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論文中文名稱:檢測Streptomyces coelicolor染色體及SCP1的ttrA同源基因在接合傳遞作用上的功能 [以論文名稱查詢館藏系統]
論文英文名稱:To examine the conjugal functions of ttrA homologs in S. coelicolor chromosome and linear plasmid SCP1 [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:99
出版年度:100
中文姓名:何志文
英文姓名:Chih-Wen He
研究生學號:98688008
學位類別:碩士
語文別:中文
口試日期:2011-07-28
論文頁數:66
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;陳月茸
口試委員英文名:Carton W. Chen;Chien-Chin Yang;Yueh-jung Chen
中文關鍵詞:鏈黴菌接合傳遞線型質體線型染色體
英文關鍵詞:conjugationStreptomyceslinear plasmidlinear chromosome
論文中文摘要:鏈黴菌具有線型的染色體與質體,其中部分的線型質體能夠進行接合傳遞。先前關於鏈黴菌接合傳遞的研究結果得知接合傳遞機制不同於研究透徹的E. coli系統,目前證實剔除S. lividans染色體或其線型質體SLP2的ttrA會降低接合傳遞的效率,並且發現這些ttrA都是位於線型染色體或質體的末端區域,以in cis方式參與接合傳遞作用。S. coelicolor染色體的ttrA同源基因無論基因序列或染色體位置與S. lividans有高度的相似性,而線型質體SCP1雖然具有兩個ttrA同源基因,但是基因位置並不是在末端區域內。
SLP2的ttrA是目前唯一證實與鏈黴菌接合傳遞有關的基因,本論文是希望瞭解S. coelicolor的染色體與質體SCP1上的ttrA同源基因是否也與接合傳遞的功能有關,本實驗將S. coelicolor染色體與線型質體SCP1上的ttrA同源基因,經由基因剔除之後,檢測這些突變株的接合傳遞能力是否有受到影響。在檢測S. coelicolor染色體上的ttrA部分,將S. coelicolor 3456與M145的ttrA同源基因(SCO0002)剔除後,分別與S. coelicolor M130及S. lividans TK54進行交配。與S. coelicolor M130的接合傳遞作用實驗,染色體的重組互換頻率下降60到176倍,而與S. lividans TK54的接合傳遞作用實驗,染色體的重組互換頻率則下降約一千倍。表示對於S. coelicolor 3456,末端染色體上的ttrA功能剔除時,同種鏈黴菌的接合作用,染色體之間重組互換的頻率沒有明顯的改變,但對於異種鏈黴菌的接合作用,染色體之間重組互換的能力有明顯下降。在檢測SCP1的ttrA同源基因的部份,分別將兩個ttrA同源基因SCP1.136及SCP1.216A剔除後,與S. coelicolor M145進行交配後染色體重組頻率約下降10倍,同時將兩個同源基因剔除後,染色體重組頻率約下降800倍。與S. lividans TK54進行交配後,單一的ttrA同源基因的剔除,染色體轉移重組頻率約下降10倍;同時剔除兩個同源基因,染色體轉移重組頻率約下降一千倍。數據顯示SCP1的ttrA基因與接合傳遞作用有關,而且兩個ttrA同源基因有互補的現象。
論文英文摘要:Streptomyces processes linear chromosomes and linear plasmids, and most of the linear plasmids can be transferred by conjugation. The mechanism of conjugal transfer for E. coli circular plasmids is not applied to the conjugal system of Streptomyces. Previous research in Streptomyces conjugation had shown that the terminally located ttrA genes on the S. lividans chromosome and SLP2 were involved in conjugation probably acting in cis. Homologs of ttrA exist in the terminal regions of most Streptomyces chromosomes and some linear plasmids. However two ttrA homologs are located in the central region on the linear plasmid SCP1.
This study was to understand whether ttrA gene on the S. coelicolor chromosomes and SCP1 were also involved in conjugal transfer. The ttrA gene (SCO0002) of S. coelicolor 3456 (containing integrated SCP1) was deleted and the mutant was mated with S. coelicolor M130 and S. lividans TK54. In mating with M130, the recombinant frequency was decreased by 60 to 176 folds. In mating with S. lividans TK54, the recombinant frequency was decreased by about 1,000 folds. The results indicated that ttrA on the S. coelicolor chromosome was more conjugatant influenced in inter-specifical transfer than intra-specifical transfer. In addition, two the ttrA homologs on SCP1 (SCP1.136 and SCP1.216Ac) were knockout individually. When these mutants were mated with S. coelicolor or S. lividans, the recombinant frequencies were decreased by about 10 folds. Double knockout mutant was also created, and the mutant was mated with S. coelicolor M145, the recombinant frequency was deceased by about 800 times. These results showed that the both ttrA homologs on SCP1 were involved in conjugal transfer.
論文目次:中文摘要 i
英文摘要 iii
誌謝 iv
目錄 vi
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1 鏈黴菌的簡介 1
1.2 細菌的接合傳遞作用 2
1.3 鏈黴菌質體和接合傳遞作用 4
1.4 與接合傳遞有關的基因ttrA 5
第二章 材料與方法 9
第三章 實驗結果 14
3.1 S. coelicolor染色體末端ttrA的剔除 14
3.2 S. coelicolor ttrA基因剔除後,接合傳遞作用的結果分析 26
3.3 SCP1.216Ac基因的剔除 29
3.4 SCP1.216Ac基因剔除後,接合傳遞作用的結果分析 32
3.5 SCP1.216Ac與SCP1.136基因的同時剔除 35
3.6 SCP1.216Ac與SCP1.136基因的同時剔除後,接合傳遞的結果分析 40
第四章 討論 43
參考文獻 46
附錄
A. Media and buffer 50
B. E. coli Competent cell preparation and transformation 53
C. Plasmid isolation from E. coli 56
D. Preparation of Streptomyces protoplast and transformation 57
E. Isolation the total DNA of Streptomyces 58
F. Polymerase Chain Reaction 59
G. Pulsed-field gel electrophoresis 60
H. Southern hybridization 63
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論文全文使用權限:同意授權於2016-08-24起公開