指導教授：張英峯臺灣大學：植物科學研究所蔡旻潔Tsai, Min-ChiehMin-ChiehTsai2014-11-282018-07-062014-11-282018-07-062013http://ntur.lib.ntu.edu.tw//handle/246246/262848bZIP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.口試委員會審定書 I 誌謝 II 中文摘要 III Abstract V Abbreviations VII Contents X List of tables XIV List of figures XV 1. Introduction 1 1.1 Environmental stresses 1 1.1.1 Biotic stresses 1 1.1.2 Abiotic stresses 2 1.2 Abscisic acid 3 1.3 Transcriptional regulatory networks in abiotic stress responses 3 1.3.1 ABA-dependent pathway 4 1.3.2 ABA-independent pathway 4 1.4 Stress-responsive transcription factors in plants 5 1.4.1 WRKY transcription factors 6 1.4.2 bZIP transcription factors 8 1.5 14-3-3 protein 10 1.5.1 14-3-3 protein and abiotic stresses 11 1.5.2 14-3-3 protein and ABA 12 1.5.3 14-3-3 protein and transcription factors 13 1.5.4 14-3-3 protein and ABFs 13 1.5.5 14-3-3 protein and WRKYs 14 1.6 Project goals 15 2. Materials and Methods 16 2.1 Plant materials and growth conditions 16 2.2 Isolation of Arabidopsis protoplasts 16 2.3 Subcellular localization of WRKY25, 33, 40, ABF3 and mutated ABF3 (T451A) 17 2.4 Bimolecular fluorescence complementation (BiFC) experiments 18 2.5 RNA extraction and q-RT-PCR analysis 19 2.6 Genomic DNA extraction and genotyping of knockout mutants 19 2.7 Transactivation assay using Arabidopsis protoplasts 20 2.8 Purification of GST-ABF3 recombinant protein 21 2.9 Electrophoretic mobility shift assay (EMSA) 22 2.10 Statistic analyses 23 3. Results 24 3.1 Induction of WRKY25, 33, 40 and ABF3 in response to salt and ABA treatments in Arabidopsis 24 3.2 Predicted 14-3-3 binding sites in the transcription factors 24 3.3 Subcellular localization of WRKY25, 33, 40, ABF3 and 14-3-3Ω 25 3.4 Protein–protein interactions between WRKY25, 33, 40, ABF3 and 14-3-3Ω were confirmed by use of BiFC 26 3.5 Protein–protein interactions between mutated ABF3 (T451A) and 14-3-3Ω revealed by BiFC 27 3.6 Subcellular localization of site-directed mutated ABF3 28 3.7 Gene expression of ABI5, DREB1A, DREB2A, MYB2 and RD22 under salt and ABA treatment 29 3.8 Gene expression of 14-3-3Ω under salt and ABA treatment 30 3.9 Transactivation assay of candidate genes regulated by WRKY25, 33, 40, ABF3 and 14-3-3Ω 31 3.10 Cis-elements in DREB1A gene promoter 32 3.11 Electrophoretic mobility shift assay (EMSA) confirmed the binding of ABF3 to the promoter regions of DREB1A 32 3.12 Specific elements in transactivation assay of DREB1A regulated by ABF3 and 14-3-3Ω 33 3.13 Specific element in transactivation assay of DREB1A regulated by mutant ABF3 (T451A) and 14-3-3Ω 34 3.14 DREB1A gene expression in abf3 mutant under salt and ABA treatment 34 3.15 Survival rate comparison under salt stress condition 34 4. Discussion 36 5. Future directions 42 6. References 44 Tables 57 Figures 60 Supplementary Table 82 Supplementary Figure 893878658 bytesapplication/pdf論文公開時間：2016/01/27論文使用權限：同意有償授權(權利金給回饋學校)bZIP轉錄因子WRKY轉錄因子14-3-3蛋白DREB1A雙分子互補螢光系統非生物逆境thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262848/1/ntu-102-R00b42018-1.pdf