抗壞血酸過氧化酶 ( ascorbate peroxidase; APX ) 是穀胱甘肽-抗壞血酸循環的主要酵素之一，可利用還原態抗壞血酸清除活性氧族達到細胞內H2O2的動態平衡，因此抗壞血酸過氧化酶及抗壞血酸在生長發育過程與環境逆境適應扮演重要角色。本實驗室先前從甘藷葉片分離出一個ethephon可誘導之抗壞血酸過氧化酶 SPAPX，外加SPAPX融合蛋白或還原態抗壞血酸皆可延緩ethephon誘導的葉片老化過程。本研究繼續先前的成果探討甘藷抗壞血酸過氧化酶SPAPX及還原態抗壞血酸與NaCl逆境下葉片老化過程是否有關。以製備的anti-SPAPX多株抗體進行西方墨點雜交結果顯示，抗壞血酸過氧化酶SPAPX表現量在自然老化、ethephon或NaCl誘導的老化葉片中稍微減少，與葉片老化程度略呈負相關。NaCl逆境下會促進甘藷葉片老化過程，可由老化相關標幟 ( marker ) 的表現包括葉片外觀形態黃化、葉綠素和Fv/Fm含量下降、H2O2及MDA含量增加證實。外加SPAPX融合蛋白則呈劑量依賴性地延緩NaCl逆境下甘藷葉片老化過程，若將SPAPX融合蛋白外加前先以95℃或anti-SPAPX抗體使之不活化，則會廢止其影響，回復NaCl逆境下促進甘藷葉片老化過程。外加SPAPX融合蛋白並無顯著影響黑暗下甘藷葉片老化過程。外加還原態抗壞血酸亦呈劑量依賴性地延緩NaCl逆境下甘藷葉片老化過程。根據這些結果結論甘藷抗壞血酸過氧化酶SPAPX及還原態抗壞血酸可延緩NaCl逆境下甘藷葉片老化過程。也建議甘藷抗壞血酸過氧化酶SPAPX需要NaCl而非黑暗可誘導的交互作用因子與之協力合作，以延緩NaCl逆境下甘藷葉片老化過程。

Ascorbate peroxidase ( APX ) is one of the key enzymes of the glutathione-ascorbate cycle. It utilizes the reduced ascorbate to scavenge the reactive oxygen species and maintains the cellular H2O2 homeostasis. Therefore, it plays an important role in association with developmental response and stress adaptation. In our laboratory, an ethephon-inducible ascorbate peroxidase SPAPX has been cloned. Exogenous application of the SPAPX fusion protein and reduced ascorbate alleviate NaCl-promoted leaf senescence. In this research the main issue focuses on whether the cloned sweet potato SPAPX and reduced ascorbate can modulate NaCl-promoted leaf senescence. Western blot hybridization with the anti-SPAPX antibody showed that the level of ascorbate peroxidase SPAPX in natural, ethephon and NaCl-mediated senescent leaves was slightly reduced and negatively correlated with the level of leaf senescence. NaCl stress promoted leaf senescence demonstrated with senescence-associated markers, including leaf yellowing, reduced chlorophyll content and Fv/Fm level, elevated H2O2 and MDA amounts. Exogenous SPAPX fusion protein mitigated NaCl-promoted leaf senescence dose-dependently, which could be abolished and reversed by inactivation of the SPAPX fusion proteins using 95℃ and anti-SPAPX antibody pre-treatment. Exogenous SPAPX fusion protein did not significantly affect leaf senescence under the dark. Exogenous reduced ascorbate, however, also attenuated NaCl-promoted leaf senescence dose-dependently. Based on these results, sweet potato ascorbate peroxidase SPAPX and reduced ascorbate attenuate NaCl-promoted leaf senescence. It also suggests that ascorbate peroxidase SPAPX requires NaCl but not dark inducible interacting components for efficient attenuation of NaCl-mediated leaf senescence.

