阿拉伯芥乙烯反應因子ERF96與GCC-boxes序列結合機制分析

DNA Binding Mechanism of Arabidopsis ERF96 to GCC boxes

林佩璇(Pei-Hsuan Lin)

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(2016年)

https://doi.org/10.6342/NTU201610069

摘要

Ethylene 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。

關鍵字

偏極螢光 ； 螢光電泳遷移率實驗 ； AP2/ERF domain ； 轉錄因子 ； 生物逆境

並列摘要

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.

並列關鍵字

Fluorescence polarization ； fEMSA ； AP2/ERF domain ； Transcription factor ； Biotic stresses

參考文獻

Allen, M.D., Yamasaki, K., Ohme-Takagi, M., Tateno, M., and Suzuki, M. (1998). A novel mode of DNA recognition by a b-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. The EMBO Journal 17, 5484–5496.

Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C., and Ecker, J.R. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657.

Berrocal-Lobo, M., Molina, A., and Solano, R. (2002). Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant Journal 29, 23-32.

Boer, D.R., Freire-Rios, A., Berg, W.A.M.v.d., Saaki, T., Manfield, I.W., Kepinski, S., pez-Vidrieo, I.L., Franco-Zorrilla, J.M., Sacco C. de Vries, R.S., Weijers, D., and Coll, a.M. (2014). Structural basis for dna binding specificity by the auxin-dependent arf transcription factors. Cell Research 156, 577–589.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.