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標題: 鑑定和分析干擾文心蘭EIN3同源蛋白質功能的小分子化合物
Identify and analyze small-molecule compounds interfering with the function of an ETHYLENE INSENSITIVE3 ortholog, OgEIL1, from Oncidium
作者: 蕭筑尹
Jhu-Yin Siao
關鍵字: 乙烯
small-molecule compound
引用: 林詩霖。2016。文心蘭EIN3同源蛋白質交互作用之研究與應用。 國立中興大學碩士論文。 陳昱安。2017。106年度農業生產目標。農政與農情:296。 黃肇家。2003。台灣文心蘭切花外銷之保鮮技術及應用。農政與農情:130。 Abeles, F., Morgan, P., and Saltveit, M. (1992). Ethylene in plant biology. 2nd edn Academic Press. New York. Alexander, L., and Grierson, D. (2002). Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. Journal of Experimental Botany 53, 2039-2055. Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proceedings of the National Academy of Sciences of the United States of America 74, 5350-5354. Ayub, R., Guis, M., Amor, M.B., Gillot, L., Roustan, J.-P., Latche, A., Bouzayen, M., and Pech, J.-C. (1996). Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nat Biotech 14, 862-866. Bapat, V.A., Trivedi, P.K., Ghosh, A., Sane, V.A., Ganapathi, T.R., and Nath, P. (2010). Ripening of fleshy fruit: Molecular insight and the role of ethylene. Biotechnology Advances 28, 94-107. Brencic, A., Angert, E.R., and Winans, S.C. (2005). Unwounded plants elicit Agrobacterium vir gene induction and T-DNA transfer: transformed plant cells produce opines yet are tumour free. Molecular Microbiology 57, 1522-1531. Burg, S.P., and Burg, E.A. (1965). Gas Exchange in Fruits. Physiologia Plantarum 18, 870-884. Chang, C., Kwok, S.F., Bleecker, A.B., and Meyerowitz, E.M. (1993). Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science 262, 539. Chen, G., Alexander, L., and Grierson, D. (2004). Constitutive expression of EIL-like transcription factor partially restores ripening in the ethylene-insensitive Nr tomato mutant*. Journal of Experimental Botany 55, 1491-1497. Chen, H., Xue, L., Chintamanani, S., Germain, H., Lin, H., Cui, H., Cai, R., Zuo, J., Tang, X., Li, X., et al. (2009). ETHYLENE INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 Repress <em>SALICYLIC ACID INDUCTION DEFICIENT2</em> Expression to Negatively Regulate Plant Innate Immunity in <em>Arabidopsis</em>. The Plant Cell 21, 2527. Chen, S.-Y., Tsai, H.-C., Raghu, R., Do, Y.-Y., and Huang, P.-L. (2011). cDNA cloning and functional characterization of ETHYLENE INSENSITIVE 3 orthologs from Oncidium Gower Ramsey involved in flower cutting and pollinia cap dislodgement. Plant Physiology and Biochemistry 49, 1209-1219. Clough Steven, J., and Bent Andrew, F. (2008). Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735-743. Deikman, J. (1997). Molecular mechanisms of ethylene regulation of gene transcription. Physiologia Plantarum 100, 561-566. Dong, C.-H., Jang, M., Scharein, B., Malach, A., Rivarola, M., Liesch, J., Groth, G., Hwang, I., and Chang, C. (2010). Molecular Association of the Arabidopsis ETR1 Ethylene Receptor and a Regulator of Ethylene Signaling, RTE1. The Journal of Biological Chemistry 285, 40706-40713. Ericsson, U.B., Hallberg, B.M., DeTitta, G.T., Dekker, N., and Nordlund, P. (2006). Thermofluor-based high-throughput stability optimization of proteins for structural studies. Analytical Biochemistry 357, 289-298. Fields, S., and Song, O.