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論文中文名稱:鏈黴菌線型質體SLP2接合傳遞的cis-acting element [以論文名稱查詢館藏系統]
論文英文名稱:Identification of a cis-acting element on Streptomyces linear plasmid SLP2 for conjugal transfer [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:97
出版年度:98
中文姓名:薛國偉
英文姓名:Kuo-Wei Hsueh
研究生學號:96688018
學位類別:碩士
語文別:中文
口試日期:2009-07-17
論文頁數:65
指導教授中文名:黃志宏
口試委員中文名:陳文盛;楊千金
中文關鍵詞:鏈黴菌SLP2同源重組接合傳遞的起始點 (oriT)
英文關鍵詞:Streptomyceshomologous recombinationSLP2oriT
論文中文摘要:革蘭氏陽性的鏈黴菌和其他細菌的基因體結構不同,是除了有環形質體外,還具有線型染色體和線型質體,在這些線型質體DNA的5’末端具有末端蛋白﹝TP﹞以共價鍵結的形式結合其上。鏈黴菌部分的線型質體具有接合生殖的能力,除了能將自己本身的線型質體﹝Donor﹞利用接合的方式傳遞到不同株的鏈黴菌中﹝Recipient﹞,也可以幫助將線型的染色體帶到Recipient中。已知細菌接合生殖的機制以大腸桿菌﹝E.coli﹞的F質體研究最為詳細,但F質體的接合生殖機制無法套用在鏈黴菌的線型質體,將線型質體完整的傳遞到recipient中。
本研究是想先藉由找出鏈黴菌線型質體的接合傳遞起始點﹝ oriT ﹞及區域,希望有助於了解鏈黴菌線型質體接合傳遞的機制。因此我選用Streptomyces lividans中已被定序完成且與接合生殖相關基因也大致瞭解的線型質體SLP2,利用實驗室開發高頻率線型質體同源重組的方法,去找出SLP2接合傳遞的起始點及區域。將構築好的同源線型質體送到具有SLP2質體的鏈黴菌中,藉由在鏈黴菌中發生一次性同源重組,會產生兩種不同的線型質體,各帶有不同段的SLP2片段,我再去測試這些帶有不同SLP2片段的線型質體其接合傳遞能力,以檢測哪個重組質體帶有SLP2的oriT。利用此策略,我把SLP2 oriT可能所在的區域縮小到約2 kb的DNA片段,位於SLP217.9 kb至19.9 kb的位置上。
論文英文摘要:The topology of Streptomyces genome is different from other bacteria, because it has not only circular plasmid, but also linear chromosome and plasmid. These linear DNA replicons carry covalent-bound terminal proteins ﹝TPs﹞ at their 5’ end of telomeric DNA. Some of the linear plasmids of Streptomyces have ability of conjugation, and sometimes they also promote transfer their linear chromosome into recipient. The current established mechanism of conjugation is mostly based on circular F plasmid in E. coli system, but this cannot be applied to linear plasmid of Streptomyces. The mechanism of conjugal transfer in Streptomyces is not clear, therefore finding the origin of conjugal transfer﹝oriT﹞ in linear plasmids may help to understand this mechanism. By a strategy of homologous recombination that we can generate different recombinant DNA carrying different parts of SLP2 DNA fragment in vivo. Then we tested the transfer ability of this recombinant DNA where SLP2 was present. A truncated SLP2 variant plasmid would be transferred to recipient if it contains oriT of SLP2. By this method, the oriT was narrowed to an internal 2 kb DNA fragment, locating at the coordinate of 17.9~19.9-kb of SLP2.
論文目次:中文摘要 ii
英文摘要 iii
誌謝 iv
目錄 v
表目錄 vi
圖目錄 vii
第一章 緒論 1
第二章 材料與方法 10
第三章 實驗結果
3.1線型質體SLP2的conjugal cis-acting element是位於15.2-kb
至23-kb之間…………………………………………………….15
3.2在本接合生殖實驗中,SLP2可被transfer到recipient中…...31
3.3 Conjugal cis-acting element位於SLP2的17.9-kb至19.9-kb
之間……………………………………………………………….32
3.4找尋SLP2與接合生殖相關的trans element基因……………..43
第四章 討論………………………………………………………...46
參考文獻 .52
附錄
Appendix 1. Media and buffer .57
Appendix 2. Competent cell preparation and transformation………….59
Appendix 3. Plasmid isolation for E. coli ……………....60
Appendix 4. Preparation of Streptomyces protoplast and transfer the plasmid DNA ………………………...61
Appendix 5. Isolation total DNA for Streptomyces ………..62
Appendix 6. Polymerase Chain Reaction﹝PCR﹞……………...63
Appendix 7. Pulsed-field gel electrophoresis﹝PFGE﹞ …. 64
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