Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97731
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
文心蘭
小分子化合物
ethylene
oncidium
small-molecule compound
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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.
URI: http://hdl.handle.net/11455/97731
文章公開時間: 10000-01-01
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