-k. (1989). A novel genetic system to detect protein protein interactions. Nature 340, 245. Fronzes, R., Christie, P.J., and Waksman, G. (2009). The structural biology of type IV secretion systems. Nature Reviews Microbiology 7, 703. Gomez-Cadenas, A., Tadeo, F.R., Talon, M., and Primo-Millo, E. (1996). Leaf Abscission Induced by Ethylene in Water-Stressed Intact Seedlings of Cleopatra Mandarin Requires Previous Abscisic Acid Accumulation in Roots. Plant Physiology 112, 401. Gray, J.E., Picton, S., Giovannoni, J.J., and Grierson, D. (1994). The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening. Plant, Cell & Environment 17, 557-571. Guzmán, P., and Ecker, J.R. (1990). Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. The Plant Cell 2, 513-523. Hiraga, S., Sasaki, K., Hibi, T., Yoshida, H., Uchida, E., Kosugi, S., Kato, T., Mie, T., Ito, H., Katou, S., et al. (2009). Involvement of two rice ETHYLENE INSENSITIVE3-LIKE genes in wound signaling. Molecular Genetics and Genomics 282, 517. Ju, C., and Chang, C. (2015). Mechanistic Insights in Ethylene Perception and Signal Transduction. Plant Physiology 169, 85-95. Kende, H. (1993). Ethylene Biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44, 283-307. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993). CTR1, a negative regulator of the ethylene response pathway in arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72, 427-441. Kleber-Janke, T., and Becker, W.-M. (2000). Use of Modified BL21(DE3) Escherichia coli Cells for High-Level Expression of Recombinant Peanut Allergens Affected by Poor Codon Usage. Protein Expression and Purification 19, 419-424. Le Masson, B., and Nowak, J. (1981). Cut-flower life of dry transported carnations as influenced by different silver form pre-treatments. Scientia Horticulturae 15, 383-390. Lelievre, J., Latche, A., Jones, B., and Bouzayen, M. (1997). M. and Pech, JC 1997. Ethylene and fruit ripening. Physiologia Plantarum 101, 727-739. Li, J., Li, Z., Tang, L., Yang, Y., Zouine, M., and Bouzayen, M. (2012). A conserved phosphorylation site regulates the transcriptional function of ETHYLENE-INSENSITIVE3-like1 in tomato. Journal of Experimental Botany 63, 427-439. Muñoz-Espinoza, V.A., López-Climent, M.F., Casaretto, J.A., and Gómez-Cadenas, A. (2015). Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions. Frontiers in Plant Science 6, 997. Picton, S., Barton, S.L., Bouzayen, M., Hamilton, A.J., and Grierson, D. (1993). Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene. The Plant Journal 3, 469-481. Pieter, B.F., and Annemarie, H. (2001). Yeast One-Hybrid Screening for DNA-Protein Interactions. Current Protocols in Molecular Biology 55, 12.12.11-12.12.12. Pirrung, M.C., Bleecker, A.B., Inoue, Y., Rodríguez, F.I., Sugawara, N., Wada, T., Zou, Y., and Binder, B.M. (2008). Ethylene Receptor Antagonists: Strained Alkenes Are Necessary but Not Sufficient. Chemistry & Biology 15, 313-321. Sakai, Y., Nakagawa, T., Shimase, M., and Kato, N. (1998). Regulation and Physiological Role of the DAS1 Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic Yeast Candida boidinii. Journal of Bacteriology 180, 5885-5890. Solano, R., Stepanova, A., Chao, Q., and Ecker, J.R. (1998). Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes & Development 12, 3703-3714. Song, J., Zhu, C., Zhang, X., Wen, X., Liu, L., Peng, J., Guo, H., and Yi, C. (2015). Biochemical and Structural Insights into the Mechanism of DNA Recognition by Arabidopsis ETHYLENE INSENSITIVE3. PLOS ONE 10, e0137439. Song, S., Huang, H., Gao, H., Wang, J., Wu, D., Liu, X., Yang, S., Zhai, Q., Li, C., Qi, T., et al. (2014). Interaction between MYC2 and ETHYLENE INSENSITIVE3 Modulates Antagonism between Jasmonate and Ethylene Signaling in Arabidopsis. The Plant Cell 26, 263-279. Theologis, A., Oeller, P.W., Wong, L.-m., Rottmann, W.H., and Gantz, D.M. (1993). Use of a tomato mutant constructed with reverse genetics to study fruit ripening, a complex developmental process. Developmental Genetics 14, 282-295. Thomma, B.P.H.J., Penninckx, I.A.M.A., Cammue, B.P.A., and Broekaert, W.F. (2001). The complexity of disease signaling in Arabidopsis. Current Opinion in Immunology 13, 63-68. Wu, H.-Y., Liu, K.-H., Wang, Y.-C., Wu, J.-F., Chiu, W.-L., Chen, C.-Y., Wu, S.-H., Sheen, J., and Lai, E.-M. (2014). AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 10, 19. Xue, C., Hsueh, Y.-P., and Heitman, J. (2008). Magnificent seven: roles of G protein-coupled receptors in extracellular sensing in fungi. FEMS Microbiology Reviews 32, 1010-1032. Yuan, J.S., Reed, A., Chen, F., and Stewart, C.N. (2006). Statistical analysis of real-time PCR data. BMC Bioinformatics 7, 85.
摘要: 文心蘭 (Oncidium orchids) 為台灣主要外銷的切花花卉,而乙烯 (Ethylene) 荷爾蒙通常在花朵採收後及運送中產生,導致文心蘭花苞脫落以及花瓣老化等問題,造成農業成本的增加和經濟損失。若能藉由調控乙烯荷爾蒙之生理功能,減少乙烯對花朵的不利影響,則能有效減少文心蘭採收及運送過程中的產品耗損。 在已建立之乙烯訊息傳遞途徑,模式植物阿拉伯芥 (Arabidopsis thaliana) 中的ETHYLENE INSENSITIVE3 (AtEIN3) 和 EIN3-like (AtEIL) 為重要的乙烯反應轉錄因子,是調控乙烯訊息傳遞不可或缺的蛋白質。AtEIN3/AtEIL1會形成蛋白質二聚體及直接結合專一之DNA序列 (EIN3 binding sequence, EBS),進而調控乙烯反應相關基因之表現。在番茄 (Solanum lycopersicum) 中之SlEIL1蛋白質中存在一個名為EPR1 (EIN3磷酸化區域1)的結構。根據雙分子螢光互補(BiFC)實驗證實,EPR1磷酸化的形成有助於S1EIL1蛋白質二聚化,而造成EPR1功能缺失的突變會使S1EIL1蛋白質失去二聚化的能力,導致S1EIL1表無法活化相關基因之轉錄。文心蘭 (Oncidium Gower Ramsey) 中有兩個EIN3同源蛋白質,分別為OgEIL1與OgEIL2,已被證實可互補阿拉伯芥ein3突變株,並且能在酵母菌中形成同源二聚體與異質二聚體結合DNA。 本篇論文藉由偵測酵母菌單/雙雜交系統 (Yeast one-/two-hybrid system) 之報導基因表現,篩選干擾OgEIL1蛋白質與DNA和蛋白質二聚體交互作用的小分子化合物。利用構築OgEIL1互補阿拉伯芥ein3-1eil1-1轉基因植物,冷光素酶活性檢測法 (luciferase activity assay)、西方墨點法 (western-blotting analysis)、即時定量擴增 (real time quantitative polymerase chain reaction, RT-qPCR) 等實驗方法,進行分析篩選所得到的小分子化合物對於轉基因植物白化苗表現型,乙烯相關基因表現量,及OgEIL1蛋白質在阿拉伯芥的功能性分析。 根據觀察和分析小分子化合物對OgEIL1轉基因植物的影響,我們發現有效之化合物可抑制白化苗三相反應表現型,降低OgEIL1結合EIN3啟動子活性表現量,以及減少EIN3 RESPONSE FACTOR1 (ERF1) 基因表現量,進而鑑定對OgEIL1蛋白質具有專一性功能抑制的化合物。接著以AtEIN3已結晶片段的結構為模板,模擬OgEIL1蛋白質同源性片段結構與化合物形成分子對接 (Molecular docking) 的模型,得到蛋白質和化合物的胺基酸的可能結合位點,藉以化合物與蛋白質結合的重要胺基酸位點。經由這些研究方法,我們從22000種化合物中得到三種具有干擾OgEIL1蛋白質在植物細胞內功能的小分子化合物。將來可進行測試此類小分子化合物調節文心蘭乙烯功能的作用,期能提升採收後觀賞花卉的貯運性良率。
Oncidium orchids are among the main high-value export cut flowers of Taiwan. Ethylene hormone is autonomously produced after harvesting and during transportation of cut flowers, which frequently results in a post-harvest issue of flower senescence and petal falling off. These issues lead to cost increases and economic loss of growers. In order to reduce the adverse effects of ethylene on cut flowers, we reason that if the ethylene physiology can be effectively controlled, the product loss after harvest and