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論文中文名稱:分析鏈黴菌線型質體SLP2的clt之接合傳遞功能 [以論文名稱查詢館藏系統]
論文英文名稱:Functional identification of clt of Streptomyces linear plasmid SLP2 in 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:化學工程與生物科技系生化與生醫工程碩士班
畢業學年度:105
畢業學期:第二學期
出版年度:106
中文姓名:陳錦憲
英文姓名:Jin-Xian Chen
研究生學號:104688010
學位類別:碩士
語文別:中文
口試日期:2017/07/19
論文頁數:90
指導教授中文名:黃志宏
指導教授英文名:Chin-Hung Huang
口試委員中文名:陳月茸;黃姿雯
中文關鍵詞:鏈黴菌接合傳遞clt
英文關鍵詞:Streptomycescltconjugation
論文中文摘要:鏈黴菌為土壤中的革蘭氏陽性菌,在大自然中扮演分解者的角色。此外,在學術研究地位也很重要。在遺傳機制的研究證明,鏈黴菌無論環形質體或線型質體,大部分具有接合傳遞(conjugation)的能力。而過去闡述質體的接合傳遞機制主要來自大腸桿菌的F質體系統,其作用機制過程中需要核酸分解酶TraI在oriT序列切出缺口後,由以單向的轉移單股DNA從供給株至接受株。與大腸桿菌相較之下,鏈黴菌的接合傳遞機制明顯不同,第一,其接合傳遞是以雙股DNA的方式進行而非單股。第二,其接合傳遞作用時間不一致,導致目前尚無法定義出接合傳遞的oriT,只能定義出一段在接合傳遞過程中,具in cis作用的一段必要DNA片段(clt)。因此,本研究想進一步探討鏈黴菌的clt是否就是接合傳遞作用起始點。本研究策略是透過將clt嵌入到鏈黴菌染色體上的不同位置,在SLP2存在下,檢測其染色體上不同的標記基因在接合重組株內出現的機率與clt所出現的位置是否有正相關,間接推論鏈黴菌的clt就是oriT。實驗結果跟預期的結果不同,供給株染色體上有插入clt的接合傳遞結果,發現絕大多數的子代都是供給株的基因表現型,接受株與重組株的數量很少,因此要直接核查每個子代的基因標記是否有重組就變得不可行。此外,供給株染色體未插入clt的接合傳遞結果,其重組株的機率為10-3。因此,本研究在染色體上插入SLP2的clt後,在SLP2的驅動下進行接合傳遞,所呈現接受株大幅消失的結果是從未被報導過的之外,也供後續研究的修正方向。
論文英文摘要:Streptomyces species are Gram-positive bacteria that play a key role on decompositim in the soil ecology. The most unique feature is that they can have both circular and linear plasmids. Most of these plasmids acquire the ability of conjugal transfer. Previously elucidated conjugation mechanisms of F plasmid from E.coli which have demonstrated a site-specific single stranded cleavage (or nicking) by a plasmid-encoded TraI protein on the cis-acting origin of transfer (oriT) as a prerequisite for DNA transfer and follow by unidirectional transfer of single stranded DNA from the donor to the recipient. Conjugation mechanisms between Streptomyces and E.coli were significantly different. First, Streptomyces conjugation is based on double- stranded DNA rather than a single-stranded DNA. Second, time of occurrence is between two systems different, so currently we still can not define oriT for conjugation. The current study can only define a necessary DNA fragments (clt) that shown in cis function during conjugal event. Therefore, this study would like to know whether the starting point of Streptomyces linear plasmid conjugation transfer was clt.
The strategy of this study is to insert clt into different positions of the chromosome. With the presence of SLP2, observing the relationship between the position of clt and the odds of different chromosomal chromosomal markers from donor appears to the recipient. From there we could try to conclude Streptomyces clt is oriT. The experi- mental data are different from the predicted results. Most of the offspring were donor with clt inserted rather than recipient or recombinant strains. This made to direct very- fycation recombinant event in offspring strains impossible. Furthermore, the recom- binant rate was 10-3 when the chromosome was not clt inserted.The disappearance of the recipient strain after clt insertion into SLP2 was never been reported.This study provides proposals to follow-up study.
論文目次:目錄

摘 要 i
ABSTRACT iii
誌 謝 v
目錄 vi
圖目錄 ix
表目錄 xi
第一章 緒論 1
1.1細菌的接合傳遞 1
1.1.1接合傳遞(conjugation)的發現 1
1.1.2 F質體的發現 1
1.1.3 Hfr菌株的發現 1
1.1.4接合菌毛(Conjugative pili) 2
1.1.5 接合傳遞起始點(Origin of transfer) 2
1.1.6 F質體的接合傳遞機制 3
1.2 鏈黴菌的接合傳遞 6
1.2.1鏈黴菌 6
1.2.2 鏈黴菌的DNA複製 7
1.2.3 鏈黴菌接合傳遞發現 7
1.2.4鏈黴菌的F(fertility) 8
1.2.5 cis-acting locus of transfer (clt) 8
1.2.6鏈黴菌線型質體SLP2上的clt 9
1.2.7鏈黴菌的接合傳遞 10
第二章 材料與方法 16
2.1 菌種與質體 16
2.2 藥品與酵素 25
2.3 培養基與緩衝溶液 25
2.4大腸桿菌質體之分離與純化 25
2.5鏈黴菌基因組DNA的分離與純化 26
2.6大腸桿菌的轉型 26
2.7菌種儲存 26
2.8南方墨點法(Southern blotting) 26
2.9限制酶、連接酶的使用方法 26
2.10聚合酶連鎖反應 PCR(polymerase chain reaction) 27
2.11 鏈黴菌原生質體的製備與轉型 27
2.12 鏈黴菌質體接合生殖頻率計算(Recombination frequency of conjugation) 27
第三章 實驗結果 28
3.1測試clt是否為oriT的實驗策略 28
3.1.1起因 28
3.1.2策略 28
3.1.3染色體位置選擇 29
3.2 鏈黴菌染色體4.3 M實驗 29
3.2.1構築含clt質體 29
3.2.2構築含clt嵌入鏈黴菌染色體4.3 M位置的自殺質體 31
3.2.3將4.3M自殺質體送入鏈黴菌M145[SLP2]、M145[SLP2 ∆ clt::aac(3)IV]並確認是否正確 34
3.2.4構築含clt嵌入鏈黴菌染色體7.7 M位置的自殺質體 40
3.2.5將7.7M自殺質體送入鏈黴菌M145[SLP2]、M145[SLP2 ∆ clt::aac(3)IV]並確認是否正確 43
3.2.6構築含clt嵌入鏈黴菌染色體900 K位置的自殺質體 48
3.2.7 將900K自殺質體送入鏈黴菌M145[SLP2]、M145[SLP2 ∆ clt::aac(3)IV] 53
3.3 對clt插入株進行接合傳遞測試 56
第四章 討論 63
參考文獻 65
第五章 附錄 73
Appendix 1. Media and buffer 73
Appendix 2. Plasmid isolation for E. coli 76
Appendix 3. Isolation total DNA for Streptomyces 77
Appendix 4. Competent cell preparation and transformation 78
Appendix 5. Southern hybridization 81
Appendix 6. Polymerase Chain Reaction(PCR) 85
Appendix 7. Preparation of Streptomyces protoplast and transfer the plasmid DNA 86
Appendix 8. REDIRECT technology: 88
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