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論文中文名稱:確認鏈黴菌線型質體SLP2接合傳遞的clt [以論文名稱查詢館藏系統]
論文英文名稱:Identification of the clt 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:生化與生醫工程研究所
畢業學年度:101
出版年度:102
中文姓名:李王民甄
英文姓名:Min-Chen Li
研究生學號:100688004
學位類別:碩士
語文別:中文
口試日期:2013-07-31
論文頁數:74
指導教授中文名:黃志宏
口試委員中文名:楊千金;陳月茸
中文關鍵詞:鏈黴菌線型質體SLP2接合傳遞clt
英文關鍵詞:Streptomyceslinear plasmid SLP2conjugal transferclt
論文中文摘要:鏈黴菌是在形態上有菌絲且具分支功能的革蘭氏陽性細菌,包含線型染色體和線型質體,且大部分質體皆有接合傳遞能力。細菌的接合傳遞機制目前是大腸桿菌環型的F質體以單股DNA單向地進行rolling-circle方式複製的傳遞研究最詳細,但仍無法說明鏈黴菌的線型質體是如何被傳遞。SLP2屬S.lividans可接合傳遞的50kb線型質體,本實驗室先前研究發現其幫助接合傳遞的clt(cis-acting locus of transfer)是在內部約450bp的一段DNA,其上有約五對正向和反向重複序列群聚,此特徵與已知clt的特徵相符。
本研究藉由剔除這些正向和反向重複序列後,對已剔除重複序列的SLP2接合傳遞能力下降10-3倍,顯示clt是在這片段內,因此繼續縮小clt的重複序列範圍。這些重複序列是由六個相同序列和其五個反向序列片段構成,發現各自分成左右同向的重複序列區域對接合傳遞效率和完整的重複序列的效率差不多,另外單獨一對或兩對重複序列的片段接到質體上對接合傳遞效果沒顯著幫助。因此分析可能至少需要超過兩對重複序列群聚的片段才能夠在線型質體SLP2接合傳遞時將本來無法傳遞的質體送到接受株。
論文英文摘要:Streptomyces are members of the Gram-positive bacteria. It is mycelium-forming bacteria,also containing the ability of branching. They contain linear chromosome and linear plasmids, many of them can be transferred to other Streptomyces via conjugation. The conjugal transfer mechanism of plasmid in E.coli system using single strand DNA rolling-circle replication for conjugal transfer is currently best-studied one. But that couldn’t explain how linear plasmids of Streptomyces proceed conjugal transfer. SLP2 in S. lividans is a 50 kb linear plasmid. Previous studies showed that the clt (cis-acting locus of transfer) is about 450 bp, carrying about five sets of repeat sequences, characteristics of some known clts.
In this study, we found that the conjugal transfer frequency of SLP2 which has knockouted all sets of repeat sequence is reduced by 103 times. To confirm its clt function in this region, therefore I narrowed down the repeat sequences region which is consist of six sequences and its complementary five sequences on the other side. We found that either half of this repeat sequences functions as good as the whole sets in conjugal transfer. However, one or two sets of repeat sequences failed to perform the conjugal transfer function. According to these results, we conclude that more than two sets of repeat sequences of the clt are required to transfer SLP2 linear plasmid to recipient.
論文目次:中文摘要 i
英文摘要 ii
致謝 iv
目錄 v
圖目錄 ix
第一章 緒論 1
1.1 細菌的接合傳遞作用 1
1.2 鏈黴菌的簡介 4
1.3 鏈黴菌的接合傳遞作用 6
1.4 鏈黴菌線型質體的clt (cis-acting locus of transfer)8
第二章 材料與方法 11
2.1 菌種與質體 11
2.2 藥品與酵素 15
2.3 培養基與緩衝溶液 15
2.4 菌種之儲存方法 15
2.5 大腸桿菌的轉型作用 15
2.6 大腸桿菌質體的分離及純化 16
2.7 鏈黴菌原生質體的製備與轉型作用 16
2.8 鏈黴菌基因組DNA的分離及純化 16
2.9 限制酵素、T4 連接酵素及各種Kit的使用方法 16
2.10 聚合酶連鎖反應(polymerase chain reaction; PCR)17
2.11 脈衝電場型膠體電泳(Pulsed-Field Gel Electrophoresis;PFGE)17
2.12 鏈黴菌質體接合生殖效率計算 17
第三章 實驗結果 18
3.1 縮小SLP2的clt範圍到正向反向重複序列群聚區域 18
3.2 剔除SLP2的R(repeat)片段後測試其接合傳遞效率 25
3.3 構築包含R重複序列內左邊的反向重複序列Rr之線型質體並測試其接合傳遞效率 27
3.4 構築包含R重複序列內右邊的反向重複序列Rl之線型質體並測試其接合傳遞效率 34
3.5 構築包含SLP2中R(Repeat)片段裡面的一對重複序列IR的線型質體並測試其接合傳遞效率 40
3.6 構築包含SLP2中R(Repeat)片段裡面的兩對重複序列IIR的線型質體並測試其接合傳遞效率 47
3.7 將本實驗構築包含SLP2中R(Repeat)片段裡面的不同重複序列範圍的線型質體送至包含剔除clt的S.lividans 1326測試其接合傳遞效率 54
第四章 討論 57
參考文獻 59
附錄 64
Appendix 1. Media and buffer 64
Appendix 2. E. coli Competent cell preparation and transformation 66
Appendix 3. Plasmid isolation from E. coli 69
Appendix 4. Preparation of Streptomyces protoplast and transformation 70
Appendix 5. Isolation the total DNA of Streptomyces 71
Appendix 6. Polymerase Chain Reaction 72
Appendix 7. Pulsed-field gel electrophoresis 73
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