during transportation can be reduced. ETHYLENE INSENSITIVE3 (AtEIN3) and EIN3-LIKE1 (AtEIL1) in the reference plant, Arabidopsis thaliana are important transcriptional factors in the ethylene response pathway.Arabidopsis EIN3/EIL1 form homodimers and bind to specific DNA sequence (EIN3 binding sequence, EBS) to regulate ethylene responsive.The ortholog of AtEIN3 in tomato (Solanum lycopersicum), SlEIL1, has a specific phosphorylation region called EPR1 (EIN3 phosphorylation region1).Results from bimolecular fluorescence complementation (BiFC) assay reveals that the presence of a phosphorylation site in the EPR1 contributes to dimer formation S1EIL1, while mutations in the phosphorylation site within the EPR1 result in loss of protein dimerization and transcriptional activity. Functional orthologs of AtEIN3 have been identified in many plant species. There are two orthologs of AtEIN3 identified in Oncidium Gower Ramsey, OgEIL1 and OgEIL2, that both have been shown to complement Arabidopsis ein3 mutant. In this study, we constructed yeast two- and one-hybrid systems to screen for small molecule compounds that interfered with the dimerization of OgEIL1 and protein-DNA interaction, respectively. Furthermore, transgenic plants expressing OgEIL1 for complementation of Arabidopsis ein3-1eil1-1, luciferase reporter gene system, western blot analysis and realtime quantitative polymerase chain reaction (RT-qPCR) were used to determine the chemical effects of hit compounds on seedling phenotype and expression of genes responsive to ethylene. The hit compounds showing significant effects in the following experiments were selected for further characterization; suppression of the triple response phenotype dependent of expression of OgEIL1 in the Arabidopsis ein3-1eil1-1 mutant, reduction of a luciferase reporter gene activity fused with multiple copies of EIN3 binding sequence, and suppression of the induced expression of ETHYLENE RESPONSE FACTOR1 (ERF1) gene by overexpression of OgEIL1 or ethylene treatment.We next used the crystallized fragment of AtEIN3 as a template to model the homologous fragment of OgEIL1 for simulated docking analysis with the hit compounds to obtain the amino acid residues as potential chemical binding sites in OgEIL1. Finally, we attempted to express and purify recombinant OgEIL1 by bacterial expression system. The purified OgEIL1 protein will be used to confirm the direct effects of small-molecule compounds on protein dimerization, DNA binding affinity by in vitro experiments. This study aims to identify and analyze small-molecule compounds modulating ethylene response by regulating OgEIL1 function in Oncidium orchids. Based on current results from the experiments in this thesis, the research will be continued to test compounds directly on protein dimerization and DNA interaction by in vitro, in vivo and in planta methods such as co-precipitation, pull-down and electrophoretic mobility shift assays and longetivity of Oncidium cut flowers. The effective small-molecule compounds on modulating ethylene function hold great potential to the postharvest quality and management of ornamental flowers of high commercial values.
文章公開時間: 10000-01-01
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