本研究探討高鹽下裂片石蓴 (Ulva fasciata Delile) ubiquitin、20S proteasome beta subunit type 1 (20sβ1)、ubiquitin-conjugating enzyme e2 (ucee2)、ATP-dependent caseinolytic protease regulatory subunit (clpC) 基因表現與蛋白質氧化之關係。Ulva fasciata在高鹽 (90‰) 0-1小時，細胞含水量、TTC (2,3,5-tripheny tetrazolium chloride) 還原能力與caseinolytic protease activity保持不變，但高鹽後回復正常鹽度下生長能力受50%抑制且其光合作用速率 (PS II) 降低並產生大量H2O2含量，推測此時有氧化逆境產生而ubiquitin基因表現增高。在1-3小時間，細胞開始失水 (損失約正常細胞水分的33%)，TTC還原能力、及光合作用速率 (PS II) 活性下降，H2O2含量增加更多而受氧化蛋白質 (其正常蛋白質含量約為起始點的20%)，20sβ1、clpC基因表現及caseinolytic protease activity增加，clpC基因表現量在3小時達最大值。6-48小時期間，細胞失水嚴重 (損失約正常細胞水分的66%) 且6、12小時形成大量的游離氨基酸 (增加量約起始點的85%)，而高鹽後回復正常鹽度下失去生長能力證實細胞已無法維持正常生理，ubiquitin、20sβ1及ucee2基因在此時的大量表現指出這些基因可能參與長期高鹽逆境造成之計畫性細胞死亡過程有關。以ROS (reactive oxygen species) 清除劑抑制高鹽ROS形成 (12 小時處理) 抑制蛋白質氧化 (carbonyl compounds) 傷害、caseinolytic proteolytic activity、及ubiquitin、20sβ1及ucee2、clpC基因表現之結果證實ROS與高鹽之protease 基因表現有關。多元胺也參與protease 基因表現之調節，但不同protease 基因不同種類多元胺的影響spermidine 及spermine 抑制ubiquitin、20sβ1及ucee2、及蛋白質氧化 (carbonyl groups) 傷害、caseinolytic protease activity，putrescine抑制clpC表現及蛋白質氧化 (carbonyl compounds) 傷害、caseinolytic protease activity卻反而增加ubiquitin、20sβ1及ucee2表現量增加。所以，裂片石蓴經由ROS及多元胺調節clpC基因表現而增加caseinolytic protease activity進行葉綠體因高鹽形成氧化蛋白質的分解。ubiquitin、20sβ1及ucee2基因可能參與長期高鹽逆境造成之計畫性細胞死亡過程有關，但須加以證明。

This study has investigated the gene expression of ubiquitin、20S proteasome beta subunit type 1 (20sβ1)、ubiquitin-conjugating enzyme e2 (ucee2)、ATP-dependent caseinolytic protease regulatory subunit (clpC) in the marine macroalga Ulva fasciata Delile in relation to the hypersalinity-induced oxidative stress and protein oxidation. During the early stage (0-1 h), the water contents and TTC (2,3,5-tripheny tetrazolium chloride) reduction ability maintained unchanged but recovery ability and photosynthetic ability (PS II activity as indicated by Fv/Fm) were decreased along with accumulated H2O2, suggesting the occurrence of oxidative stress. Only ubiquitin expressed at this stage. During 1-3 h, water lost (approximately 33% of the control) with a further decrease in recovery ability, TTC reduction ability、PS II activity but more H2O2 accumulation and protein carbonyl compound. The transcripts of 20sβ1 and clpC and caseinolytic protease activity increased at this stage with the maximum of clpC at hour 3. In the 6-48 h, water lost seriously with high accumulated free amino acid at 6-12 h but low recovery ability. The transcript amounts of ubiquitin、20sβ1 and ucee2 increased marked during this stage, in which these might be related to programmed cell death caused by long-term exposure to hypersalinity. Reactive oxygen species (ROS) scavengers inhibited H2O2 accumulation, caseinolytic proteolytic activity increase, carbonyl compound formation and gene expression of ubiquitin、20sβ1、ucee2、clpC, indicating a role of ROS in the regulation of protease genes. A role of polyamines in the regulation of protease gene expression was tested. Spermidine and spermine inhibited the gene expression of ubiquitin、20sβ1 and ucee2, the oxidation of proteins (carbonyl groups) and the induction of caseinolytic protease activity in 90‰-treated thalli, whereas putrescine inhibited clpC expression, the oxidation of proteins and caseinolytic protease activity but enhanced the gene expression of ubiquitin、20sβ1 and ucee2. In conclusion, the results of the present investigation show that the degradation of oxidatively damaged proteins under hypersalinity conditions by increased caseinolytic protease activity is driven by the up-regulation of clpC gene expression via ROS and polyamines. It seems likely that the induction of ubiquitin、20sβ1 and ucee2 gene expression might be associated with the hypersalinity-mediated programmed cell death.

謝辭-------------------------------------------------- i
中文摘要---------------------------------------------- ii
英文摘要---------------------------------------------- iv
目錄-------------------------------------------------- vi
圖目錄------------------------------------------------ vii
表目錄------------------------------------------------ x
一、前言---------------------------------------------- 1
二、材料與方法---------------------------------------- 6
三、結果---------------------------------------------- 21
四、討論---------------------------------------------- 67
參考文獻---------------------------------------------- 73
附錄-------------------------------------------------- 81

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