水稻幼苗於不同淹水程度下之生理特性及NADPH氧化酶表現分析

隨著全球氣候改變，造成世界各地不同的自然災害。而豪大雨所造成的淹水逆境，使許多作物的產量下降或直接造成死亡。已知在淹水逆境下會產生活化氧族 (reactive oxygen species, ROS)之累積，而NADPH氧化酶為活化氧族來源之一。本實驗室先前以模式植物阿拉伯芥研究中指出NADPH氧化酶參與植物淹水逆境下之調控機制，因此本研究以水稻台稉九號為材料，觀測在不同淹水程度下，水稻之生理及分子變化。實驗結果顯示，於半淹或全淹處理下台稉九號幼苗地上部高度皆增加，且第三葉鞘長度快速增加。葉綠素含量則隨淹水時間增加而下降，又以全淹處理之葉綠素含量下降幅度更為明顯。DAB和NBT染色結果顯示，不同淹水程度下，過氧化氫和超氧陰離子均在葉尖累積，又以全淹處理累積程度較明顯。Evans blue染色結果呈現，全淹處理下對細胞之損害程度較半淹處理明顯。抗氧化酵素活性分析結果顯示，過氧化氫酶、抗壞血酸過氧化酶及超氧化物歧化酶活性於淹水六天和回氧一天後皆下降，且抗壞血酸過氧化酶活性於全淹處理較半淹處理明顯下降。過氧化物酶無論是否回氧活性皆明顯上升。即時定量聚合酶鏈鎖反應結果顯示，不論半淹或全淹處理下，OsRbohH及I表現量皆上升，而OsRbohA及G表現量皆下降。OsRbohC、D及F表現則於半淹處理上升而於全淹處理則下降。顯示水稻Rboh基因成員參與不同程度淹水逆境之調控。為進一步釐清淹水下，缺氧訊號傳遞路徑與滲透壓逆境之差異，以即時定量聚合酶鏈鎖反應結果顯示，不論半淹或全淹處理下，OsRbohA、G、H及I基因於淹水下外加300 mM氯化鈉及300 mM甘露醇處理下，表現量皆較淹水處理高。綜合以上實驗結果顯示，於不同淹水程度下，活化氧族之過氧化氫可能調控台稉九號幼苗之生理反應，及參與缺氧訊號途徑下相關基因之分子調控，且於淹水逆境施加滲透壓物質，透過OsRbohs之表現得知缺氧訊號與滲透壓訊號具有「crosstalk」之關係。Many nature disasters were due to climate change in the world, such as heavy rain resulting in reduced crop yield or death. Many stresses cause reactive oxygen species (ROS) accumulation in plant such as flooding stress. The plasma membrane NADPH oxidase (respiratory burst oxidase homologs, Rbohs) is a main source of H2O2 in cells. Previous studies display AtRbohs involving in regulation mechanism under flooding stress. However, little is known about the regulation mechanism of rice Rbohs under flooding stress. In this study, we characterized the physiological response and the transcript profiles of Rbohs under different levels of flooding treatments in rice (Oryza sativa L. japonica, Tai-keng 9). The plants height was significant increased under partial submergence (PS) and full submergence (FS) compared to control. The 3rd leaf sheath plays an important role during plant growth under different levels of flooding stress. The contents of total chlorophyll and chlorophyll a, b were decreased with the increase in the flooding days; especially in FS treatment was shown significant reduced compare with control. DAB and NBT staining were shown that the accumulation of H2O2 and O2- in leaves tip under FS treatment were higher than PS treatment. The antioxidant activity was shown that CAT, SOD, APX activity were decreased under FS and PS treatment for 6 days or recovery for 1 day. The peroxidase activity was presented significant increased under FS and PS treatment for 6 days or recovery for 1 day. The qRT-PCR results were revealed that the transcript levels of OsRbohH and I were increased; OsRbohA and G were decreased in PS and FS conditions. The levels of OsRbohC, D and F were increased under PS condition, and decreased under FS condition. The NADPH oxidase were involved in hypoxia signaling pathway under different levels of flooding stress. Furthermore, the transcript levels of OsRbohA, G, H and I were increased under submergence combined with 300 mM NaCl or 300 mM Mannitol compare with submergence only. Taken together, our results present that under different levels of submergence, the effect on the length of plants, the contents of total chlorophyll and the activity of antioxidants. The transcription profiles of 9 Rbohs in TK9 under different levels of submergence were characterized. This studies also providing evidence of differences between hypoxia-signaling and osmotic-stress signaling, and of cross-talk between them.中文摘要………………………….…….………………….…………………..i 英文摘要………………………………….….……………………………..….ii 目錄………………………………………………………………………..…..iv 壹、 前言…………...……………………...…………………….…...………...1 一、 水稻之耐淹水機制…………………………………………………….….2 二、 淹水逆境下作物之生理代謝反應…………………………………….….4 三、 植物NADPH氧化酶參與逆境之反應……………..………….........……7 四、 滲透壓逆境對作物生長發育之影響……………………….……...……10 貳、 材料方法 一、 水稻種子消毒及幼苗生長條件……………………………….………..12 二、 幼苗淹水處理………………………………………………….………..12 三、 幼苗滲透壓逆境之處理……………………………………….………..12 四、 幼苗株高及葉鞘長度之測量…………………………………….……..13 五、 葉綠素含量之測量…………………………………………….....……..13 六、 DAB染色………………………………………………………….……14 七、 NBT染色…………………………………………………………….….15 八、 Evans blue 染色…………………………………………………….…. 16 九、 抗氧化酵素測定………………………………………………………...17 十、 Rboh基因同源樹狀圖………………………………………….……….19 十一、 總RNA之萃取………………………………………………….….20 十二、 DNase處理………………………………………………………..…20 十三、 第一股cDNA之合成 (reverse transcription, RT)……………….…20 十四、 即時定量聚合酶連鎖反應分析 (real-time quantitative polymerase chain reaction, qRT-PCR)……………………………………………......21 參、 結果 一、 台稉九號於不同淹水程度下之株高及葉鞘長度變化………...........…22 二、 台稉九號幼苗於全淹處理下葉綠素含量下降更為顯著……..........….23 三、 台稉九號幼苗於全淹水逆境下具較多活化氧族累積………....……...24 四、 台稉九號幼苗於淹水逆境下抗氧化酵素活性測定………….........…..25 五、 水稻NADPH氧化酶於不同淹水程度下之表現………………..….…..27 六、 水稻NADPH氧化酶於滲透壓逆境下之表現………………………….28 肆、 討論 一、 不同程度淹水逆境下台稉九號幼苗之生理反應……………...……….30 二、 淹水逆境下台稉九號幼苗葉綠素含量之影響……………...………….30 三、 不同程度淹水逆境下台稉九號幼苗氧化平衡之影響…………............31 四、 水稻Rboh基因參與淹水逆境……………………………...…………...32 伍、 參考文獻………………………………………………………...………35 陸、 圖表………………………………………………………………...……4