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論文中文名稱:鏈黴菌環型質體pIJ303如何在接合過程協助傳遞線型染色體 [以論文名稱查詢館藏系統]
論文英文名稱:How does streptomyces circular plasmid pIJ303 transfer linear chromosome by conjugation [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:98
出版年度:99
中文姓名:李晟榮
英文姓名:Sheng-Rong Li
研究生學號:96688004
學位類別:碩士
語文別:中文
口試日期:2010-01-29
論文頁數:58
指導教授中文名:黃志宏
指導教授英文名:Chih-Hung Huang
口試委員中文名:陳文盛;楊千金
口試委員英文名:Carton Wen-sheng Chen;Chien-Chin Yang
中文關鍵詞:鏈黴菌環型質體同源重組線型染色體
英文關鍵詞:Streptomycescircular plasmidhomologous recombinationlinear chromosomes
論文中文摘要:鏈黴菌是存在於土壤中的革蘭氏陽性菌,具有線型的染色體、質體以及環型質體。其線型DNA的5’末端位置有蛋白質以共價鍵與DNA結合。已知鏈黴菌的線狀或環狀質體,具有發生接合時促進質體本身的傳遞,也會協助染色體傳遞。
鏈黴菌發生接合時,環型質體如何協助線型染色體傳遞的詳細機制尚不明瞭,因此我們假設鏈黴菌可能是利用類似大腸桿菌F質體發生Hfr現象,利用帶有染色體同源序列的環型重組質體,測試發生接合時環型質體是否需要嵌入染色體以協助染色體傳遞?另外,如果環型質體的傳遞是從固定端點開始單方向的傳遞,應該會失去起始位置另一端的DNA,造成染色體傳送不完全。因此傳遞過程質體是否採雙向傳遞的方式進行?這部分我們利用接受株染色體上的基因標記,觀察鏈黴菌的接合傳遞是否與大腸桿菌模型一樣是單方向進行。目前的實驗結果,以嵌入染色體的方式進行傳遞的供給株,沒有造成比帶有獨立質體的供給株更高的傳遞效率,這點與大腸桿菌F質體模型不同。比較R76、R79帶有M145同源片段的環型質體,發生染色體同源重組的頻率與帶有pIJ303質體的對照組差異不大。雖然目前仍不能排除發生嵌入染色體的形式的可能性,但是這樣的結果很可能暗示了染色體發生同源重組不需要質體嵌入到染色體中。至於環型質體傳遞的方向性,還需要進一步觀察。
論文英文摘要:Streptomyces, gram-positive bacteria, are rich in soil and harbor linear and circular replicons. These linear replicons share the same feature that the terminal proteins are covalently capped at 5’ end of DNA. Both linear and linear conjugative plasmids drive themselves and linear chromosome (in a lower frequency) into recipient, but the mechanism is still unclear.
The simple model for conjugal circular plasmids drive linear chromosome transferring into recipients is the well studied F plasmids, which integrate into chromosome by homologous recombination then mobilize plasmids and covalent-bounded chromosome into recipients. While the tra has shown to be an only required gene for conjugation of circular plasmids, a kor-clt-tra DNA fragment integrated into chromosome did not facilitate the chromosome transfer. That experiment still can’t rule out a possibility that the other factors of circular plasmids might involve in chromosome transfer. To address the question, we integrated conjugative plasmid, pIJ303, into chromosome and analyzed the conjugation result. Our results showed the integration of whole plasmid did not facilitate chromosome transfer and indicated the circular plasmids driven linear chromosome transfer did not required plasmids integrated into chromosome as a conjugal intermediate.
論文目次:目 錄

中文摘要 .i
英文摘要 .ii
誌謝 .iii
目錄 .iv
表目錄 .v
圖目錄 .vi
第一章 緒論 .1
第二章 材料與方法 .11
第三章 實驗結果
3.0 設計引子從定序完成的pIJ101拼湊出pIJ303圖譜….......16
3.1 pIJ303功能區域之測試…………………………….…...…18
3.2 構築帶有S. coelicolor M145染色體同源序列的pIJ303衍生質體…………………………………………….………........ 20
3.3 分析pIJ303衍生型經由接合傳遞驅動染色體重組分析…. 32
第四章 討論………………………………………………………………...37
參考文獻… .41
附錄
Appendix 1. Media and buffer .45
Appendix 2. Competent cell preparation and transformation………….49
Appendix 3. Plasmid isolation for E. coli……………………………....51
Appendix 4. Preparation of Streptomyces protoplast and transfer the plasmid DNA………………………………...52
Appendix 5. Isolation total DNA for Streptomyces……………………..54
Appendix 6. 煮沸法萃取鏈黴菌環型質體……....…........55
Appendix 7. Polymerase Chain Reaction (PCR)……….….56
Appendix 8. Southern Transfer and Hybridization….……….57
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論文全文使用權限:同意授權於2012-02-05起公開