跳到主要內容

臺灣博碩士論文加值系統

(34.204.180.223) 您好!臺灣時間:2021/08/05 14:59
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:曾筱筑
研究生(外文):Hsiao-Chu Tseng
論文名稱:探討Haloarcula marismortui之光感受體表現
論文名稱(外文):Characterization of the expression of rhodopsins in Haloarcula marismortui
指導教授:吳韋訥
指導教授(外文):Wailap Victor Ng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:55
中文關鍵詞:嗜鹽古生菌視紫質光感受體
外文關鍵詞:Haloarcula marismortuirhodopsinphotoreceptor
相關次數:
  • 被引用被引用:1
  • 點閱點閱:146
  • 評分評分:
  • 下載下載:17
  • 收藏至我的研究室書目清單書目收藏:0
本論文主題將探討Haloarcula marismortui環境反應調控機制中與光照有關的六個光感受器基因層面 (mRNA) 的表現,在光照與無光照的情況下,觀察這些個基因mRNA表現程度和差異,以及用5’RACE (Rapid amplification of 5' complementary DNA ends) 找出光感受器的轉錄起始位置,以利未來基因調控機制的研究。本論文發現,離子幫浦基因bop、hop、sop1在光照及無光照情況下RNA表現趨勢相似,RNA表現也會因光的存在而誘發,直到細菌生長進入衰退期,RNA表現才會開始下降,但在缺乏光照的情況下,RNA表現量表現量較低,直到生長曲線到達指數期後期,此時環境中含氧量逐漸消耗降低,表現量才會上升。而sop1、 sop2之mRNA表現情形的不同,推測與其不同的趨光性有關。在親緣關係的預測上,xop2和sop1的相似度很高,故具有類似性質的可能性相當高,實驗結果也顯示RNA表現上有相似之處。另外本論文也找出了光感受器基因之轉錄起始點,希望透過對這些感光受器轉錄起始點位置的確認,幫助更加了解基因結構外,也希望藉由轉錄起始點上游序列之分析,找出轉錄訊息有關的序列,了解更多轉錄調控機制。
In this study, we had analyzed the mRNA expression profiles of the six photoreceptors of Haloarcula marismortui, the growing in light and dark conditions. We discovered the transcript levels of the ion pumps: Bop,Hop and Sop1 in light and dark culture conditions. They are increase by light but decrease at late log phase. In the dark culture condition, Bop, Hop and Sop1 gene transcript levels remain low until late log phase (a condition of decreased oxgen tension) in which the transcript levels increased. The expression of the mRNA of Sop1 and Sop2 is different, this might due to different phototaxis response between SRI and SRII. For Xop2 and Sop1, the trend of expression is alike. We had also performed 5’RACE to identify the transcription start site of these photoreceptors for analyzing the upstream sequences to shed light on their gene regulatory mechanism.
目次
目次…………………………………………………………………………………….i
圖 目次……………………………………………………………………………….iii
表 目次……………………………………………………………………………….iv
摘要………………………………………………………….………………………...v
Abstract………………………………………………………….……………………vi

第一章 導論……………………………………………….………………………….1
1.1 光感受器(photoreceptors)……………………………..………………………….1
1.2 古生菌轉錄機制………………………………………………………………….2
1.3 Haloarcula marismortui …………………………………………………………..3
1.4 Haloarcula marismortui 基因體…………………………. ……………………...4
1.5 研究動機及目的………………………………………………………………….6

