快速鑑定X. vesicatoria,而引子對bsta1/a2與Rxv1/2則可分別用來鑑定番茄菌群及番茄第一型生理小種。
Bacterial spot, caused by Xanthomonas axonopodis pv. vesicatoria(Xav)and X. vesicatoria(Xv) is one of the most important diseases of tomato and pepper plants. The disease may severely affect the quality and yield of the tomato and pepper fruits. The primary inoculum source of the causal pathogens is usually from the infected or contaminated seeds or transplants. Specific DNA primer pairs for Xav strains has been designed by Jones et al.(1993) In this study, sixty different random primers were used to obtain specific DNA fragments of Xv by using the random amplified polymorphic DNA (RAPD) analysis. A specific DNA fragment (about 500 bp in size) of Xv amplified by the primer OPC-02 was cloned into the yT&A vector, and sequenced to design primer pair C-2-2L/2R. The primer pair could amplified a 407 bp specific DNA fragment from Xv strains but not from strains Xav and other Xanthomonas bacteria tested. The minimum amount of DNA from Xv that could be amplified by PCR was 1 pg. Sensitivity of PCR to detection of cells of Xv with primer C-2-2L/2R was 1.5 cfu. PCR technique with primer pair C-2-2L/2R could detect Xv from 1000 artificial infected seeds (0.1% infestation rate). Multiplex PCR with primer pairs RST13/14 and c-2-2L/2R could simultaneously detect Xav and Xv from pepper leaf tissues inoculated with both Xav and Xv. The results indicated that the primer pair C-2-2L/2R could be used for rapid identification and detection of Xv in diseased tissues infected with Xv, and could also be potentially used for detection of contaminated seeds. In this study we designed other two specific primer pairs bsta1/a2 and Rxv1/2 according to the avrbsT and avrRxv of X. campestris pv. vesicatoria(XCV). The primer pairs bsta1/a2 and Rxv1/2 could be used to identify strains of the XCV T group and strains of XCV tomato race 1, respectively.
壹、前言…………………………………………………………………….1
貳、材料方法……………………………………………………………….7
一、供試菌株來源、培養及保存…………………………………………7
二、細菌全DNA的抽取與濃度測定………………………..…..7
1.細菌全DNA的抽取……………………………………7
2.DNA濃度及純度測定…………………………………….8
三、以RST2/3、RST9/10引子對應用PCR技術檢測茄科植物細菌性斑點病菌即其他種之Xanthomonas 植物病原細菌……………………………………………………..8
四、利用RAPD技術篩選Xanthomonas vesicatoria之專一性片段…………………………….……………………9
五、Xanthomonas vesicatoria 專一性DNA片段之回收及純化……………………………………………………………….10
六、利用南方雜合法(Southern hybridization)測試所篩選之專一性DNA片段……..…………………………..10
(一)轉漬(transfer)及聯結(UV-linking)…….…………..10
(二)核酸探針之製備………………………………………..11
(三)核酸雜合反應(hybridization)及偵測反應(detection)………..………………………………..11
七、專一性核酸片段之選殖……………………………………………12
八、小量質體DNA(plasmid DNA)的製備及選殖株從組質體DNA崁入片段之電泳分析………..………………………………………………….13
九、重組質體DNA嵌入片段之核酸定序及專一性引子對之設計……14
十、聚合酵素連鎖反應及引子對專一性測試及靈敏度測試………………14
(一)聚合酵素連鎖反應之引子對粘合溫度測試及專一性測定…………14
(二)梯度聚合酵素連鎖反應測試………………………………………………..15
(三)靈敏度測試…………………………………………….………..15
十一、以引子對RST13/14與C-2-2L/2R應用multiplex PCR鑑定茄科植物細菌性斑點病菌……………………………………………… .....……...15
十二、應用PCR技術偵測人工帶菌種子… … … … … … … …………16
十三、茄科植物細菌性斑點病菌防治藥劑對以PCR檢測甜椒種子之影響……………………………………………………………………..17
十四、葉片組織內之茄科植物細菌性斑點病菌之快速偵測………………18
(一)甜椒葉片之接種………………….……………………………………18
(二)PCR反應樣品的製備與茄科植物細菌性斑點病菌………………..18
十五、無毒力基因之搜尋及引子對之設計…………………………………18
十六、無毒力基因引子對之聚合酵素連鎖反應……...…………………..19
參、結果……………………………………………………………………..20
一、利用引子對RST2/3、RST9/10測試茄科植物細菌性斑點病菌.....…..20
二、利用RAPD篩選Xanthomonas vesicatoria之專一性片段………..…20
三、利用南方雜合法 ( Southern hybridization )測試以隨機引子OPC-02所增幅出之Xanthomonas vesicatoria專一性DNA片段…………...20
四、Xanthomonas vesicatoria專一性DNA片段選殖……………….…21
五、選殖株重組質體DNA嵌入片段之序列分…………………………………21
六、Xanthomonas vesicatoria專一性引子對之設計…………..………….22
七、聚合連鎖反應之引子黏合溫度、專一性及靈敏度…………………22
(一)聚合酵素連鎖反應之引子黏合溫度及專一性………………………22
(二)引子對C-2-2L/2R之梯度聚合酵素連鎖反應測試………………………23
(三)引子對C-2-2L/2R之靈敏度測試…………………….…………..23
八、以引子對RST13/14與C-2-2L/2R進行multiplex PCR鑑定茄科植物細 菌性斑點病菌…………………………………………………………..24
九、應用PCR技術偵測人工帶菌種子………………………………………24
十、銅劑對以PCR檢測甜椒種子之影響………………………………… 24
十一、葉片組織內之茄科植物細菌性斑點病菌之偵測……………………25
十二、無毒力基因之搜尋及引子對之設計…………………………………25
十三、無毒力基因引子對之聚合酵素連鎖反應……………………………26
肆、討論………………………….…………………………………27
伍、參考文獻……………………...…………………………………………..33
陸、中文摘要………………...………………………………………………..39
柒、英文摘要…………………………………………………………………40
捌、圖表………………………………………………………………………41
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