分析水稻 RING 鋅手指蛋白質 OsRZFP34 與其正向調控蛋白質之交互作用;Analysis of the interaction between OsRZFP34, a rice RING zinc-finger protein, with its up-regulated proteins

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Title:	分析水稻 RING 鋅手指蛋白質 OsRZFP34 與其正向調控蛋白質之交互作用;Analysis of the interaction between OsRZFP34, a rice RING zinc-finger protein, with its up-regulated proteins
Authors:	連珮君;Lian,Pei-Jyun
Contributors:	生命科學系
Keywords:	RING 鋅手指蛋白質;水稻 RING 鋅手指蛋白質34;熱逆境;離層酸;氣孔開闔;蛋白質交互作用;RING zinc finger proteins;OsRZFP34;Heat Stress;ABA;Stomatal movement;Protein-protein interaction
Date:	2015-05-27
Issue Date:	2015-07-30 18:45:17 (UTC+8)
Publisher:	國立中央大學
Abstract:	RING 鋅手指蛋白質 (RZFPs) 在植物生長過程、生物及非生物逆境調控上扮演重要的角色，實驗室先前依據 oligo microarray 實驗篩選出水稻中受高溫及 ABA 誘導的 RZFP 基因，我們稱之為 OsRZFP34。此外，在生理功能研究上發現 OsRZFP34可能參與蒸散冷卻作用並且具有調控 Ca2+ 感應、K+ 調節、ABA 反應等與水稻氣孔開啟過程相關基因表現的功能 (Hsu et al., 2014)。因此我們進一步探討 OsRZFP34 與攜鈣素結合蛋白質 OsCaMBP (RAP-DB 基因編號：Os12g0556200)、鉀離子運輸蛋白質OsHAK5 (RAP-DB 基因編號：Os01g0930400)、ABA 反應轉錄因子 OsWRKY80 (RAP-DB基因編號：Os09g0481700) 三者間的蛋白質交互作用並研究 OsRZFP34 調控氣孔開啟的相關機制。首先，我們利用洋蔥表皮短暫表現系統觀察蛋白質表現位置並利用雙分子螢光互補實驗 (Bimolecular fluorescence complementation; BiFC) 分析 OsRZFP34 與三個蛋白質之間的交互作用。由綠螢光蛋白質6 (green fluorescent protein 6; GFP6) 表現位置實驗之結果顯示 OsCaMBP-GFP6 及 OsWRKY80-GFP6 融合蛋白質主要表現在細胞核中；而 OsHAK5-GFP6 融合蛋白質則表現於細胞膜上，此結果與 Horie et al., 2011 文獻相符。根據 BiFC 分析結果發現 OsRZFP34 能夠與 OsCaMBP 及 OsWRKY80 產生蛋白質交互作用且僅於細胞核中，亦能夠與 OsHAK5 於細胞核及細胞膜上產生蛋白質交互作用；反之，當 OsRZFP34 與 OsHAK5 於高溫逆境下產生的蛋白質交互作用則集中於細胞膜上。接著我們利用對 Ca2+ 敏感的螢光染劑 Fluo-3/AM 偵測保衛細胞於高溫、ABA、H2O2、Ca2+、K+ 等條件處理後保衛細胞中 [Ca2+]cyt。由實驗結果發現經過逆境處理後 OsRZFP34 過量表現轉殖株中保衛細胞的 [Ca2+]cyt 低於 WT。綜合以上結果顯示 OsRZFP34 能與 OsCaMBP、OsHAK5、OsWRKY80 產生蛋白質交互作用，並透過改變保衛細胞中 [Ca2+]cyt 進而影響水稻氣孔開啟。;RING zinc-finger proteins (RZFPs) are known to be essential in the regulation of plant processes, including responses to biotic and abiotic stress. By oligo microarray expression profiling, we identified a rice RZFP gene, OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. Our previous study has shown that OsRZFP34 is involved in transpiration cooling and may modulate the genes implicated in Ca2+ sensing, K+ regulator, and ABA response to control stomata opening in rice (Hsu et al., 2014). Here we further investigate the interaction between OsRZFP34 and a calmodulin binding protein OsCaMBP (RAP-DB accession no. Os12g0556200), a potassium transporter OsHAK5 (RAP-DB accession no. Os01g0930400), or an ABA response transcriptional factor OsWRKY80 (RAP-DB accession no. Os09g0481700) and OsRZFP34-regulated mechanism of stomatal opening. Firstly, we determined subcellular localization of these proteins and used bimolecular fluorescence complementation (BiFC) to analyze the interaction between OsRZFP34 and each of the 3 proteins. The results of green fluorescent protein 6 (GFP6) localization experiment showed that OsCaMBP-GFP6 and OsWRKY80-GFP6 fusion proteins were mainly associated with nucleus, while OsHAK5-GFP6 fusion protein was localized in the plant plasma membrane of onion epidermal cells that corresponded with Horie et al., 2011. By BiFC analysis, we found that our OsRZFP34 physically interacted with OsCaMBP and OsWRKY80 only in the nucleus, and with OsHAK5 in the nucleus and plasma membrane. In contrast, the fluorescent signals for co-expression of OsRZFP34 and OsHAK5 were found on the plasma membrane under high temperature. Then we used the Ca2+-sensitive fluorescent dye Fluo-3/AM to detect the [Ca2+]cyt of guard cells treated with heat, ABA, H2O2, Ca2+, or K+. The results indicated that the guard cells of OsRZFP34-overexpressing plants showed lower [Ca2+]cyt than that of the WT after treatment. Taken together, these results demonstrate that OsRZFP34 can interact with OsCaMBP, OsHAK5, and OsWRKY80 and modulate [Ca2+]cyt of guard cells to regulate stomatal opening in rice.
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