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研究生:李逸洹
研究生(外文):I-Huan Li
論文名稱:Xanthomonas axonopodis pv. glycines 噬菌體ΦXag15S 之分析
論文名稱(外文):Characterization of Xanthomonas axonopodis pv. glycines phage ΦXag15S
指導教授:林瑞文林瑞文引用關係
指導教授(外文):Juey-Wen Lin
口試委員:翁淑芬曾義雄
口試委員(外文):Shu-Fen WengYi-Hsiung Tseng
口試日期:2016-07-26
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:70
中文關鍵詞:豆科植物葉燒病噬菌體
外文關鍵詞:Xanthomonas axonopodis pv. glycinesphage
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Xanthomonas axonopodis pv. glycines (Xag) 是一好氧、無胞外夾膜與內孢子之革蘭氏陰性的植物病原菌,主要感染豆科類植物並造成葉燒病。本實驗室前人採集 28 個環境樣本,以 Xag12609 為宿主,分離出 2 株無法溶裂菌株的溫和型噬菌體,分別命名為 ΦXag6S 與 ΦXag15S。利用限制酶切割噬菌體 DNA,顯示兩者為不同分離株。經過純化與多次增殖培養,ΦXag6S 效價仍然偏低,因此選擇感染力較強的 ΦXag15S 進一步分析。以電子顯微鏡觀察 ΦXag15S 外型,具有六角形頭部 (54 × 54 nm),以及較長且不可收縮的尾部構造 (167 nm in length)。依其外型特徵分類,屬於 Siphoviridae 科。噬菌體 ΦXag15S 樣品保存於 4℃ 環境下,8 個月後其效價減至為原本的 46%。利用 one-step growth assay,結果獲知 ΦXag15S 的 latent period 為 60 分鐘,在感染後 135 分鐘觀察到其 burst size 約為 30 PFU/cell。ΦXag15S 屬於窄寄主(narrow host range) 噬菌體,只能感染 Xag12609 與 Xag12620。比較 ΦXag15S 感染兩者的生長曲線、存活菌數及噬菌體顆粒增殖情形,發現以 Xag12620 為宿主之溶裂及增殖效果較佳。噬菌體 ΦXag15S 的遺傳物質為雙股環型 DNA,基因體大小為 43,028 bp,G+C content 為 53.8%。以軟體可預測到共有 58 個 ORFs,其中有 33 個 ORFs 可比對到已知蛋白質,其他 25 個則為 hypothetical protein。依照蛋白質功能可區分成溶裂相關、結構相關與複製、重組和修復之相關基因及其他這四大類型。比對結果顯示,ΦXag15S基因體 與 P. aeruginosa phage vB_Pae-Kakheti25 (PA25) 具有高度相似性,比較基因間之相同度多為 90% 以上,其次則為 P. aeruginosa phage 73 (PA73) 與實驗室前人研究的 ΦXag25。另外,實驗室前人發現 ΦXag25 感染 Xag12609 時所認辨的受體之一為 PilA1 蛋白質;以 ΦXag15S 感染 Xag12609 之 pilA1 突變株也無法產生溶菌圈,顯示 ΦXag15S 亦認辨 PilA1 為感染受體。基因體比較結果顯示 ΦXag15S 與 PA25 更相近,卻無法感染 P. aeruginosa 的菌株。可進一步測試更多 P. aeruginosa 菌株並分析探討這些噬菌體與宿主之間的認辨機制。

Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative plant pathogenic bacterium, which is aerobic and with no capsules and endospores. It can cause the scorch in legumes cultivars. Two of Xag12609 temperate phages isolated from 28 environmental samples, named ΦXag6S and ΦXag15S, were obtained in our lab. Results of restriction analysis of the genomic DNA indicated that these 2 phages are different. After serveral times of purification and incubation, Proliferation ability of ΦXag6S is poor. Therefore, ΦXag15S is selected for further study, which has better ability to infect Xag12609. Electron microscopy revealed that ΦXag15S possesses the typical morphology of a siphovirus, with a hexagonal head (54 × 54 nm) and a long non-contractile tail (167 nm in length). After storing at 4℃ for 8 months, about 46% of the phage particles were retained infected ability. One step growth assay suggested that ϕXag15S has a latent period and burst size of approximately 60 mins and 30 PFU per infected cell, respectively. ΦXag15S only can infect Xag12609 and Xag12620, suggesting that ΦXag15S is a narrow host range phage. Comparing the growth curves, quantity of remaining bacteria, and the phage proliferations by infection of Xag12609 and Xag12620, the result indicated that as a host for ΦXag15S, Xag12620 had more obvious lysis and higher titers, suggesting that Xag12620 might be the better host for ΦXag15S. ϕXag15S has a genome of circular double-stranded DNA. Sequencing results revealed that ϕXag15S genome consists of 43,028 bp with a G+C content of 53.8%. Of the 56 ORFs identified, 33 are similar to known proteins which can be classified into four different functional categories (lysis, head, tail and DNA replication/repair), 25 are similar to hypothetical proteins. Similarity analysis showed that ΦXag15S has high identity to P. aeruginosa phage vB_Pae-Kakheti25 (PA25). Identities of most ORF pairs are higher than 90%. ΦXag15S also gets close to P. aeruginosa phage 73 (PA73), and ΦXag25, a Xag phage that has been studied by our lab. In addition, our laboratory founded that Xag PilA1 is one of the receptor responsible for phage attachment, ΦXag15S also attached PilA1 to infect Xag. ΦXag15S is similar to many P. aeruginosa phages, by contrast, it cannot infect P. aeruginosa, suggesting that the major host anti-receptor is significantly different.

摘要..........i
Abstract..........ii
縮寫字對照表..........iii
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
七、試劑及緩衝溶液 ..........6
方法..........9
一、細菌之培養與保存..........9
二、噬菌體的篩選..........9
三、噬菌體之增殖與保存..........10
四、溶菌圈點測試 (spot test)..........10
五、Plaque assay..........10
六、噬菌體 DNA 之抽取..........11
七、洋菜膠電泳分析 (agarose gel electrophoresis)..........11
八、SDS 聚丙醯胺凝膠蛋白質電泳分析 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)..........12
九、生長曲線測定..........13
十、一步生長曲線 (one step growth curve) 測試..........13
十一、聚合酶連鎖反應 (Polymerase chain reaction,PCR)..........13
十二、限制酶切割 (Restriction enzyme digestion)..........14
十三、DNA 片段之回收..........14
十四、DNA 之黏接反應 (Ligation)..........15
十五、DNA 定序..........15
十六、基因體序列分析..........15
結果..........16
一、Xag 菌株之基本生理特性..........16
二、Xag 噬菌體 ΦXag15S 之純化..........16
三、噬菌體 ΦXag15S 之特性分析..........17
四、Xag 噬菌體 ΦXag15S 之核酸分析..........22
1.噬菌體 ΦXag15S 之溶裂相關基因..........25
2.噬菌體 ΦXag15S 之結構基因..........26
3.噬菌體 ΦXag15S 之複製、重組和修復相關基因..........28
4.其他..........28
討論..........30
參考文獻..........37
圖表..........41


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