超氧化物歧化酶 (superoxide dismutase, SOD),為催化超氧化物經由反應生成O2和H2O2的酵素,在植物生長發育過程及環境逆境反應中扮演重要角色。本研究利用甘藷台農57號為材料分析探討葉片SOD同功酵素表現型態及其與生長發育及環境逆境誘導的葉片老化之關聯性,結果顯示甘藷葉片可偵測到明顯的四條SOD 同功酵素分別為SOD1、SOD2、SOD3、及SOD4,利用H2O2、ethanol/chloroform、及銅離子螯合劑diethyldithiocarbamate (DDTC)處理顯示SOD1/SOD3/SOD4可能為Cu/Zn SOD同功酵素,而SOD2可能為Fe SOD同功酵素。甘藷SOD總活性及Cu/Zn SOD同功酵素含量於自然葉片老化過程顯著下降且呈負相關性。甘藷葉片分別處理DTTC、ethephon (釋放乙烯的化合物)、或NaCl逆境亦顯著減少SOD總活性及Cu/Zn SOD同功酵素含量,與誘導的葉片老化亦呈負相關性,外加CuSO4 (DTTC處理)或還原態抗壞血酸(reduced ascorbate) (ethephon或NaCl逆境處理)前處理可以拮抗抑制此影響,回復至DTTC、NaCl逆境、或ethephon單獨處理的結果。外加proteasome抑制劑MG132前處理亦可以顯著減緩ethephon或NaCl逆境下誘導的葉片老化、SOD總活性及Cu/Zn SOD同功酵素含量下降。根據這些結果顯示甘藷SOD總活性及Cu/Zn SOD同功酵素含量變化與發育過程、銅礦物元素缺乏、ethephon、及NaCl逆境下葉片老化過程呈負相關性。

Superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the superoxide radicle into the products of O2 and H2O2. In plants, it plays an important role in cope with developmental processes and environmental responses. In this research, sweet potato (cv. Tainong 57) leaves are used as the material to study its isoform patterns and their correlation with leaf senescence. There are four SOD isoforms identified as SOD1, SOD2, SOD3 and SOD4. The SOD1/SOD3/SOD4 and SOD2 are likely the Cu/Zn SOD and Fe SOD isoforms, respectively, based on the results of H2O2, ethanol/chloroform, and copper chelator diethyldithiocarbamate (DDTC) treatments. Sweet potato total SOD activity and Cu/Zn SOD isoform level are significantly decreased and exhibit a negative correlation with natural leaf senescence. Leaves treated with DTTC, ethephon (an ethylene-releasing compound), or NaCl stress also remarkably reduce their total SOD activity and Cu/Zn SOD isoform level and display a negative association with induced leaf senescence, which could be antagonized and repressed by the exogenous CuSO4 (for DTTC) and reduced ascorbate (ethephon or NaCl stress) pretreatments, respectively, and reverse it to the status of the DTTC, ethephon or NaCl stress alone. Pretreatment of exogenous proteasome inhibitor MG132 also significantly attenuates the ethephon and NaCl stress-mediated leaf senescence, decrease of total SOD activity and Cu/Zn SOD isoform level. Based on the results, sweet potato total SOD activity and Cu/Zn SOD isoform level exhibits a negative association with leaf senescence under developmental processes, copper mineral deficiency, ethephon, and NaCl stress.

壹、緒論 1
1.1 甘藷 1
1.2 葉片老化 1
1.3 乙烯 2
1.4 鹽分逆境 3
1.5 抗氧化物 4
1.6 超氧化物歧化酶 (SOD) 4
1.7 Proteasome 6
贰、研究材料和方法 8
實驗架構圖 8
2.1 實驗材料 9
2.11 不同發育階段葉片選取 9
2.2 實驗方法 9
2.2.1 甘藷葉片不同生長發育階段 9
2.2.2 鑒別SOD類型的染色定位法 10
2.2.3 外加DDTC及Cu2+回復處理 10
2.2.4 DDTC處理 11
2.2.5 DDTC及Cu2+回復處理 11 
2.2.6 Ethephon處理 12
2.2.7 Ethephon及抗壞血酸(ASA)處理 12
2.2.8 NaCl處理 13
2.2.9 NaCl及抗壞血酸(ASA)處理 13
2.2.10 Ethephon及MG132處理 14
2.2.11 NaCl及MG132處理 15
2.2.12 SOD活性染色 15
2.2.13 總SOD活性測定 16
2.2.14 H2O2含量測定 16
2.2.15 軟體image J分析 17
2.2.16 葉綠素含量的測定 17
2.2.17 光化學效率的測定 18
2.2.18 統計分析 19
2.2.19 藥品配製 20
叁、結果 22
3.1 自然老化之下SOD活性表現 22
3.2 SOD不同isoform 種類與區別 22
3.3 在DDTC處理下缺失銅離子所產生葉片老化過程中的SOD 同功酵素表現型態 23
3.4 在DDTC處理及外加銅離子回復下的SOD 同功酵素表現型態 24
3.5 在乙烯誘導的葉片老化下之SOD 同功酵素表現型態 25
3.6 在使用乙烯及ASA處理之下葉片中的SOD同功酵素表現型態 26
3.7 在鹽分逆境所產生葉片老化過程中的SOD同功酵素表現型態 27
3.8 在鹽分逆境及ASA處理所產生葉片老化過程的SOD同功酵素表現型態 28
3.9 在使用乙烯及MG132處理之下葉片中的SOD同功酵素表現型態 29
3.10 在鹽分逆境及MG132處理所產生葉片老化過程中的SOD同功酵素表現型態 29
肆、討論 32
參考文獻 35

圖一:Cu/Zn SOD在不同生長發育時期的活性表現 45
圖二:SOD不同isoform 種類與區別 47
圖三:Cu/Zn SOD在DDTC處理下缺失銅離子所產生葉片老化過程中的同功酵素表現型態 49
圖四:Cu/Zn SOD在DDTC處理下外加銅離子回復 51
圖五:Cu/Zn SOD在乙烯誘導的葉片老化下之同功酵素表現型態 53
圖六:Cu/Zn SOD在使用乙烯及ASA處理之下葉片中的同功酵素表現型態 55
圖七: Cu/Zn SOD在鹽分逆境所產生葉片老化過程中的同功酵素表現型態 57
圖八: Cu/Zn SOD在鹽分逆境及ASA處理所產生葉片老化過程的同功酵素表現型態 59
圖九: Cu/Zn SOD在使用乙烯及MG132處理之下葉片中的同功酵素表現型態 61
圖十:Cu/Zn SOD在鹽分逆境及MG132處理所產生葉片老化過程中的同功酵素表現型態 63

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