阿拉伯芥乙烯反應因子ERF96 N端之GCC box結合功能探討

Functional analysis of Arabidopsis ERF96 N-terminal region in GCC box binding

陳昆宏(Kun-Hong Chen)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201704201

摘要

乙烯反應因子(ethylene response factors, ERFs)在植物體內為一重要的轉錄因子(transcription factors)，最早發現在阿拉伯芥(Arabidopsis thaliana)的ERF1會受到乙烯的誘導而大量表現，以獨特的AP2/ERF domain與目標序列上的GCC box (AGCCGCC)結合，調控下游基因表現，影響植物生理反應，包含植物生長發育與抵抗逆境；ERF96，分類於ERFs家族第IX群中，在病原菌侵襲時，會大量表現，正向調控PDF1.2、PR-3及 PR-4的轉錄，對植物防禦反應有一定貢獻。在過去實驗室的研究中，發現ERF96對於GCC box有二配位結合(two-site binding)，而近期實驗室所解出的ERF96 - GCC11複合體結構，可觀察到N端形成的螺旋結構，結合至GCC box的小溝(minor groove)。由於N端結合對於ERF轉錄因子的功能不確定，因此本研究將探討ERF N端在DNA結合時所扮演的功能。
根據ERF96-DNA複合體結構，N端之極性胺基酸Asp2、Gln3、Arg6位置做點突變，並利用螢光電泳遷移率實驗及偏極螢光實驗測量其重組蛋白結合能力的差異，進一步利用原生質體轉錄活性分析去觀察ERF96的胺基端突變以及AP2/ERF domain突變對轉錄活性的影響；在螢光電泳遷移率實驗中得知胺基端突變並不影響對GCC box的結合，而在偏極螢光實驗發現胺基端突變會造成第二解離常數的消失，顯示胺基端是ERF96影響GCC box的第二步驟結合；以原生質體進行轉錄活性實驗，結果顯示胺基端突變會造成部分轉錄活性的下降，但影響不大，但是AP2/ERF domain突變則會造成ERF96轉錄活性的完全消失，尤其是在R16、R19、R21、R31、R39這些會與GCC box有緊密交互作用的胺基酸。

關鍵字

轉錄因子 ； 螢光電泳遷移率實驗 ； 偏極螢光 ； 原生質體轉錄活性分析

並列摘要

Ethylene response factors (ERFs) play a major role in regulating gene expression against biotic and abiotic stress such as drought and pathogen infection. ERF1, the earliest ERFs found in Arabidopsis thaliana, can be induced by ethylene signal, and regulates the downstream gene expression by recognizing the GCC box with unique AP2/ERF domain. ERF96, a transcription activator belonged to the group IX of AP2/ERF family, is involved in plant defense for necrotrophic pathogen infection. Previous study indicated that ERF96 activates the defense genes PDF1.2, PR-3 and PR-4, by recognizing the GCC box located on the promoters of these genes. In previous studies, we found that ERF96 have two-site binding mechanism on GCC box. Recently, we resolve the structure of ERF96-GCC11 complex. We found that N-terminal region forms a α- helix and bind to the minor groove of GCC box. However, the detailed mechanism of how ERF96 recognize the specific DNA sequence via N-terminal region need more explanations. Our objectives aim to determine the binding mechanism of the N-terminal region of ERF96 to GCC box.
We identified several amino acids which directly interact with the GCC box. Most of those residues contain polar side chain, located in AP2 domain and N-terminal region. In our results, Protoplast transactivation assay showed that the mutation of polar amino acid located in AP2 domain for alanine (R16, R19, R21, R31, R39) would significantly reduce the transactivation activity of ERF96; Nevertheless, the mutations of polar residues at N-terminal site only slightly repress the transactivation efficiently. In addition, we noticed that mutation of ERF96 in N-terminal region will eliminate the second dissociation constant via fluorescence polarization, indicate that N-terminal region of ERF96 have an important role in two-site binding mechanism. The protein structure and biophysical assays of ERF96 reveal the mechanism of DNA recognition of ERF TFs and provide the basis for further research on ERFs TFs.

並列關鍵字

AP2/ERF domain ； Transcription factors ； fEMSA ； Fluorescence polarization ； Protoplast transactivation assay

參考文獻

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