第二章 材料與方法…………………………………………………………………..7
2.1菌種與培養條件………………………………………….……………………….7
2.2確認視紫紅質 (rhodopsin) 及觸發器 (transducer)基因轉譯起始點 (translation start site)……………………………………………………………………………….7
2.3利用RT-PCR測量Haloarcula marismortui之RNA表現………………………7
2.3.1 萃取total RNA………………….................………………………………7
2.3.2 Reverse transcription reaction...........................…………………………...8
2.3.3 PCR………………………………………………………………………...8
2.4以5’RACE (Rapid amplification of 5' complementary DNA ends)找出轉譯起始點………………………………………………………………………………………9
2.4.1 First-strand cDNA synthesis…………………….………………………….9
2.4.2 RACE PCR Reaction………………………………..……………………...9
2.4.3 純化萃取PCR產物…………………………………………………….....9
2.4.4 TA-cloning……………………………………………………...................10
2.4.5 E.coli轉型反應……………………………………………………...........10
2.4.6 菌落篩選……………………………………………………....................10
2.4.7 微量重組質體製備………………………………………………………11
2.4.8 重組DNA定序…………………………………………………………..11
2.5 MEME分析………………………………………………………………………12

第三章 實驗結果……………………………………………………………………13
3.1野生型H. marismortui在光照及無光照下生長曲線…………………………13
3.2確認視紫紅質(rhodopsin)及觸發器(transducer)基因轉譯起始點……………...13
3.3六個視紫紅質(rhodopsin)及三個觸發器(transducer)分別在光照及無光照情況下之基因表現………………………………………………………………………..14
3.4找出視紫紅質(rhodopsin)及觸發器(transducer)基因之轉錄起始點…………...15
3.5分析轉錄起始點上游保留性區域………………………………………………18

第四章 討論………………………………….. …………………………………….20
4.1野生型H. marismortui在光照及無光照下之生長曲線…. …………………..20
4.2六個視紫紅質(rhodopsin)及三個觸發器(transducer)分別在光照及無光照情況下之基因表現………………………. ………………………………………………20
4.3紫紅質(rhodopsin)及觸發器(transducer)基因之轉錄起始點.............................23
4.4轉錄起始點上游保留性區域…………………………………………………….24
參考文獻……………………………………………………………………………..26
圖表…………………………………………………………………………………..30

圖 目次

圖一 野生型H. marismortui生長曲線…………………………………………......30
圖二 視紫紅質及觸發器基因之胺基酸序列………………………………………31
圖三 bop基因在有光照及無光照下的表現………………………………………..33
圖四 hop基因在有光照及無光照下的表現………………………………………..34
圖五 sop1基因在有光照及無光照下的表現………………………………………35
圖六 sop2基因在有光照及無光照下的表現………………………………………36
圖七 xop1基因在有光照及無光照下的表現………………………………………37
圖八 xop2基因在有光照及無光照下的表現………………………………………38
圖九 htr1基因在有光照及無光照下的表現……………………………………….39
圖十 htr2基因在有光照及無光照下的表現……………………………………….40
圖十一 htrM基因在有光照及無光照下的表現……………………………………41
圖十二 5’RACE PCR 產物藉由1.5% agarose gel electrophoresis分析結果……..42
圖十三 Nest PCR產物藉由1.5% agarose gel electrophoresis分析結果…………..43
圖十四 Nested PCR primer設計……………………………………………………44
圖十五 以PCR確認質體成功轉形E.coli DH5α,將PCR產物進1.5 % 洋菜膠電泳……………………………………………………………………………………..45
圖十六 以MEME分析bop基因前之保留序列 (motif)…………………………...47
圖十七 以MEME分析hop基因前之保留序列 (motif)…………………………...48
圖十八 以MEME分析sop1基因前之保留序列 (motif)…………………………..49
圖十九 以MEME分析sop2基因前之保留序列 (motif)………………………….50
圖二十 以MEME分析xop1基因前之保留序列 (motif)………………………….51
圖二十一 以MEME分析xop2基因前之保留序列 (motif)…………………….....52



目次
表1 5’RACE各基因之primer序列及位置………………………………………53
表2 Nested PCR各基因Primer之序列及位置…………………………………...54
表3 視紫紅質及觸發器基因轉錄起始點位置……………………………………55
參考文獻

