摘要或動機

隨著迅速的工業化，重金屬汙染已是嚴重的環境問題。在植物中，當植物體內累積過量的重金屬，對於植物根部、葉部等器官的生長與發育有嚴重的影響或傷害。銅離子為植物生長所必須之重金屬，但是過量銅離子會導致細胞死亡，生長受到抑制。本實驗以大豆( Glycine max )及田菁( Sesbania roxburghii )為植物材料，藉由Evans blue 染色法、螢光染色、西方墨點法、反轉錄聚合?鏈鎖反應等，觀察過量銅離子影響植物根部生長、細胞死亡和細胞訊息傳遞物質變化之情形，並探討過量銅離子影響大豆根部細胞死亡的訊息傳遞路徑。過量銅離子會限制植物根部的生長及造成根部細胞死亡。以螢光染色觀察根尖(ROS, reactive oxygen species)、Ca²⁺累積情形，根尖細胞Ca²⁺、ROS 累積隨處理銅濃度的增加而上升，可能影響細胞死亡程度。以Ca²⁺螯合劑EGTA 和W-7(CDPK(calcium-dependent protein kinase)、Calmodulin 抑制劑)前處理發現可以降低過量銅離子對大豆根部的細胞死亡程度，推測Ca²⁺、CDPK 參與銅引發大豆根部細胞死亡的途徑。為檢驗MAPK 參與根部細胞死亡的途徑，以西方墨點法偵測根部細胞MAPK 的TEY 或TDY 磷酸化，實驗結果發現，隨著過量銅離子濃度的升高，田菁、大豆根部42-kDa MAPK磷酸化情形有上升之趨勢。以RT-PCR 分析大豆MAPK1 及MAPK2 基因之表現量，發現在銅處理時大豆之MAPK1 和MAPK2 基因的轉錄情形增加。大豆( Glycine max )及田菁( Sesbania roxburghii )皆屬於豆科植物，可作為綠肥植物。探討銅影響大豆、田菁細胞死亡之訊息傳遞路徑，希望進而控制生物體所受的毒害情形及訊息傳遞途徑，加強生物體對重金屬的防禦機制，未來可以以基因轉殖等基因工程技術，轉入抗重金屬基因或增強植物體對抗重金屬的能力等，作為綠肥植物、抗重金屬植物吸
附重金屬來復育土地達綠色淨化等用途。 Many heavy metals are necessary for plants, but excessive quantities directly affect plant growth and survival of organisms, cause cell death, or even affect human life indirectly. Cu (copper ion) is a heavy metal, which is one of micronutrients essential for normal growth and development of plants. The purpose of this experiment is to study the effect of excessive copper on Glycine max and Sesbania roxburghii root tips. I conducted some experiments by means of Evans blue staining (analysis of cell death), western blot analyses, and fluorescence microscope in order to examine the way copper results in plant death. Measurement of root length and analysis of cell death showed that excessive copper could bring about the inhibition of plant growth as well as cell death. With fluorescence microscope, I found that excessive copper might increase the level of copper-caused reactive oxygen species (ROS) in both the root tips of Glycine max and Sesbania roxburghii. In addition, I used Oregon Green 488 BAPTA-1 so as to assess the accumulation of calcium ions in root tips and found that the exposure of root tips to excessive copper results in the accumulation of calcium ions. To investigate whether calcium ions and calcium-dependent
protein kinase (CDPK) play a role in the cell death caused by excessive copper, I tested W-7, calmodulin and CDPK inhibitors, and EGTA, Ca²⁺ chelating agents, before copper treatment – immersing copper in CuCl2. In this way, plant cells would be effectively prevented from copper-caused death. Besides, to find out whether copper activates MAPKs in plant cells, I took advantage of western blot analysis with Phosphor-Map kinase Antibody and Map kinase Antibody. The
results revealed that excessive copper might lead to TEY or TDY motif phosphorylation of approximate 42- and 64-kDa MAPKs in the cells of Glycine max root-tip and approximate 42-kDa MAPKs in the cells of Sesbania roxburghii root-tip. Furthermore, with RT-PCR, I found that the transcription of Glycine max MAPK1 and MAPK2 mRNA happens more
frequently in root cells after copper treatment. In addition, this study suggested that the MAPK cascade CDPK pathway may function in the heavy-metal-signaling pathway in plant, and that calcium ions and ROS might get involved in the copper-caused death of plant cells. By studying signal transduction against heavy-metal toxicity in the plants, we can know how the organisms protect themselves. Sesbania roxburghiivv (or Glycine max), as green manure, could be used for metal-hyper-accumulator with the help of genetic engineering in the future.