現在位置首頁 > 博碩士論文 > 詳目
論文中文名稱:鏈黴菌線型質體SLP2的接合傳遞基因之研究 [以論文名稱查詢館藏系統]
論文英文名稱:Identification of the conjugation related genes of Streptomyces linear plasmid [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:104
畢業學期:第一學期
中文姓名:徐銘妤
英文姓名:Ming-Yu Hsu
研究生學號:102688030
學位類別:碩士
語文別:中文
口試日期:2016/01/29
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:SLP2;trans acting element;接合傳遞
英文關鍵詞:Streptomyces, SLP2;trans acting element; conjugation
論文中文摘要:鏈黴菌是一種土壤中常見的細菌,其中最獨特的是它具有環形質體以及線型質體。這些質體大部分具有接合傳遞(conjugation)的能力。在細菌接合傳遞機制的研究中,以大腸桿菌環形F質體研究較為透徹,其經TraI在oriT切出缺口後,由單向單股的方式傳遞到接合傳遞接受株中,但此作用機轉無法套用於線型DNA的接合傳遞現象,除非是其oriT位於最末端上。目前對鏈黴菌線型DNA的接合傳遞作用機制仍不清楚。因此本實驗即是以鏈黴菌線形質體SLP2為研究對象,希望尋找出參與接合傳遞相關基因進而更了解線形質體的接合傳遞機制。
鏈黴菌S. lividans中的線形質體SLP2大小約50 kb,先前本實驗室以線型DNA同源重組的方式,分別得到不同DNA片段缺失的衍生質體,測試其接合傳遞效率以找尋相關的基因。先前我們已知SLP2末端的SLP2.01基因缺失的重組株造成接合傳遞效率下降10倍,接下來,本論文研究中為了更確定SLP2.01於接合傳遞中的角色,因此將SLP2.01剔除,測試其接合傳遞效率。接著為了證明重組株的接合傳遞效率下降是因SLP2.01基因缺失,計畫將SLP2.01重新放回已剔除SLP2.01的SLP2重組株中,測試效率以證明SLP2.01是否參與線型質體接合傳遞的基因。另外,本實驗使用螢光蛋白與SLP2.01在基因層次進行融合,希望藉由觀察螢光蛋白了解SLP2.01於接合傳遞過程的不同時間點的位置及扮演的角色。另一方面,先前證明,SLP2 25.2 kb到右邊末端中的ttrA基因與接合傳遞相關,接著計畫以同源重組的方式使SLP2 25.2 kb~右邊末端只含有ttrA基因,並測試其接合傳遞效率以證明ttrA基因是否為SLP2自25.2 kb到右邊末端中唯一與接合傳遞相關基因。
論文英文摘要:Streptomyces is a common soil bacterium. The most unique feature is that they can have both circular and linear plasmids. Most of these plasmids acquire the ability of conjugal transfer. Currently, the conjugal transfer mechanism of E.coli F plasmid is the most clear in bacteria. The oriT of F plasmid is cut by TraI, then leads the single-strand DNA transfer to recipient by uni-direction. But this mechanism is only applied to linear plasmid to be transferred with the oriT at the end. Currently, the transfer mechanism of linear plasmid in Streptomyces is still unclear. Thus, we plan to find the conjugation related genes of Streptomyces linear plasmid SLP2.
The size of Streptomyces S. lividans linear plasmid SLP2 is about 50 kb. Our lab used different SLP2 partial-deleted mutants generated by homologous recombination to find the conjugation related genes. Previously, we knew that the recombinant of deleted SLP2.01 gene reduced transfer frequency with a decrease by 100 times than wild type SLP2. Thus, we deleted the SLP2.01 gene directly in order to make sure the role of SLP2.01 gene in conjugation. Following, the reason we plan to put SLP2.01 gene back to recombinant to prove the decrease in frequency is caused by lack of SLP2.01 gene. In addition, the experiment with the use of fluorescent fusion protein at the genetic level, we plan to fuse SLP2.01 with a fluorescent protein to verify the cellular localization of SLP2.01 during conjugation. On the other hand, ttrA gene,a conjugation gene,was identified in the 25.2 kb to right arm of SLP2. We plan to let the right arm of SLP2 25.2kb to only have ttrA gene by using homologous recombination to prove that the ttrA is the only gene related to conjugation between SLP2 25.2kb to right arm of SLP2.
論文目次:中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
圖目錄 vii
表目錄 ix
第一章 緒論 1
1.1 鏈黴菌的簡介 1
1.2 細菌的接合傳遞 2
1.3 鏈黴菌的環形接合傳遞 3
1.4 鏈黴菌的線型質體及線型染色體 5
1.5 SLP2線型質體 6
第二章 材料與方法 9
2.1 菌種與質體 9
2.2 藥品與酵素 12
2.3 培養基與緩衝溶液 13
2.4 菌種與儲存 13
2.5 大腸桿菌的轉型 13
2.6 大腸桿菌質體的分離與純化 13
2.7 鏈黴菌原生質體的製備與轉型 13
2.8 鏈黴菌基因組DNA的分離與純化 14
2.9 限制酵素、T4 連接酵素及各種Kit的使用方法 14
2.10 聚合酶連鎖反應(PCR, polymerase chain reaction) 14
2.11 脈衝電場型膠體電泳(Pulsed-Field Gel Electrophoresis) 15
2.12南方墨點法(Southern blotting) 15
2.13 鏈黴菌質體接合生殖頻率計算(Recombination frequency of linear plasmid conjugation) 15
第三章 實驗結果 16
3.1 在 SLP2 上剔除SLP2.01 並測試其接合傳遞的效率 16
3.2 將 SLP2.01 重新放回pMY02並測試其接合傳遞效率 26
3.3 建構 SLP2.01 - 綠色螢光融合蛋白 39
3.4 使SLP2 25.2kb~右邊末端只含有ttrA基因 46
第四章 討論 52
參考文獻 55
第五章 附錄 60
Appendix 1. Media and buffer 60
Appendix 2. Competent cell preparation and transformation 62
Appendix 3. Plasmid isolation for E. coli 63
Appendix 4. Preparation of Streptomyces protoplast and transfer the plasmid DNA 64
Appendix 5. Isolation total DNA for Streptomyces 65
Appendix 6. Polymerase Chain Reaction﹝PCR﹞ 67
Appendix 7. REDIRECT technology: 68
Appendix 8. Pulsed-field gel electrophoresis﹝PFGE﹞ 70
論文參考文獻:1. Bao K, Cohen SN: Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes Dev 2001, 15(12):1518-1527.