1. Spudich, W.R.B.a.J.L. Handbook of Photosensory Receptors (2005).
2. Oesterhelt D, Stoeckenius W. Functions of a new photoreceptor membrane. Proc Natl Acad Sci U S A 97, 2853-7 (1973).
3. Schobert B, Lanyi JK. Halorhodopsin is a light-driven chloride pump. J Biol Chem. 257, 10306-13 (1982).
4. Lanyi JK, Luecke H. Bacteriorhodopsin. Curr Opin Struct Biol. 11, 415-9 (2001)
5. Váró G. Analogies between halorhodopsin and bacteriorhodopsin. Biochim Biophys Acta.1460, 220-9 (2000).
6. Spudich EN, Spudich JL. Control of transmembrane ion fluxes to select halorhodopsin-deficient and other energy-transduction mutants of Halobacterium halobium. Proc Natl Acad Sci U S A. 79, 4308-12 (1982).
7. Bogomolni RA, Spudich JL. Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proc Natl Acad Sci U S A. 79, 6250-4 (1982).
8. Takahashi T, Tomioka H. Photic driving evoked by hemifield flickering dot pattern stimulation in a patient with brain tumor. Electroencephalogr Clin Neurophysiol. 61, 381-4 (1985).
9. Spudich JL, Luecke H. Sensory rhodopsin II: functional insights from structure. Curr Opin Struct Biol. 12, 540-6 (2002).
10. Sasaki J, Spudich JL. The transducer protein HtrII modulates the lifetimes of sensory rhodopsin II photointermediates. Biophys J. 75, 2435-40 (1998).
11. Mennes N. et al. Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli. FEBS Lett. 581, 1487-94 (2007).
12. Hoff WD, Jung KH, Spudich JL. Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annu Rev Biophys Biomol Struct. 26, 223-58 (1997).
13. Langer D, Hain J, Thuriaux P, Zillig W. Transcription in archaea: similarity to that in eucarya. Proc Natl Acad Sci U S A. 92, 5768-72 (1995).
14. Hausner W, Wettach J, Hethke C, Thomm M. Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase. J Biol Chem. 271, 30144-8 (1996).
15. A cell-free transcription system for the hyperthermophilic archaeon Pyrococcus furiosus. Hethke C, Geerling AC, Hausner W, de Vos WM, Thomm M. Nucleic Acids Res. 24, 2369-76 (1996).
16. TAFs revisited: more data reveal new twists and confirm old ideas.Albright SR, Tjian R. Gene. 242, 1-13 (2000).
17. Brown, J.W., Daniels, C.J., Reeve, J.R. Gene structure, organization, and expressionin archaeabacteria. CRC Crit. Rev. Microbiol. 16, 287–338 (1989).
18. Reiter, W-D., Hudelpohl, U., Zillig, W. Mutational analysis of an archaebacterial promoter; essential role of a TATA box for transcription efficiency and start-site selection in vitro. Proc. Natl. Acad. Sci. USA 87, 9509–9513 (1990).
19. Soppa, J. Transcription initiation in archaea: facts, factors and future aspects. Mol. Microbiol. 31, 1295–1305 (1999).
20. Soppa J. Transcription initiation in Archaea: facts, factors and future aspects. Mol Microbiol. 31, 1295-305 (1999).
21. Soppa J. Normalized nucleotide frequencies allow the definition of archaeal promoter elements for different archaeal groups and reveal base-specific TFB contacts upstream of the TATA box. Mol Microbiol. 31,1589-92 (1999).
22. Beelman CA, Parker R. Degradation of mRNA in eukaryotes. Cell. 81, 179-83 (1995).
23. Jäger S, Fuhrmann O, Heck C, Hebermehl M, Schiltz E, Rauhut R, Klug G. An mRNA degrading complex in Rhodobacter capsulatus. Nucleic Acids Res. 29, 4581-8 (2001).
24. Omer AD, Ziesche S, Ebhardt H, Dennis PP. In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex. Proc Natl Acad Sci U S A. 99, 5289-94 (2000).
