阿拉伯芥DREB1A基因表現受ABF3及14-3-3蛋白 調控機制之研究

Abstract

bZIP和WRKY轉錄因子在阿拉伯芥中皆屬於大族群轉錄因子。先前研究發現bZIP與WRKY參與許多非生物性逆境的調控反應，bZIP與WRKY這類型的轉錄因子皆藉由調節下游目標基因之表現抵抗逆境。然而，轉錄因子的調控反應機制非常複雜，截至目前並不十分明確。14-3-3蛋白為一種鷹架蛋白，能與其他磷酸化後的蛋白質結合，並改變其蛋白的表現位置、穩定度與活性等，14-3-3蛋白也可與某些轉錄因子進行交互作用。本論文藉由轉活化分析的方式，找到潛在ABF3與WRKYs的目標基因。ABF3與WRKYs會誘導DREB1A，MYB2，ABI5及RD22等基因之表現。首先，這些轉錄因子存在於細胞核中。同時，利用雙分子螢光互補系統在雷射共軛焦顯微鏡下觀察14-3-3蛋白分別可與WRKY25,-33,40及ABF3有交互作用。再由轉活化分析的方式，經冷光的偵測，證實ABF3轉錄因子會藉由與ABRE結合而活化DREB1A基因表現，且共轉錄14-3-3基因會增強ABF3轉錄因子對於下游基因DREB1A的轉錄活性，此結果指出，DREB1A是ABF3的目標基因，且14-3-3蛋白可能會與轉錄因子交互作用後，影響轉錄因子的轉錄活性。

Basic region/leucine zipper (bZIP) and WRKY transcription factors (TFs) belong to a large gene family in plants. Previous researches showed that WRKYs and bZIPs participate in many abiotic stress responses. WRKYs and bZIPs can regulate many downstream genes, which are responsive to stresses. However, the transcriptional network and the molecular mechanism in plants are still not very clear. 14-3-3 protein is a scaffold protein, which can interact with other target proteins to change the subcellular localization, stability or activity. 14-3-3 protein was reported to interact with TFs. In this study, by using protoplast transactivation assay, the potential target genes of ABF3 and WRKYs, including DREB1A, MYB2, ABI5 and RD22 gene expression. In addition, to investigate the relationship between WRKYs, ABF3 and 14-3-3 protein in the transcriptional regulation in Arabidopsis thaliana, confocal microscope was used to observe the subcellular localization of these TFs are localized to the nucleus. The bimolecular fluorescence complementation (BiFC) assay demonstrated that WRKY25, -33, 40 and ABF3 proteins physicaly interacted with 14-3-3in protoplast cells. Moreover, the transactivation assay, showed that ABF3 up-regulated a reporter gene driven by the DREB1A promoter, possibly via the direct binding to the ABRE cis-elements, co-expression of 14-3-3 enhanced the transcriptional activity of ABF3. Taken together this study demonstrated that DREB1A is a target gene of ABF3, and 14-3-3 has a role in participating with ABF3 to regulate the downstream genes expression.