現在位置首頁 > 博碩士論文 > 詳目
  • 同意授權
論文中文名稱:找尋鏈黴菌線型質體SLP2接合傳遞的trans-acting element [以論文名稱查詢館藏系統]
論文英文名稱:Identification of trans-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:101
出版年度:102
中文姓名:蔡宜臻
英文姓名:Yi-Chen Tsai
研究生學號:99688020
學位類別:碩士
語文別:中文
口試日期:2013-01-31
論文頁數:72
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:楊千金;陳月茸
口試委員英文名:Chien-Chin Yang;Yueh-Jung Chen
中文關鍵詞:鏈黴菌SLP2同源重組接合傳遞trans-acting element
英文關鍵詞:Streptomyceshomologous recombinationconjugationSLP2trans
論文中文摘要:鏈黴菌擁有線型染色體和線型質體,其線型DNA的5’末端有末端蛋白(TP)以共價方式連接於其上。大部分的鏈黴菌質體都具有接合傳遞的能力,但是其線型質體的傳遞機制仍不清楚,而研究較清楚的大腸桿菌F質體,由TraI蛋白在oriT上產生單股DNA切口以進行單股DNA單向複製傳遞的模式,也明顯無法套用於線型DNA。本實驗室先前在找尋S. lividans的50 kb線型質體SLP2接合傳遞所需之cis-acting element (clt)的研究過程中,得到許多不同大片段缺失的SLP2衍生質體,這些質體在缺乏完整SLP2的狀況下仍具有接合傳遞的能力,但是效率較完整的SLP2降低約100倍,顯示缺失的片段中有基因參與接合傳遞。Xu等人在2006年的研究指出除了traSLP2以外尚有transglycosylase及ATP/GTP binding protein參與接合傳遞,但我們先前所觀察到的SLP2接合傳遞功能下降的DNA缺失片段並不在此內,所以本論文即是找尋其他參與SLP2接合傳遞的基因。藉由實驗室建構的線型DNA間同源重組之方法,我得到其他不同DNA片段缺失的SLP2線型衍生質體,藉由觀察其接合傳遞的能力來找尋相關基因,經過檢測比對發現,包含SLP2 581 bp~50 kb的衍生質體的接合傳遞能力比其他較大片段缺失的衍生質體增加10倍,且多出的DNA片段只含有SLP2.01基因,其蛋白質序列比對顯示沒有與已知功能蛋白質的序列相似,因此可能為一全新的參與線型質體接合傳遞的基因。
論文英文摘要:Streptomyces harbors linear chromosome and linear plasmid, which a terminal protein covalently-bound at their 5’ ends. The most plasmids of Streptomyces have the ability of conjugal transfer, but the transfer mechanism of linear plasmid is still unclear. Due to its structure, the single-stranded, uni-direction transfer mechanism of E. coli F plasmid is not applied to this linear DNA system. Thus, a novel mechanism is required to explain this process. Previously, we have found several SLP2 partial-deleted mutants generated by homologous recombination between a constructed linear plasmids and SLP2 during the study of SLP2 clt, resulted in transfer frequency decrease by 100 times than wild-type SLP2. It suggest that there has something involved in conjugal transfer in the deleted region. Xu et al (2006) showed three genes encoding TraSLP2, Transglycosylase, and ATP/GTP bind protein are involving in SLP2 transfer. However, all mutants in the previous study contained identified three genes. This result hints the absent region from SLP2 in those plasmids might contain other genes involving in SLP2 transfer. My work is trying to identify those novel genes involved in conjugal transfer. By the same strategy to generated a series partial-deleted SLP2 derivatives and tested their transfer frequency, we generated series of partial-deleted SLP2 mutants. Comparing to those generated partial-deleted SLP2 derivatives, a 10 times increased in transfer frequency had been observed only when it contains 581 bp~50 kb region of SLP2 which containing only one gene, SLP 2.01 in that region. There is no similar protein sequence of SLP2.01 on the data base, it might be a novel protein involved in conjugal transfer of linear plasmid.
論文目次:中文摘要 …i
英文摘要……………………………………………………………………...iii
致謝…………………………………………………………………….………v
目錄 .........vi
表目錄 ....viii
圖目錄 ..ix
第一章 緒論
1.1 鏈黴菌的簡介………………………………………………………1
1.2 細菌的接合傳遞……………………………………………….……3
1.3 鏈黴菌的環形接合傳遞……………………….……………….……6
1.4 SLP2線型質體………….……………………………...……….……7
第二章 材料與方法
2.1 菌種與質體 ……………………………………………………....10
2.2 藥品、酵素…………………………………………………………13
2.3 培養基及緩衝溶液……………………………….………………13
2.4 菌種之儲存……………………………………….………………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……………………..………………14
2.7 鏈黴菌原生質體的製備與轉型……………..……………………14
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….…………………………………….….15
2.12南方墨點法(Southern blot)……………………………….….15
2.13 鏈黴菌質體接合生殖頻率計算……………………………..….15
第三章 實驗結果
3.1測試SLP2 1~13 kb和1~17 kb缺失的衍生質體之接合傳遞效率來
找尋相關trans-element…………………………..……….…..…....17
3.2構築SLP2 4.5 kb~50 kb以及8.5 kb~50 kb的線型質體並測試其
接合傳遞效率………………………………………..……..………24
3.3構築SLP2 2 kb~50 kb以及581 bp~50 kb的線型質體並測試其接
合傳遞效率……..………………………….……………………….35
3.4測試SLP2 25.2~50.4 kb和37.5~50.4 kb缺失的衍生質體之接合
傳遞效率來找尋相關trans-element…………………….…..…....45
3.5將ttrA放回pCU32………………………………………………….48
3.6測試同樣帶有SLP2的clt片段但end不同的線形質體是否具有相
同的傳遞效率………………………………………….………….52
第四章 討論………………………………………….……………………...54
參考文獻 ……..58
附錄
Appendix 1. Media and buffer………………………………………… .63
Appendix 2. Competent cell preparation and transformation…………..65
Appendix 3. Plasmid isolation for E. coli……………………………....66
Appendix 4. Preparation of Streptomyces protoplast and transfer the
Plasmid DNA……………………………………………...68
Appendix 5. Isolation total DNA for Streptomyces................................79
Appendix 6. Polymerase Chain Reaction (PCR)..…………....………...70
Appendix 7. Pulsed-field gel electrophoresis (PFGE)….…………..…. 71
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:同意授權於2013-02-19起公開