2. Bao K, Cohen SN: Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication. Genes Dev 2003, 17(6):774-785.

3. Bao K, Cohen SN: Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex. Proc Natl Acad Sci U S A 2004, 101(40):14361-14366.

4. Bey SJ, Tsou MF, Huang CH, Yang CC, Chen CW: The homologous terminal sequence of the Streptomyces lividans chromosome and SLP2 plasmid. Microbiology 2000, 146 ( Pt 4):911-922.

5. Challis GL, Hopwood DA: Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci U S A 2003, 100 Suppl 2:14555-14561.

6. Chang PC, Cohen SN: Bidirectional replication from an internal origin in a linear streptomyces plasmid. Science 1994, 265(5174):952-954.

7. Chen CW, Yu TW, Lin YS, Kieser HM, Hopwood DA: The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule. Mol Microbiol 1993, 7(6):925-932.
8. Claverys JP, Martin B, Polard P: The genetic transformation machinery: composition, localization, and mechanism. FEMS Microbiol Rev 2009, 33(3):643-656.

9. Clewell DB: Bacterial sex pheromone-induced plasmid transfer. Cell 1993, 73(1):9-12.

10. Frost LS, Bazett-Jones DP: Examination of the phosphate in conjugative F-like pili by use of electron spectroscopic imaging. J Bacteriol 1991, 173(23):7728-7731.

11. Frost LS, Ippen-Ihler K, Skurray RA: Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev 1994, 58(2):162-210.

12. Gravius B, Glocker D, Pigac J, Pandza K, Hranueli D, Cullum J: The 387 kb linear plasmid pPZG101 of Streptomyces rimosus and its interactions with the chromosome. Microbiology 1994, 140 ( Pt 9):2271-2277.