25.Brow,J. W., C. J. Daniels, and J. N. Reeve. Gene structure, organization, and expression in archarbacteria. Crit. Rev. Microbiol. 16, 287-338 (1989).
26. Bousquet-Antonelli C, Henry Y, G'elugne JP, Caizergues-Ferrer M, Kiss T. A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs. EMBO J. 16, 4770-6 (1997).
27. Charron C, Manival X, Cléry A, Senty-Ségault V, Charpentier B, Marmier-Gourrier N, Branlant C, Aubry A. The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity. J Mol Biol. 342, 757-73 (2004).
28. Jäger S, Fuhrmann O, Heck C, Hebermehl M, Schiltz E, Rauhut R, Klug G. An mRNA degrading complex in Rhodobacter capsulatus. Nucleic Acids Res.29, 4581-8 (2001).
29. Kjems, J., and R. A. Garrett. An intro in the 23S ribosomal RNA gene of the archaebacterium Desulfurococcus mobolis. Nature 318, 675-677 (1985).
30. Kjems J, Garrett RA. Ribosomal RNA introns in archaea and evidence for RNA conformational changes associated with splicing. Proc Natl Acad Sci U S A. 88, 439-43 (1991).
31. Falb M, Müller K, Königsmaier L, Oberwinkler T, Horn P, von Gronau S, Gonzalez O, Pfeiffer F, Bornberg-Bauer E, Oesterhelt D. Metabolism of halophilic archaea. Extremophiles. 12, 177-96 (2008).
32. Woese CR, Fox GE. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci U S A.;74, 5088-90 (1977).
33. Bell SD, Jackson SP. Transcription and translation in Archaea: a mosaic of eukaryal and bacterial features. Trends Microbiol. 6, 222-8 (1998).
34. Dennis PP.Ancient ciphers: translation in Archaea. Cell. 89, 1007-10 (1997).
35. Edgell DR, Doolittle WF. Archaea and the origin(s) of DNA replication proteins. Cell. 89, 995-8 (1997).
36. Joo WA, Kim CW. J Chromatogr B. Proteomics of Halophilic archaea. Analyt Technol Biomed Life Sci. 815, 237-50 (2005).
37.Russell NJ,Nichols DS. Polyunsaturated fatty acids in marine bacteria--a dogma rewritten. Microbiology. 145, 767-79 (1999).
38. Ike A, Murakawa T, Kawaguchi H, Hirata K, Miyamoto K. Photoproduction of hydrogen from raw starch using a halophilic bacterial community. J Biosci Bioeng. 88, 72-7 (1999).
39. Margesin R, Schinner F. Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles. 5, 73-83 (2001).
40. Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, Lasky SR, Baliga NS, Thorsson V, Sbrogna J, Swartzell S, Weir D, Hall J, Dahl TA, Welti R, Goo YA, Leithauser B, Keller K, Cruz R, Danson MJ, Hough DW, Maddocks DG, Jablonski PE, Krebs MP, Angevine CM, Dale H, Isenbarger TA, Peck RF, Pohlschroder M, Spudich JL, Jung KW, Alam M, Freitas T, Hou S, Daniels CJ, Dennis PP, Omer AD, Ebhardt H, Lowe TM, Liang P, Riley M, Hood L, DasSarma S. Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci U S A. 97, 12176-81 (2000).
41. Baliga NS, Bonneau R, Facciotti MT, Pan M, Glusman G, Deutsch EW, Shannon P, Chiu Y, Weng RS, Gan RR, Hung P, Date SV, Marcotte E, Hood L, Ng WV. Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res. 14, 2221-34 (2004).
42. Sharma AK, Walsh DA, Bapteste E, Rodriguez-Valera F, Ford Doolittle W, Papke RT. Evolution of rhodopsin ion pumps in haloarchaea. BMC Evol Biol. 18, 7:79 (2007).
43. Dassarma S, Rajbhandary UL, Khorana HG. Bacterio-opsin mRNA in wild-type and bacterio-opsin-deficient Halobacterium halobium strains. Proc Natl Acad Sci U S A. 81, 125-129 (1984).
44. Gropp F, Gropp R, Betlach MC. Effects of upstream deletions on light- and oxygen-regulated bacterio-opsin gene expression in Halobacterium halobium. Mol Microbiol. 16, 357-64 (1995).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