臺灣博碩士論文加值系統

　　從兩隻頭足類動物體內分離出三株海洋發光細菌品系，透過表型特徵的檢驗，以及分析其16S ribosomal RNA基因序列後，鑑定出此三株品系均為Photobacterium leiognathi。
　　本研究中所分離出來的三株P. leiognathi品系的發光強度以肉眼觀察便可辨識出有明顯差異，故當其受到特定理化因子的影響時，不同品系間的發光強度所受到的影響程度亦可能有所差異。實驗結果顯示，不同的溫度、pH值、鹽度、滲透壓、Zn2+濃度、H2O2濃度，以及紫外光強度等理化因子，對於不同品系的發光強度確實有不同程度的影響，但由於這三株P. leiognathi品系的發光強度差異甚大，即使是在不同理化因子處理下，發光強度的排位上並不會發生改變，故在生態上的意義並不十分明顯。
　　此外，傳統上在定量發光細菌的發光程度時，大多使用昂貴的光度計，但此類儀器對於未開發國家或是落後地區來說是一大負擔。因此，本研究嘗試採用數位單眼相機擷取細菌的發光訊號，並在電腦軟體的協助下進行發光強度的定量，結果顯示這樣的方法確實可在某些試驗條件下取代價格不斐的光度計。

中文摘要 ………………………………………………………………………P. 2
英文摘要………………………………………………………………………P. 3
壹、前言 ………………………………………………………………………P. 4
　一、海洋發光細菌簡介……………………………………………………P. 4
　二、海洋發光細菌的鑑定方法……………………………………………P. 5
　三、海洋發光細菌在生態學研究上的應用………………………………P. 6
　四、Photobacterium leiognathi簡介………………………………P. 8
　五、研究目的………………………………………………………………P. 8
貳、研究材料與方法 ………………………………………………………P. 10
　一、菌種的分離與保存……………………………………………………P. 10
　二、菌種的鑑定……………………………………………………………P. 10
　三、發光細菌發光強度的測定……………………………………………P. 15
　四、不同理化因子對於不同細菌品系發光強度的影響…………………P. 16
　五、數位訊號的定量………………………………………………………P. 20
參、結果 ……………………………………………………………………P. 22
肆、討論 ……………………………………………………………………P. 31
伍、參考文獻 ………………………………………………………………P. 37
陸、圖表………………………………………………………………………P. 43

Applegate, B.M., Kehrmeyer, S.R., Sayler, G.S., 1998. A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing. Appl. Environ. Microbiol. 64: 2730-2735.

Baumann, P. and Baumann, L., 1984. Genus II Photobacterium Beijerinck 1889, 401AL. In Bergey’s Manual Systematic Bacteriology, vol. 1, pp. 539-545. Edited by N.R. Krieg & J.G. Holt. Baltimore: Williams & Wilkins.

Belkin, S., Smulski, D.R., Vollmer, A.C., Vandyk, T.K., LaRossa, R.A., 1996. Oxidative stress detection with Escherichia coli harboring a katG'::lux fusion. Appl. Environ. Microbiol. 62: 2252-2256.

Caccamo, D., Di Cello, F., Fani, R., Gugliandolo, C., Maugeri, T.L., 1999. Polyphasic approach to the characterization of marine luminous bacteria. 150: 221-230.

Czyz, A., Wrobel, B., Wegrzyn, G., 2000. Vibrio harveyi bioluminescence plays a role in stimulation of DNA repair. Microbiol. 146: 283-288.

Eckstein, J.W., Cho, K.W., Colepicolo, P., Ghisla, S., Hastings, W., Wilson, T., 1990 A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: Energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1. Proc. Natl. Acad. Sci. USA 87: 1466-1470

Ehlers, L.J., Luthy, R.G., 2003. Contaminant bioavailability in soil and sediment. Environ. Sci. Technol. 37: 295A-302A.

Holt, J.G., Krieg, N.R., Sneath, P.H., Staley, J.T., Williams, S.T. (Editors). 1994. Group 5 Facultatively anaerobic Gram-negative rods. Subgroup 2: family Vibrionaceae. In Burgey’s Manual of Determinative Bacteriology. 9th edn., pp. 190-194. Baltimore: Williams & Wilkins.

Irwin, R.J. (Editors). 1997. Environmental Contaminants Encyclopedia Zinc Entry. National Park Service Water Resources Divisions, Water Operations Branch.

Ivask, A., Hakkila, K., Virta, M., 2001. Detection of organomercurials with sensor bacteria. Anal. Chem. 73: 5168-5171.

Kita-Tsukamoto, K., Oyaizu, H., Nanba, K., Simidu, U., 1993. Phylogenetic relationships of marine bacteria mainly members of the family Vibriobaceae determined on the basis of 16S rRNA sequences. Int. J. Syst. Bacteriol. 1: 8-19.

