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標題: Xanthomonas campestris 噬菌體 phiXc10 之分離與分析
Isolation and characterization of Xanthomonas campestris phage phiXc10.
作者: 郭雅竫
Ya-Jing Guo
關鍵字: 溶裂型噬菌體;lytic phage;Xanthomonas campestris
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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。
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