Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92217
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dc.contributor林瑞文zh_TW
dc.contributor.author郭雅竫zh_TW
dc.contributor.authorYa-Jing Guoen_US
dc.contributor.other生物化學研究所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-15T05:30:05Z-
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Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection. Mol. Microbiol. 37(2):345-355. Park, T., Struck, D. K., Deaton, J. F., and Young, R. 2006. Topological dynamics of holins in programmed bacterial lysis. Proc. Natl. Acad. Sci. USA 103:19713-19718. Raaijmakers, H., Vix, O., Toro, I., Golz, S., Kemper, B., and Suck, D. 1999. X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture. EMBO J. 18(6):1447-1458. Ramanculov, E., and Young, R. 2001. Genetic analysis of the T4 holin: timing and topology. Gene 265:25–36. Reese, J. F., Dimitracopoulos, G., and Bartell, P. F. 1974. Factors influencing the adsorption of bacteriophage 2 to cells of Pseudomonas aeruginosa. J. Virol. 13:22-27. Rothwell, P. J., and Waksman, G. 2005. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71:401-440. Rountree, P. M. 1955. 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The requirement for calcium in infection with Lactobacillus phage. J. Gen. Virol. 17:19-30. West, D., Lagenaur, C. and Agabian, N. 1976. Isolation and characterization of Caulobacter crecentus bacteriophage phi Cd1. J. Virol. 17(2):568-575. Xu, M., Struck, D., Deaton, J., Wang, I. N., and Young, R. 2004. A signal-arrest-release sequence mediates export and control of the phage P1 endolysin. Proc. Natl. Acad. Sci. USA 101:6415-6420. Young, R. 2002. Bacteriophage holins: deadly diversity. J. Mol. Microbiol Biotechnol. 4:21-36. Young, R., Wang, I. N., and Roof, W. D. 2000. Phages will out: strategies of host cell lysis. Microbiology 8:120-128.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/92217-
dc.description.abstract摘要 Xanthomonas campestris 為革蘭氏陰性嗜氧之植物病原菌,可感染多種重要經濟作物,造成經濟損失。本研究從收集獲得的28個樣品中分離到4株可感染X. campestris pv. campestris (Xcc) P20H之溶裂型噬菌體,分別命名為ϕXc6L、ϕXc6S、ϕXc10L和ϕXc10S。依據限制酶切割圖譜及SDS-PAGE分析結果,推測此四株噬菌體應為相同或極為相似,因此針對ϕXc10S (以下稱為ϕXc10)進行特性分析。電子顯微鏡下觀察 ϕXc10噬菌體,具有六角形頭部 (55 × 55 nm)及短小的尾部結構 (10 nm in length),依外型特徵分類屬於Podoviridae科。將此噬菌體置於4℃冰箱中保存11個月,其效價仍有51%,顯示其為相當穩定之噬菌體。利用one-step growth assay方法,得知噬菌體 ϕXc10之latent period約為20分鐘,burst size約為35 PFU/cell。ϕXc10屬於廣宿主(broad-host-range) 噬菌體,除了可感染P20H外,亦可感染不同strain的Xcc菌株Xc17fhrΔ8361和Xc11,不同X. campestris pv. glycines分離株Xcg12609和Xcg12620,以及X. campestris pv. citri分離株Xci60;但無法感染Xc17、Xcg YR32、Escherichia coli及Pseudomonas aeruginosa。在液體培養基中,ϕXc10對Xc17fhrΔ8361及P20H的溶裂效果最好,其次為Xc11和Xci60;感染Xcg12609和Xcg12620時,則需要較高的MOI (=10)才有明顯的溶裂現象。在培養液中加入Mg2+或Ca2+,可幫助噬菌體裂解宿主及增加噬菌體效價。噬菌體 ϕXc10的遺傳物質為線型雙股DNA,序列末端具有399 bp同向重複序列 (direct terminal repeat),基因體大小為44,597 bp。