高溫逆境會使植物產生熱逆境反應。熱逆境反應使熱休克蛋白質的表現量提升，保護植物內重要的生理反應免於受到熱逆境的傷害外，也可讓氣孔開啟，使植物利用蒸散作用散熱。在乾旱、低溫或高鹽逆境下，植物體內會累積大量的ABA，引發由ABA調節的逆境反應。本實驗室以microarray與RT-PCR結果鑑定出在水稻葉片中受熱與ABA共同調控的基因— OsRZFP34。為深入研究熱與ABA逆境在阿拉伯芥中的反應機制，利用胺基酸序列比對，由阿拉伯芥基因庫中找到OsRZFP34的同源基因— At5g22920，命名為AtRZFP33；AtRZFP33具有RING鋅手指結構，被歸類為鋅手指蛋白質家族。首先以quantitative RT-PCR分析發現，AtRZFP33受熱與ABA抑制；以RT-PCR分析也發現AtRZFP33主要表現在植株的地上部。由AtRZFP33-GFP在洋蔥表皮細胞中的表現情形發現結合蛋白質表現在細胞核與細胞質中。我們利用AtRZFP33的T-DNA插入突變株—SALK_017562，分析其在各種逆境下的抗性。在種子時期，atrzfp33突變株對於高溫較為敏感；在幼苗時期則發現在ABA及mannitol逆境下，atrzfp33突變株具有較佳的耐受性；另一方面，我們也證實在低溫逆境或水分散失速率實驗中，atrzfp33突變株的抵抗能力較佳，推測AtRZFP33可能與氣孔調節有關。觀察植株的氣孔開闔情形後發現，atrzfp33突變株的氣孔打開比例較野生型低。進一步觀察AtRZFP33過量表現轉殖株，發現轉殖株的氣孔打開比例較野生型高約20%，證實AtRZFP33確實參與在調控氣孔開闔的機制中。另外，我們也將AtRZFP33於nced3、abi1、abi2突變株中過量表現，發現nced3/AtRZFP33OX與abi2/AtRZFP33OX轉殖株中的氣孔開啟程度分別較nced3、abi2突變株高；但在abi1/AtRZFP33OX轉殖株中，其氣孔開啟程度與abi1突變株的差異並不明顯，推測AtRZFP33可能藉由調節abi1，達到控制氣孔開啟的目的。以quantitative RT-PCR分析與氣孔相關之離子通道蛋白KAT1、OST2與MRP5的表現情形，發現在atrzfp33突變株中，這些基因的表現量下降25%~50%，在過量表現轉殖株中的表現量上升50%左右。我們證實AtRZFP33可能參與在由熱與ABA共同調節的訊息傳遞路徑中，藉由調節氣孔開闔的機制使植物處於多重逆境下，仍能維持體內的生理平衡。

Plants respond to evaluated temperature by inducing the heat shock response. The heat shock response (HSR) protects plants from cellular damage not only by accumulation of heat shock protein, but also by accelerated cooling down of biological system mediated by stomatal opening. The plant hormone abscisic acid (ABA) normally accumulates in response to drought, cold and salt stresses. Previous study indicated that rice OsRZFP34 was identified as a heat- and ABA- responsive gene according to analyses of microarray expression profile and reverse transcriptase polymerase chain reaction (RT-PCR). The complex cascades of gene expression in various stresses can contribute to stress response, which further enhances the stress tolerance. To identify the crosslink between high temperature and ABA-related response in Arabidopsis, we characterized the Arabidopsis At5g22920, also named AtRZFP33, which is a homolog of rice OsRZFP34. AtRZFP33 belongs to zinc finger subfamily, containing a deduced RING zinc finger signature. Using quantitative RT-PCR to analyze tissue specificity and expression pattern of AtRZFP33, we found that AtRZFP33 was significantly reduced by heat and ABA treatments. Although zinc finger protein is usually characterized as a transcription factor, subcellular localization of AtRZFP33-GFP in onion epidermal cells showed that AtRZFP33 was localized not only in nucleus but in the cytosol. To study the role of AtRZFP33 in resistance to stresses, we examined the loss of function of AtRZFP33 in a T-DNA insertion mutant, SALK_017562. Thermotolerance was decreased in atrzfp33 mutant line in seed stage; in seedling, we found atrzfp33 mutant line was resistant to drought and chilling stresses, resulting from its stomatal closure. The stomatal aperture was smaller in atrzfp33 mutant line, but 20% higher in transgenic plants overexpressing AtRZFP33 relative to wild type. It reflects that AtRZFP33 may play a negative role in stomatal closure controlling mechanism. Using quantitative RT-PCR to analyze the expression of ion channels related to stomata opening, we found that KAT1, OST2 and MRP5,were upregulated in atrzfp33 mutant, and downregulated in overexpression plants. Moreover, we transformed 35S::AtRZFP33 construct into nced3, abi1 and abi2 mutant lines. Stomatal aperture of nced3/AtRZFP33OX and abi2/AtRZFP33OX were higher than single mutants, but there was no difference between abi1/AtRZFP33OX and its single mutant. Collectively, these results indicated that AtRZFP33 may functions in heat and ABA stress response by regulating the stomatal mechanism.

摘要 ........ III
縮寫對照表 .......................... V
Abstract....... VI
壹、緒論............ 1
植物與環境逆境.........1
熱休克（Heat shockHeat) 反應................ 2
離層酸（Abscisic acid)反應 .... 2
氣孔的調節............... 4
RING(Ring Interesting New Gene）鋅手指蛋白質....... 5
OsRZFP34 .... 6
研究起源與目的....................... 7
貳、材料與方法............................ 9
一、基因表現分析 ............... 9
二、AtRZFP33蛋白質在細胞中的表現位置 ........ 12
三、阿拉伯芥農桿菌轉殖...................... 17
四、植株生理功能分析........................ 19
叁、結果................................ 22
AtRZFP33在阿拉伯芥中的表現部位 ............... 22
AtRZFP33突變株的鑑定 ............. 23
突變株之逆境抗性分析........................ 24
AtRZFP33過量表現轉殖株之鑑定 .................. 26
轉殖株之氣孔開闔分析....................... 27
基因表現量之分析 .......................... 30
肆、討論 .................................... 32
AtRZFP33在阿拉伯芥的地上部表現且受高溫與ABA抑制表現..... 32
AtRZFP33-GFP結合蛋白質表現在細胞質與細胞核中.................... 33
AtRZFP33之生理功能分析 ..................... 33
水稻OsRZFP34與AtRZFP33之生理功能相似...................... 36
AtRZFP33參與的訊息傳遞路徑 ................. 37
未來研究方向.............................. 37
伍、參考文獻 ........................... 39

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