論文審定書………………………………………………………………… i
誌謝………………………………………………………………………… ii
中文摘要…………………………………………………………………… iii
英文摘要…………………………………………………………………… iv
目錄………………………………………………………………………… v
圖次………………………………………………………………………… viii
縮寫表……………………………………………………………………… x
壹、緒論…………………………………………………………………… 1
甘藷………………………………………………………………………… 1
葉片老化…………………………………………………………………… 1
NaCl逆境與葉片老化……………………………………………………… 2
乙烯與NaCl逆境………………………………………………………… … 4
穀胱甘肽–抗壞血酸循環…………………………………………………… 5
抗壞血酸過氧化酶與還原態抗壞血酸…………………………………… 6
研究動機與目的…………………………………………………………… 7
貳、實驗材料與方法……………………………………………………… 8
A. 實驗材料………………………………………………………………… 8
甘藷………………………………………………………………………… 8
相關基因、融合蛋白及抗體……………………………………………… 8
B. 實驗方法………………………………………………………………… 9
實驗流程…………………………………………………………………… 9
NaCl處理…………………………………………………………………… 10
SPAPX融合蛋白處理……………………………………………………… 10
還原態抗壞血酸處理……………………………………………………… 11
Ethephon、SPAPX融合蛋白及anti-SPAPX抗體處理………………… 12
葉綠素測量………………………………………………………………… 13
Fv/Fm測量………………………………………………………………… 13
DAB染色分析……………………………………………………………… 13
H2O2測量…………………………………………………………………… 14
MDA測量…………………………………………………………………… 14
SPAPX融合蛋白的誘導與純化…………………………………………… 15
SPAPX融合蛋白的誘導及萃取…………………………………………… 15
以Ni-NTA His*Bind Resin親和性管柱層析純化SPAPX融合蛋白……… 15
Anti-SPAPX抗體製備……………………………………………………… 16
葉蛋白質粗萃取…………………………………………………………… 17
蛋白質定量………………………………………………………………… 17
SDS-PAGE電泳分析……………………………………………………… 18
西方墨點雜交 ( Western blot hybridization ) …………………………… 20
Total RNA extraction……………………………………………………… 21
RT-PCR……………………………………………………………………… 23
PCR………………………………………………………………………… 23
Image J定量分析…………………………………………………………… 24
統計方法…………………………………………………………………… 24
參、結果…………………………………………………………………… 25
SPAPX融合蛋白的純化及anti-SPAPX多株抗體製備…………………… 25
甘藷抗壞血酸過氧化酶SPAPX表現量與自然的、ethephon或NaCl誘導的
葉片老化程度呈負相關性………………………………………………… 25
外加SPAPX融合蛋白延緩鹽分誘導的甘藷葉片老化…………………… 28
失活的SPAPX融合蛋白喪失延緩鹽分逆境誘導的甘藷葉片老化過程… 29
外加SPAPX融合蛋白亦延緩ethephon誘導的甘藷葉片老化…………… 30
外加SPAPX融合蛋白對黑暗誘導的甘藷葉片老化過程無顯著影響…… 31
外加還原態抗壞血酸延緩NaCl逆境誘導的甘藷葉片老化過程………… 31
肆、討論…………………………………………………………………… 33
NaCl逆境促進甘藷葉片老化過程及老化相關指標表現………………… 33
甘藷葉片抗壞血酸過氧化酶SPAPX表現量與自然葉片老化、ethephon或
NaCl逆境誘導的葉片老化程度呈負相關………………………………… 33
甘藷抗壞血酸過氧化酶SPAPX只有一個isoform………………………… 34
外加SPAPX融合蛋白顯著降低老化相關指標表現並且有效延緩NaCl逆境
誘導的甘藷葉片老化……………………………………………………… 34
SPAPX融合蛋白有效延緩ethephon誘導的甘藷葉片老化，可作為NaCl逆
境處理的正對照組………………………………………………………… 35
SPAPX需要與ethephon或NaCl可誘導的因子交互作用以延緩甘藷葉片老
化…………………………………………………………………………… 35
外加還原態抗壞血酸有效延緩NaCl逆境誘導的甘藷葉片老化………… 36
伍、參考文獻……………………………………………………………… 37

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