生命科學院: 植物科學研究所指導教授: 鄭貽生林佩璇Lin, Pei-HsuanPei-HsuanLin2017-03-032018-07-062017-03-032018-07-062016http://ntur.lib.ntu.edu.tw//handle/246246/274964Ethylene response factors (ERFs)是一群在植物中參與生物與非生物逆境的特有轉錄因子，ERF96屬於其中分類第九族的成員，擁有一個會結合GCC box (AGCCGCC)的高保守性DNA binding domain名為AP2/ERF domain，前人研究ERF96在病原菌感染後，被大量誘導，以參與植物防禦反應，由於植物ERF蛋白之功能分析不多，本論文分析ERF96重組蛋白及其關鍵胺基酸與特定DNA序列結合，探討植物ERFs在對抗逆境時的可能分子機制。 本研究利用膠體過濾層析法與動態光散色儀分析，確認ERF96在溶液中為單體，且均勻穩定存在於溶液中。利用螢光電泳遷移率實驗分析ERF96與不同長度的GCC box親和性，結果顯示核心序列外的核酸長度與序列影響ERF96 R16A、W23A、R31A、W41A的結合能力。利用定點突變分析ERF96上的R16A、R19A、R21A、W23A、R31A、R39A、W41A與GCC box結合，經由螢光電泳遷移率實驗與偏極螢光實驗，發現單點突變仍具有結合能力，雙點突變(R19AR21A、R31AR39A)後之結合能力則大幅下降。在不同DNA序列的研究中，發現ERF96與其他非GCC box序列(P box、CS1 box)具有結合能力。總結以上結果，ERF96在溶液中，以單體存在，主要以Arg19、Arg21、Arg31、Arg39與GCC box 結合，除此之外，ERF96亦會與P box、CS1 box、DRE box結合，推測ERFs將可能參與調控相關基因的轉錄起始。Ethylene response factors (ERFs) belong to a large family of plant-specific transcription factors which participate in biotic and abiotic stresses in plants. ERF96 has been classified as group IX and contains one conserved DNA binding domain named AP2/ERF domain, which specifically binds to GCC box (AGCCGCC). Previous study demonstrated that ERF96 involved in plant defense response. Since functional analysis for ERF proteins is still rare, we aim to study the molecular mechanism of ERF96 protein binding to GCC box or other non-GCC box sequences. Size-exclusive chromatography and dynamic light scattering identified that full-length recombinant ERF96 is monomer and mono-disperse in the solution. Fluorescein-based electrophoretic mobility shift assay (fEMSA) showed the binding ability of ERF96 to different lengths of GCC box, and the results indicated that flanking region of GCC box influence binding affinity of R16A, W23A, R31A and W41A. Site-directed mutagenesis of ERF96 with R16A, R19A, R21A, W23A, R31A, R39A, and W41A indicated that there is no significant change of binding ability in ERF96 single mutation. However, the binding of ERF96 was abolished in double mutation with R19AR21A and R31AR39A. This results showed that Arg19, Arg21, Arg31, and Arg39 are the important residues for DNA binding. Furthermore, we examined binding ability of ERF96 to non-GCC box sequence, such as P box, CS1 box, and DRE box. The results indicated that ERF96 can bind to P box, CS1 box and DRE box. Taken together, recombinant ERF96 showed the monomer form in solution and Arg19, Arg21, Arg31, and Arg39 of ERF96 are the key residues to bind to GCC box DNA. In addition, ERF96 not only bound to GCC box, but also could bind to P box, CS1 box and DRE box. We proposed that ERFs transcription factor might involve in the transcription activation via non-GCC box binding ability.5630874 bytesapplication/pdf論文公開時間: 2016/3/8論文使用權限: 同意有償授權(權利金給回饋學校)偏極螢光螢光電泳遷移率實驗AP2/ERF domain轉錄因子生物逆境Fluorescence polarizationfEMSATranscription factorBiotic stressesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/274964/1/ntu-105-R02b42011-1.pdf