強光逆境下，植物對光的吸收大於其光合作用所能使用及排除的限度時，過多的能量會由氧氣接收，使細胞內產生大量的活性氧化物質 (reactive oxygen species, ROS)，造成細胞內大分子結構改變進而導致生理代謝及生長分化變異。本研究發現在 TAP 混營之可存活強光 (1,400 E m-2 s-1) 下，單胞藻 (Chlamydomonas reinhardtii, CC400) 在短時間會有ROS、 ascorbic acid (AsA) 含量與Ascorbate peroxidase ( APX ) 活性增加，並使其AsA/DHA 快速上升，單胞藻 CrAPX1 (CrAPX1; Cre02.g087700.t1.1)、CrAPX2 (CrAPX2; Cre06.g285150.t1.1)及CrAPX4 (CrAPX4; Cre05.g233900.t1.1)，在其強光反應下RNA表現量可見CrAPX4高於CrAPX1與CrAPX2。因此本研究先針對 APX4 對於單胞藻強光耐性之重要性進行探討，主要實驗以抑制表達 (downregulation)轉殖株探討強光耐性。抑制表達 CrAPX4 之轉殖株，使用之空載體為 pChlamyiRNA_3 vector，接上 PSAD::CrAPX-amiRNA。抑制表達轉殖株經強光處理 (1,400 E m-2 s-1) 後，APX活性下降進而造成ASA明顯增多，DHA於強光也可見其增多(可能是AsA直接與H2O2作用形成)。其存活率低於野生型，以2-thiobarbituric acid reacting substances (TBARS) 測定脂質過氧化程度含量高於野生型；綜合結果，缺乏 CrAPX 能夠會使單胞藻 APX活性下降，在強光下ROS無法有效的受APX去除，進而降低單胞藻對強光逆境的抗性。

When light absorption is higher than photosynthetic utilization under high light stress, excess energy will be accepted by O2 and in turn a large reactive oxygen species (ROS) produce, then leads to alteration of macromolecule structure and subsequently physiological and metabolic changes. This studydiscovered Chlamydomonas reinhardtii (CC400) can survive under 1,400 E m-2 s-1 , and ROS, ascorbic acid (AsA) contents, AsA/DHA ratio increase, and ascorbate peroxidase ( APX ) activity increased in short time. C. reinhardtii has CrAPX1 (CrAPX1;Cre02.g087700.t1.1), CrAPX2 (CrAPX2; Cre06.g285150.t1.1) and CrAPX4 (CrAPX4; Cre05.g233900.t1.1). Under the high light stress, the amount of RNA expression showed that CrAPX4 was higher than CrAPX1 and CrAPX2. The main experiment used the study of mutant strains inhibiting CrAPX4 to study the tolerance of high light stress.

The downregulation CrAPX4 transformants were made through the transformation of pClamyiRNA_3 vector by PSAD::CrAPX4-amiRNA. Uner high light condition (1,400 E m-2 s-1), the APX activity of downregulation transformants was significantly inhibited, and the ASA increased significantly. DHA also showed an increase in high light stress (AsA may be directly oxidized in response to H2O2 role).The survival rate is lower than that of the wild type, and the level of lipid peroxidation is higher than that of the wild. As a result, the downregulation of CrAPX4 can decrease the APX activity of C. reinhardtii. Under high light stress, ROS can not be effectively removed by decreased APX, which reduces the resistance of unicellular algae to high light stress.

誌謝 ................................ ................................ ................................ .............................. iii
摘要 ................................ ................................ ................................ ............................... iv
Abstract AbstractAbstract ................................ ................................ ................................ .......................... v
目錄 ................................ ................................ ................................ ............................... vi
圖目錄 ................................ ................................ ................................ ........................ viii viii
表目錄 ................................ ................................ ................................ ............................ x
壹、 前言 ................................ ................................ ................................ ............... 1
貳、 材料方法 ................................ ................................ ................................ ....... 5
一、 藻類栽培與處理.................................................................................... 5
二、 螢光分析細胞中活性氧化物質 (reactive oxygen species, ROS) 含量 6
三、 蛋白質含量分析.................................................................................... 7
四、 Ascorbate peroxidase (APX, EC 1.11.1.11) 之抽取及活性分析 ........ 7
五、 Ascorbic acid (AsA)/Oxidized AsA(DHA) 含量測定 ......................... 8
六、 脂質過氧化 (lipid peroxidation) 程度之測定 .................................... 9
七、 Total RNA 萃取 .................................................................................... 9
八、 cDNA 合成 ......................................................................................... 10
九、 Real-time PCR 測定 ........................................................................... 11
十、 CrAPX1, CrAPX2, CrAPX4 amiRNA 表達載體 (pChlamiRNA3-CrAPX1, pChlamiRNA3-CrAPX2, pChlamiRNA3-CrAPX4) 構建 ................................................................... 12
接合反應物成分為膠體純化之 pChlamyiRNA3、磷酸化處理之 ................ 15
十一、 藻類轉殖.......................................................................................... 18
十二、 藻類篩選.......................................................................................... 19
參、 結果 ................................ ................................ ................................ ............. 21
一、 強光 (1,400 E m-2 s-1) 對單胞藻 (Chlamydomonas reinhardti) 生理表現之影響 ............................................................................................. 21
二、 單胞藻 CrAPX 分子特性分析 .......................................................... 23
vii
三、 CrAPX1, CrAPX2, CrAPX4 抑制表達 (downregulation) 轉殖載體 (vector) 構築與篩選 .................................................................................. 25
四、 抑制表達轉殖株說明APX 是強光適應之因子............................... 26
肆、 討論 ................................ ................................ ................................ ............. 27
伍、 參考文獻 ................................ ................................ ................................ ..... 29

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