經由電腦分析比對,推測有54個ORFs,其中有26個ORFs可比對到已知蛋白質,22個ORFs比對到hypothetical protein,其餘 6個ORFs則未比對到任何相似的蛋白質。依蛋白質功能可區分為複製、轉錄、頭部組裝、尾部組裝及溶裂之相關基因。比對結果顯示噬菌體 ϕXc10與Xylella phage Prado、Xylella phage Paz及Caulobacter phage Cd1之基因體極相似;因此,推測噬菌體 ϕXc10亦屬於 ϕKMV-like phage。本論文實驗證明噬菌體ϕXc10無法感染P20H pilA1突變株,顯示宿主受體包含了type IV pilus。為何有些Xcc及Xcg的分離株無法被 ϕXc10感染?其type IV pilus結構是否不同或其表現受到調控?亦或尚有其他因子與感染有關?值得進一步探討。未來期望能將這些噬菌體發展成生物藥劑,以達到防治之功效。zh_TW
dc.description.tableofcontents目錄 摘要…………………………………………………………………………………………………………………………………………i Abstract……………………………………………………………………………………………………………………………ii 目錄………………………………………………………………………………………………………………………………………iii 縮寫字對照表…………………………………………………………………………………………………………………………vi 前言……………………………………………………………………………………………………………………………………………1 材料……………………………………………………………………………………………………………………………………………5 一、菌種、噬菌體及質體……………………………………………………………………………………………………5 二、藥品…………………………………………………………………………………………………………………………………5 三、酵素……………………………………………………………………………………………………………………………………5 四、引子……………………………………………………………………………………………………………………………………5 五、抗生素使用濃度………………………………………………………………………………………………………………6 六、培養基………………………………………………………………………………………………………………………………6 (一)液體培養基…………………………………………………………………………………………………………………6 (二)固體培養基……………………………………………………………………………………………………………………6 七、試劑及緩衝溶液………………………………………………………………………………………………………………6 (一)抽取質體 DNA 試劑…………………………………………………………………………………………………6 (二)抽取噬菌體 DNA 之試劑…………………………………………………………………………………………7 (三)DNA 電泳試劑……………………………………………………………………………………………………………7 (四)SDS-聚丙烯醯胺凝膠試劑………………………………………………………………………………………8 方法…………………………………………………………………………………………………………………………………………10 一、細菌之培養與保存………………………………………………………………………………………………………10 二、噬菌體的篩選…………………………………………………………………………………………………………………10 (一)樣品的處理…………………………………………………………………………………………………………………10 (二)噬菌體的分離……………………………………………………………………………………………………………10 (三)噬菌體的純化……………………………………………………………………………………………………………10 三、噬菌體之增殖與保存……………………………………………………………………………………………………11 四、點測試 (spot test) …………………………………………………………………………………………11 五、Plaque assay………………………………………………………………………………………………………11 六、DNA 之製備………..……………………………………………………………………………………………………12 (一)小量質體 DNA 之製備……………………………………………………………………………………………12 (二)噬菌體 DNA 之抽取………………………………………………………………………………………………13 七、洋菜膠電泳分析 (agarose gel electrophoresis)……………………………13 八、質體之選殖……………………………………………………………14 (一)聚合酶連鎖反應 (Polymerase chain reaction,PCR) …………………14 (二)限制酶切割 (Restriction enzyme digestion) ……………………………15 (三)DNA 片段之回收………………………………………………………………………………………………………15 (四)DNA 之黏接反應 (Ligation) ……………………………………………………………………15 九、轉形作用 (transformation) …………………………………………………………………………15 (一)E. coli 勝任細胞 (competent cell) 之製備……………………………………16 (二)E. coli 之轉形作用 (transformation) …………………………………………16 十、X. campestris 之電穿孔法 (Electroporation)……………………………16 十一、質體快速篩選法 (Rapid screen)………………………………………………………………17 十二、SDS 聚丙醯胺凝膠蛋白質電泳分析 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) …………………17 (一)SDS-PAGE 