Kita-Tsukamoto, K., Yao, K., Kamiya, A., Yoshizawa, S., Uchiyama, N., Kogure, K., Wada, M., 2006. Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis. FEMS Microbiol. Lett. 256: 298-303.

Kudryasheva, N.S., 2006. Bioluminescence and exogenous compounds: Physico-chemical basis for bioluminescent assay. J. Photochem. Photobiol. B. 83: 77-86.

Łyżeń, R., Węgrzyn, G., 2005. Sensitivity of dark mutants of various strains of luminescent bacteria to reactive oxygen speices. Arch. Microbiol. 183: 203-208.

MacDonell, M.T. and Colwell, R.R., 1985. Phylogeny of the Vibrionaceae and recommendation for two new genera, Listonella and Shewanella. Syst. Appl. Microbiol. 6: 171-182.

Meighen, E.A., 1994. Genetics of bacterial bioluminescence. Annu. Rev. Genet. 28: 117-139.

Nealson, K.H. 1979. Alternative strategies of symbiosis of marine luminous fishes harboring light-emitting bacteria. North-Holland Biochemical Press 105-110.

Nealson, K.H., Wimpee, B. and Wimpee, C., 1993. Identification of Vibrio splendidus as a member of the planktonic luminous bacteria from the Persian Gulf and Kuwait region with luxA probes. Appl. Environ. Microbiol. 59: 2684-2689.

Ramesh, A.B., Loganathan, G., Venkateswaran, K., 1990. Ecological dynamics of marine luminous bacteria. J. Basic Microbiol. 30: 686-703.

Rees, J., De Wergifosse, B., Noiset, O., Dubbisson, M., Janssens, B., Thompson, E.M., 1998. The origin of marine bioluminescence: turning oxygen defense mechanisms into deep-sea communication tools. J. Exp. Biol. 201: 1211-1221.

Reichelt, J.L. and Baumann, P., 1973. Taxonomy of the marine luminous bacteria. Arch. Mikrobiol. 94: 283-330.

Ribo, J.M., Kaiser, K.L.E., 1987. Photobacterium phosphoreum, toxicity bioassay. Toxic. Assess. 2: 305-323.

Ruimy, R., Breittmayer, V., Elbaze, P., Lafay, B., Boussemart, O., Gauthier, M., Christen, R., 1994. Phylogenetic analysis and assessment of the genera Vibrio Photobacterium Aeromonas and Plesiomonas deduced from small-subunit rRNA sequences. Int. J. Syst. Bacteriol. 44: 416-426.

Semple, K.T., Doick, K.J., Jones, K.C., Burauel, P., Craven, A., Harms, H., 2004. Defining bioavailability and bioaccessibility of contaminated soil and sediment is complicated. Environ. Sci. Technol. 38: 228A-231A.

Shilo, M., Yetinson, T., 1979. Physiological Characteristics Underlying the Distribution Patterns of Luminous Bacteria in the Mediterranean Sea and the Gulf of Elat. Appl. Environ. Microbiol. 38: 577-584.

Simpson, M.L., Sayler, G.S., Fleming, J.T., Applegate, B., 2001. Whole-cell biocomputing. Trends Biotechnol. 19: 317-323.

Tamminen, M.V., Virta, M.P.J., 2006. Quantification of ecotoxicological tests based on bioluminescence using Polaroid film. Chemosphere. doi:10.1016/j.chemosphere.2006.07.034.

Tauriainen, S., Karp, M., Chang, W., Vitra, M., 1997. Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite. Appl. Environ. Microbiol. 63: 4456-4461.

van der Lelie, D., Regniers, L., Borremans, B., Provoost, A., Verschaeve, L., 1997. The VITOTOX test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics. Mutat. Res. 389: 279-290.

Wellauer, P.K., Reeder, R.H., Carroll, D., Brown, D.D., Deutch, A., Higashinakagawa, T., Dawid, I.B., 1974. Amplified ribosomal DNA from Xenopus laevis has heterogeneous spacer lengths. Proc. Natl. Acad. Sci. USA 71: 2823-2827.

Wilson, T., Hastings, J.W., 1998. Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14: 197-230.

Wimpee, C.F., Nadeau, T.L., Nealson, K.H., 1991. Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification. Appl. Environ. Microbiol. 57: 1319-1324.

Wood, K.V., DeLuca, M., 1987. Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase. Anal. Biochem. 161: 501-507.

陳秀娟. 1998. 污水中分離外分泌脂質酵素細菌的鑑定與其生理特性之研究. 國立臺灣師範大學生物研究所碩士論文.

邱秀慧. 2006. 台灣沿岸海洋弧菌之收集、分離與分類. 國立台灣大學海洋研究所博士論文.

王孟群. 1983. 實用微生物學實驗 pp. 60-124. 九州圖書公司.