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研究生:游晴翔
研究生(外文):You, Cingsiang
論文名稱:檢測十字花科黑腐病菌生理小種之技術開發
論文名稱(外文):The Development Of Detecting Technique To Identify The Races Of Xanthomonas campestris pv. campestris
指導教授:朱木貴
指導教授(外文):Chu, mukuei
口試委員:陳瑞祥李佩芳
口試委員(外文):Chen, RueyshyangLi, Peifang
口試日期:2012-07-04
學位類別:碩士
校院名稱:中華醫事科技大學
系所名稱:生物醫學研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:45
中文關鍵詞:十字花科黑腐病菌生理小種PCR生物資訊
外文關鍵詞:Xanthomonas campestrisRaceBioinformatics
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Xanthomonas屬之植物病原菌為世界性的植物病害,係台灣地區常見之植物病原菌,造成許多經濟性蔬果之重要病害。其中之十字花科黑腐病菌Xanthomonas campestris pv. Campestris (XCC)主要感染十字花科作物如蕓苔屬(Brassica)、阿拉伯芥(Arabidopsis)等,並造成十字花科黑腐病,其具致病性差異的生理小種早已存在於XCC。在XCC生理小種的研究上,Fargier 及Manceau已成功利用六個鑑別品種,將黑腐病菌之菌株分為9個生理小種(race 1~9)。植物病原菌在感染宿主前利用分泌系統將蛋白質通過內膜及外膜分泌至特定細胞,需要養分的取得、毒性分子的表現等;本研究擬利用生物資訊學比對分析XCC中致病系統中相關之基因,希望能從致病系統篩選致病基因出可鑑別生理小種間之些微差異。本次研究中從NCBI Genomes資料庫中匯出Xanthomonas campestris pv. campestris str. B100及ATCC33913等菌株分泌系統之毒力基因與無毒基因序列,做為XCC生理小種的分析依據。本研究已篩選出8個基因可用於生理小種的鑑別,分別為pilA 、pilD 、pilMN、avrA2及4組vir基因。本研究進一步利用上述基因進行PCR選殖,將本研究的Race5’、Race6、Race6’和Race7做出區分。本研究發現pilA基因可輔助鑑定Race 6與Race 6’的差異,此外XCC不同之生理小種可依據本研究利用上述基因來做初步之區別。
Xanthomonas genus is considered to be one of the most important diseases of crucifers worldwide. Economically, the most important of X. campestris pv. campestris (XCC) causes a serious disease, black rot on crucifers that results in reduced yield and crop quality. The pathogenicity differences in the races already exist in the XCC. In the XCC pathogenic variants research, Fargier and Manceau success using six differential cultivars to differentiate the XCCs into nine physiological races. In this study, we will utilize bioinformatics from NCBI to screen the genes that could be used to identify the differences between the races. This study from the NCBI Genomes database to export the plant pathogen Xanthomonas campestris pv. campestris, the str. B100, and ATCC33913 strains secrete virulence genes and avirulence gene sequence analysis as the XCC Physiological Races basis. This study have been screened out of the eight genes can be used for the identification of physiological races. The genes pilA, pilD, pilMN, avrA2 and four vir gene would be used to differentiate the Race5’, Race6, Race6’ and Race7 in the preliminary judgment of races.
目錄
英文摘要…………………………………………………………………….…1
中文摘要………………………………………………………………….……2
壹、 前人研究…………………………………………………………..……3
一、 十字花科黑腐菌介紹………………………………………...…3
二、 病原菌診斷…………………………………………………...…4
三、 病原致病系統………………………………………………...…5
四、 生物資訊學…………………………………………………...…6

貳、 材料與方法…………………………………………………………..…7
一、材料……………………………………………………………………..…7
二、染色體DNA的抽取………………………………………………………7
三、聚合酶酵素連鎖反應…………………………………………………..…8
四、PCR產物之純化……………………………………………………….…8
五、PCR產物與質體之接合作用………………………………………….…8
六、核酸轉型作用………………………………………………………….…9
七、DNA定序與引子組設計…………………………………………………9
八、生物資訊分析………………………………………………………….…9
參、結果………………………………………………………………………10
一、第三型分泌系統相同病原菌不同基因之多序列比對…………………10
二、第四型分泌系統相同病原菌不同基因之多序列比對……………….…10
三、第四型纖毛基因相同病原菌不同基因之多序列比對……………….…10
四、virB1基因序列之分析與應用…………………………………………..11
五、virB4基因序列之分析與應用…………………………………………..11
六、virB10基因序列之分析與應用…………………………………………12
七、virB11基因序列之分析與應用…………………………………………12
八、virD4基因序列之分析與應用………………………………………….13
九、第四型纖毛pilA基因序列之分析與應用………………………………13
十、XCC病原菌基因race typing分析表……………………………………14
肆、討論………………………………………………………………………15
伍、參考文獻…………………………………………………………………18
陸、圖表………………………………………………………………………25
表一 本研究所使用XCC菌株名稱、宿主、生理小種及來源……………25
表二 本研究所使用引子之名稱及其核酸序列………………………….26
表三 XCC str.B100和ATCC 33913兩條序列比對結果………………..27
表四 XCC初步type表…………………………………………………….29
圖一 virB1引子組進行PCR增幅XCC染色體DNA之結果……………30
圖二 多序列比對軟體ClustalW分析virB1結果…………………………31
圖三 virB4引子組進行PCR增幅XCC染色體DNA之結果……………32
圖四 多序列比對軟體ClustalW分析virB4結果…………………………33
圖五 virB10引子組進行PCR增幅XCC染色體DNA之結果………….34
圖六 多序列比對軟體ClustalW分析virB10結果………………….…….35
圖七 virB11引子組進行PCR增幅XCC染色體DNA之結果………….36
圖八 多序列比對軟體ClustalW分析virB11結果…………………….…37
圖九 virD4引子組進行PCR增幅XCC染色體DNA之結果………..…38
圖十 多序列比對軟體ClustalW分析virD4結果…………………………39
圖十一 pilA引子組進行PCR增幅XCC染色體DNA之結果……….…40
圖十二 多序列比對軟體ClustalW分析pilA結果……………………..…41
圖十三 pilA引子組進行PCR分析pattern之結果………………….……42
圖十四 pilD引子組進行PCR分析pattern之結果………………….……43
圖十五 pilMN引子組進行PCR分析pattern之結果………………….…44
圖十六 avrA2引子組進行PCR分析pattern之結果

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