之製配…………………………………………………………………………………………………17 (二)蛋白樣本之製備…………………………………………………………………………………………………………16 (三)電泳分析及染色………………………………………………………………………………………………………18 十三、電子顯微鏡樣品製備………………………………………………………………………………………………18 十四、生長曲線測定……………………………………………………………………………………………………………18 十五、單步生長曲線 (one step growth curve) 測試………………………………19 十六、噬菌體基因體之定序………………………………………………………………………………………………19 十七、質體構築………………………………………………………………………………………………………………………19 (一)P20H pilA1基因突變株構築………………………………………………………………………………19 (二)pilA1互補質體構築………………………………………………………………………………………………20 結果…………………………………………………………………………………………………………………………………………21 一、Xanthomonas campestris菌株之基本生理特性…………………………………………21 二、Xcc溶裂型噬菌體之分離與純化………………………………………………………………………………21 (一)噬菌體之篩選……………………………………………………………………………………………………………21 (二)噬菌體分離株之初步篩選…………………………………………………………………………………………21 三、噬菌體 ϕXc10 之特性分析……………………………………………………………………………………22 (一)噬菌體 ϕXc10 之外型……………………………………………………………………………………………22 (二)噬菌體 ϕXc10之穩定性………………………………………………………………………………………22 (三)宿主範圍 (Host range) ………………………………………………………………………………22 (四)噬菌體 ϕXc10 之一步生長曲線…………………………………………………………………………23 (五)不同MOI之噬菌體感染P20H生長之影響……………………………………………………………23 (六)ϕXc10感染不同宿主之效應…………………………………………………………………………………24 (七)二價陽離子對於噬菌體 ϕXc10 感染不同宿主之影響……………………………………25 四、溶裂型噬菌體 ϕXc10 之基因體分析……………………………………………………………………27 (一)ϕXc10基因體 DNA 之限制酶切割圖譜……………………………………………………………27 (二)基因體之定序………………………………………………………………………………………………………………27 (三) 基因體末端序列之確定……………………………………………………………………………………28 (四) 基因體之ORF組成………………………………………………………………………………………………28 (五)噬菌體 ϕXc10 不同類型基因分析………………………………………………………………………30 1. 早期基因…………………………………………………………………………………………………………………30 2. DNA複製及轉錄相關基因……………………………………………………………………………………30 3. 結構與溶裂基因……………………………………………………………………………………………………32 五、噬菌體 ϕXc10之蛋白體分析……………………………………………………………………………………33 六、噬菌體 ϕXc10吸附宿主受體(receptor)之探討……………………………………………34 討論…………………………………………………………………………………………………………………………………………36 一、噬菌體 ϕXc10 之特性分析……………………………………………………………………………………36 二、噬菌體 ϕXc10 之基因體分析…………………………………………………………………………………39 (一)噬菌體 ϕXc10基因體 ORFs 之組成………………………………………………………………39 (二)噬菌體 ϕXc10基因體之複製及轉錄相關基因…………………………………………………40 (三)噬菌體 ϕXc10 基因體之結構蛋白……………………………………………………………………41 (四)噬菌體 ϕXc10 之溶裂相關基因………………………………………………………………………43 (五)其他……………………………………………………………………………………………………………………………45 參考文獻………………………………………………………………………………………………………………………………46 圖表………………………………………………………………………………………………………………………………………52zh_TW
dc.language.isozh_TWzh_TW
dc.rights不同意授權瀏覽/列印電子全文服務zh_TW
dc.subject溶裂型噬菌體zh_TW
dc.subjectlytic phageen_US
dc.subjectXanthomonas campestrisen_US
dc.titleXanthomonas campestris 噬菌體 phiXc10 之分離與分析zh_TW
dc.titleIsolation and characterization of Xanthomonas campestris phage phiXc10.en_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-02-03zh_TW
dc.date.openaccess10000-01-01-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
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item.languageiso639-1zh_TW-
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