13. Grohmann E, Muth G, Espinosa M: Conjugative plasmid transfer in gram-positive bacteria. Microbiol Mol Biol Rev 2003, 67(2):277-301, table of contents.

14. Grote M: Hybridizing Bacteria, Crossing Methods, Cross-checking Arguments: The Transition from Episomes to Plasmids (1961-1969). Hist Phil Life Sci 2008, 30(3-4):407-430.

15. Hopwood DA: Soil to genomics: the Streptomyces chromosome. Annu Rev Genet 2006, 40:1-23.

16. Hopwood DA, Kieser T, Wright HM, Bibb MJ: Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J Gen Microbiol 1983, 129(7):2257-2269.

17. Hsu CC, Chen CW: Linear plasmid SLP2 is maintained by partitioning, intrahyphal spread, and conjugal transfer in Streptomyces. J Bacteriol 2010, 192(1):307-315.

18. Huang CH, Chen CY, Tsai HH, Chen C, Lin YS, Chen CW: Linear plasmid SLP2 of Streptomyces lividans is a composite replicon. Mol Microbiol 2003, 47(6):1563-1576.

19. Kendall KJ, Cohen SN: Complete nucleotide sequence of the Streptomyces lividans plasmid pIJ101 and correlation of the sequence with genetic properties. J Bacteriol 1988, 170(10):4634-4651.

20. Kosono S, Kataoka M, Seki T, Yoshida T: The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP-binding motifs and is localized to the cytoplasmic membrane. Mol Microbiol 1996, 19(2):397-405.

21. Lederberg J, Tatum EL: Gene recombination in Escherichia coli. Nature 1946, 158(4016):558.

22. Lin YS, Kieser HM, Hopwood DA, Chen CW: The chromosomal DNA of Streptomyces lividans 66 is linear. Mol Microbiol 1993, 10(5):923-933.

23. Pandza K, Pfalzer G, Cullum J, Hranueli D: Physical mapping shows that the unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end of the linear chromosome. Microbiology 1997, 143 ( Pt 5):1493-1501.
24. Panicker MM, Minkley EG, Jr.: DNA transfer occurs during a cell surface contact stage of F sex factor-mediated bacterial conjugation. J Bacteriol 1985, 162(2):584-590.

25. Possoz C, Ribard C, Gagnat J, Pernodet JL, Guerineau M: The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer. Mol Microbiol 2001, 42(1):159-166.

26. Reuther J, Gekeler C, Tiffert Y, Wohlleben W, Muth G: Unique conjugation mechanism in mycelial streptomycetes: a DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip. Mol Microbiol 2006, 61(2):436-446.

27. Siefert HM, Maruhn D, Maul W, Forster D, Ritter W: Pharmacokinetics of ciprofloxacin. 1st communication: absorption, concentrations in plasma, metabolism and excretion after a single administration of [14C]ciprofloxacin in albino rats and rhesus monkeys. Arzneimittelforschung 1986, 36(10):1496-1502.

28. Thoma L, Muth G: Conjugative DNA transfer in Streptomyces by TraB: is one protein enough? FEMS Microbiol Lett 2012, 337(2):81-88.

29. Wang J, Pettis GS: The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon. Microbiology 2010, 156(Pt 9):2723-2733.

30. Wellington EM, Cresswell N, Saunders VA: Growth and survival of streptomycete inoculants and extent of plasmid transfer in sterile and nonsterile soil. Appl Environ Microbiol 1990, 56(5):1413-1419.

31. Willetts N, Maule J: Specificities of IncF plasmid conjugation genes. Genet Res 1986, 47(1):1-11.
32. Xu M, Zhu Y, Zhang R, Shen M, Jiang W, Zhao G, Qin Z: Characterization of the genetic components of Streptomyces lividans linear plasmid SLP2 for replication in circular and linear modes. J Bacteriol 2006, 188(19):6851-6857.

33. Yang CC, Huang CH, Li CY, Tsay YG, Lee SC, Chen CW: The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins. Mol Microbiol 2002, 43(2):297-305.
論文全文使用權限:不同意授權