臺灣博碩士論文加值系統

植物界常見現象—「逆境共耐性」意指當植物體在面對一個短暫且不致命的逆境後，會對另一個逆境產生較好的防禦力。
衣藻細胞 Chlamydomonas reinhardtii 經過 40℃ 加熱兩小時後給予銅處理，並使用 H2DCFDA 為染劑，以流式細胞儀偵測細胞內 ROS 的含量，發現未熱處理的細胞會隨著銅濃度梯度產生大量 ROS，而事先經熱處理的細胞則可大幅降低此 ROS 的產生。以掃描式共軛焦顯微鏡，也觀察到相同結果。
為更深入探討熱處理是否透過一新合成蛋白質，使衣藻在遭受銅逆境時減少 ROS 產生，我們以 cycloheximide 進行前處理，發現熱處理降低 ROS 產生的效果消失。以 HSP70 單株抗體 3a3 進行 western blot，發現熱處理細胞的 HSP70 表現量較未熱處理細胞高。
此外，在我們的研究中也發現，事先熱處理可降低銅離子對光合作用活性的傷害，但卻無法挽回葉綠素含量的受損，同時細胞死亡程度也會增加。
綜合以上結果，雖然事先熱處理可誘發衣藻產生大量 HSP70，進而減少衣藻在銅逆境下 ROS 的產生，但細胞終究還是會走向死亡一途。說明熱逆境所產生的防禦力不足以使衣藻能夠完全地抵抗銅逆境的毒害。

摘要-------------------------------------------------------1
前言-------------------------------------------------------4
材料與方法--------------------------------------------8
結果-------------------------------------------------------14
討論-------------------------------------------------------18
圖表-------------------------------------------------------23
附表-------------------------------------------------------55
參考文獻------------------------------------------------59

Balczun C, Bunse A, Schwarz C, Piotrowski M, Kuck U. 2006. Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intronspecific RNA-binding protein. FEBS Letters 580: 4527-4532.
Berman SA, Wilson NF, Haas NA, Lefebvre PA. 2003. A novel MAP kinase regulates flagellar length in Chlamydomonas. Current Biology 12: 1145-1149.
Boston RS, Viitanen PV, Vierling E. 1996. Molecular chaperones and protein folding in plants. Plant Molecular Biology 32: 191-222.
Boswell C, Sharma NC, Sahi SV. 2002. Copper tolerance and accumulation potential of Chlamydomonas reinhardtii.
Bulletin of Environmental Contamination and Toxicology 69: 546-553.
Button KS, Hostetter HP. 1977. Copper sorption and release by Cyclotella meneghiniana (Bacillariophyceae) and Chlamydomonas reinhardtii (Chlorophyceae). Journal of Phycology 13: 198-202.
Chen PY, Lee KT, Chi WC, Hirt H, Chang CC, Huang HJ. 2008. Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants. Planta 228: 499-509.
Eisenberg-Domovich Y, Kloppstech K, Ohad I. 1994. Reversible membrane association of heat-shock protein 22 in Chlamydomonas reinhardtii during heat shock and recovery. European Journal of Biochemistry 222: 1041-1046.
Franklin NM, Adams MS, Stauber JL, Lim RP. 2001. Development of an improved rapid enzyme inhibition bioassay with marine and freshwater microalgae using flow cytometry. Archives of Environmental Contamination and Toxicology 40: 469-480.
Garvey JE, Owen HA, Winner RW. 1991. Toxicity of copper to the green alga, Chlamydomonas reinhardtii (Chlorophyceae), as affected by humic substances of terrestrial and fresh water origin. Aquatic Toxicology 19: 89-96.
Gerloff-Elias A, Barua D, Molich A, Spijkerman E. 2006. Temperature- and pH-dependent accumulation of heat-shock proteins in the acidophilic green alga Chlamydomonas acidophila. FEMS Microbiology Ecology 56: 345-354.
Hoffmann JH, Linke K, Graf PC, Lilie H, Jakob U. 2004. Identification of a redox-regulated chaperone network. The EMBO Journal 23: 160-168.
Jamers A, Van der Ven K, Moens L, Robbens J, Potters G, Guisez Y, Blust R, De Coen W. 2006. Effect of copper exposure on gene 61 expression profiles in Chlamydomonas reinhardtii based on microarray analysis. Aquatic Toxicology 80: 249-260.
Jimenez C, Berl T, Rivard CJ, Edelstein CL, Capasso JM. 2004. Phosphorylation of MAP kinase-like proteins mediate the response of the halotolerant alga Dunaliella viridis to hypertonic shock. Biochimica et Biophysica Acta-Molecular Cell Research 1644: 61-69.
Juneau P, El Berdey A, Popovic R. 2002. PAM fluorometry in the determination of the sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper. Archives of Environmental Contamination and Toxicology 42: 155-164.
Lage OM, Sansonetty F, O'Connor JE, Parente AM. 2001. Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae. Cytometry 44: 226-235.
Liu YD, Ren DT, Pike S, Pallardy S, Gassmann W, Zhang SQ. 2007. Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogenactivated protein kinase cascade. Plant Journal 51: 941-954.
Luis P, Behnke K, Toepel J, Wilhelm C. 2006. Parallel analysis of transcript levels and physiological key parameters allows the 62 identification of stress phase gene markers in Chlamydomonas reinhardtii under copper excess. Plant Cell and Environment 29: 2043-2054.
Macfie SM, Welbourn PM. 2000. The cell wall as a barrier to uptake of metal ions in the unicellular green alga Chlamydomonas reinhardtii (Chlorophyceae). Archives of Environmental Contamination and Toxicology 39: 413-419.
Muller FW, Igloi GL, Beck CF. 1992. Structure of a Gene Encoding Heat-Shock Protein Hsp70 from the Unicellular Alga Chlamydomonas reinhardtii. Gene 111: 165-173.
Nishikawa K, Yamakoshi Y, Uemura I, Tominaga N. 2003. Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism. FEMS Microbiology Ecology 44: 253-259.
Pospisil P. 2009. Production of reactive oxygen species by photosystem II. Biochimca et Biophysica Acta 1787: 1151-1160.
Prasad MNV, Drej K, Skawinska A, Strzalka K. 1998. Toxicity of cadmium and copper in Chlamydomonas reinhardtii wild-type (WT 2137) and cell wall deficient mutant strain (CW 15). Bulletin of Environmental Contamination and Toxicology 60: 667-667.
Schroda M, Kropat J, Oster U, Rudiger W, Vallon O, Wollman FA, Beck CF. 2001. Possible role for molecular chaperones in assembly and repair of photosystem II. Biochemical Society Transactions 29: 413-418.
Schulz-Raffelt M, Lodha M, Schroda M. 2007. Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas. Plant Journal 52: 286-295.
Shao N, Beck CF, Lemaire SD, Krieger-Liszkay A. 2008. Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii. Planta 228: 1055-1066.
von Gromoff ED, Treier U, Beck CF. 1989. Three light-inducible heat shock genes of Chlamydomonas reinhardtii. Molecular and Cell Biology 9: 3911-3918.
Wang WX, Dei RC. 2006. Metal stoichiometry in predicting Cd and Cu toxicity to a freshwater green alga Chlamydomonas reinhardtii. Environmental Pollution 142: 303-312.
Yu Y, Kong FX, Wang ML, Qian LL, Shi XL. 2007. Determination of short-term copper toxicity in a multispecies microalgal population using flow cytometry. Ecotoxicology and Environmental Safety 66: 49-56.