利用ethephon處理甘藷分離的葉片會導致SPCP3表現 (Chen et al., 2006)、葉綠素含量及Fv/Fm值下降、H2O2量增加及細胞死亡、葉片快速老化。外加還原態glutathione、EGTA或cycloheximide等調節物質可減緩ethephon處理所導致的葉片老化及細胞死亡影響。此部份研究結果顯示氧化性逆境、鈣離子的流入及新合成蛋白質會影響ethephon誘導葉片老化及細胞死亡的效應。Ethephon誘導葉片老化及細胞死亡過程，granulin-containing cysteine protease SPCP3亦受它所誘導，因此本研究利用阿拉伯芥轉殖植株探討SPCP3可能扮演的生理功能與角色。實驗結果發現外源表現SPCP3於轉殖植株導致植株提早開花、開花時含較少的rosette leaves、較高的黃化果莢百分率及較低的種子發芽率。在乾旱實驗亦發現轉殖植株的Fv/Fm值和相對含水量明顯減少、H2O2含量增加，並且有較高程度的細胞死亡。此結果顯示外源表現SPCP3改變轉殖植株發育特徵與對乾旱逆境敏感性。先前研究指出granulin-like domain在酵素活性調控上扮演重要的角色 (Yamada et al., 2001)。因此本研究預先移除SPCP3的granulin-like domain，結果發現對於轉殖植株在乾旱逆境敏感性的反應無顯著影響。根據這些結果結論氧化逆境、鈣離子的流入及新合成蛋白質參與乙烯訊息傳導，活化功能性基因SPCP3表現及葉片老化，且外源表現SPCP3導致轉殖植株發育特徵與對乾旱逆境敏感性改變，granulin-like domain存在與否對外源表現SPCP3造成對乾旱敏感性的生理功能似乎無太大影響。

Ethephon treatment caused SPCP3 gene expression (Chen et al., 2006), reduction of chlorophyll content, decrease of Fv/Fm value, increase of H2O2 amount, and more cell death, and accelerated leaf senescence in detached sweet potato leave. Exogenous application of modulators such as reduced glutathione, EGTA or cycloheximide delay leaf senescence and cell death caused by ethephon. These data suggest that oxidative stress, calcium influx and de novo synthesized protein may influence ethephon-mediated leaf senescence and cell death. When ethephon induced leaf senescence and cell death, granulin-containing cysteine protease SPCP3 gene was induced. Transgenic Arabidopsis system was used to explore the possible physiological role and function of SPCP3. The results showed that ectopic expression of SPCP3 in transgenic Arabidopsis plants caused earlier flowering, less rosette leaves when flowering, higher yellowing silique percentage during harvest, and lower germination percentage than that in control. During drought treatment, transgenic plants also exhibited reduction of Fv/Fm value and relative water content, but an increase in H2O2 content and cell death. These data suggest that ecopic expression of SPCP3 caused altered developmental characteristics and drought stress sensitivity. Previous report suggests that granulin-like domain may play a role in regulating enzymatic activity of granulin-containing cysteine protease (Yamada et al., 2001). In this report we demonstrate that pre-removal of granulin-like domain of SPCP3 does not affect significantly drought stress sensitivity compared to full-length SPCP3 in transgenic Arabidopsis plants. Based on these data we conclude that oxidative stress, calcium influx, and de novo synthesized proteins may be involved in ethylene signaling leading to leaf senescence and SPCP3 gene expression in detached sweet potato leaves, and ectopic SPCP3 expression in transgenic Arabidopsis plants caused altered developmental characteristics and enhanced drought sensitivity. Granulin-like domain may have no significant influence on SPCP3-mediated effect on drought stress sensitivity.

壹、
緒論……………………………………………………… 1
甘藷……………………………………………………… 1
阿拉伯芥……………………………………………… 2
葉片細胞老化生理……………………………………… 2
細胞程式死亡…………………………………………… 4
乙烯的生理功能研究…………………………………… 7
Granulin-containing半胱胺酸蛋白分解酶 ………… 9
貳、材料與方法………………………………………… 14
A.實驗材料……………………………………………… 14
B.實驗方法……………………………………………… 15
I、調節物(modulator)對ethephon誘導甘藷葉片老化及細胞死亡的影響……… 15
Ethephon的處理………………………………… 16
調節物的前處理…………………………………… 17
Fv/Fm測量 ……………………………………… 18
葉綠素含量測量 ………………………………… 18
DAB染色分析 ……………………………………18
Evans blue染色分析…………………………… 19
II、利用轉殖阿拉伯芥探討甘藷半胱胺酸蛋白酶SPCP3的生理功能…………………………………………… 19
(A) 轉殖阿拉伯芥植株發育性狀定量分析…………19
對照組及轉殖植株之相對開花天數測量…………20
對照組及轉殖植株於開花時之rosette leaves數
目測量……………………………………………20
對照組及轉殖植株之黃化果莢百分比測量……20
對照組及轉植植株採收時葉片之葉綠素含量測量20
(B) 對照組及轉殖後代種子發芽性狀分析……………21
無菌培養基配製…………………………………22
種子發芽率、發芽曲線及發芽速率的測量………22
(C) 轉殖阿拉伯芥植株耐乾旱逆境分析……………23
Fv/Fm測量 ………………………………………23
相對含水量測量………………………………… 24
DAB染色分析……………………………………… 24
Evans blue染色分析………………………………24
轉殖阿拉伯芥植株genomic PCR分析…………… 25
RT-PCR分析……………………………………28
西方墨點雜交分析………………………………… 33
参、結果……………………………………………… 41
I、Ethephon誘導分離的甘藷葉片老化及細胞死亡受還原態 glutathione、EGTA及cycloheximide影響 ……41
ethephon誘導分離甘藷葉片老化及細胞死亡 ……41
還原態glutathione處理減緩ethephon誘導分離甘藷葉片的老化及細胞死亡…………………………………．42
EGTA處理減緩ethephon誘導分離甘藷葉片的老化及細胞死亡…………………………………………………43
Cycloheximide處理減緩ethephon誘導分離甘藷葉片的老化及細胞死亡……………………………………．44
II、甘藷半胱胺酸蛋白分解酶SPCP3於轉殖阿拉伯芥45
植株之功能性研究外源表現SPCP3改變轉殖阿拉伯芥植株發育性狀………………………………………… 45
外源表現SPCP3降低轉殖阿拉伯芥種子存活率……47
外源表現SPCP3增加轉殖阿拉伯芥植株對乾旱逆境的敏感性……………………………………………………47
外源表現含或不含granulin-like domain之SPCP3對轉殖阿拉 伯芥植株於乾旱逆境的敏感性無顯著影響…49
肆、討論……………………………………………… 52
參考文獻……………………………………………… 62

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