2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/701682摘要：WRKY 是植物特有的轉錄因子，可以結合到目標基因啟動子的W-框(W-box)行轉錄調控。在以前的研究 中，AtWRKY發現參與鹽逆境的反應和抗性。此外，AtWRKY 發現也參與ABA 信號傳導。在我們的初步 結果，我們發現 AtWRKY25 基因的過度表達之阿拉伯芥植株是耐鹽的。此外，AtWRKY25基因的過度表 達阿拉伯芥植株側根生長受到抑制。我們也發現AtWRKY25與14-3-3 蛋白質有交互作用。我們推測， 14-3-3 蛋白質與WRKY25交互作用對鹽逆境的反應發揮重要作用，並參與植物體內的離層酸的信號傳 導，以及調控側根之生長發育。在此計畫中我們使用阿拉伯芥作為模式生物。此計畫的最終目標是要 闡明WRKY25 參與鹽逆境的反應及抗性機制，和在離層酸信號傳導中的作用，及參與根生長發育之功 能。35S 啟動子驅動的AtWRKY25過量表達植株將建立，並測試受到鹽、滲透壓力或離層酸處理之回應。 特別是，我們想觀察根的外表型。我們將測量AtWRKY25過量表達植株中生長素及離層酸之含量及分布 是否受影響。為了確定AtWRKY25受鹽或滲透壓力影響之下游基因，我們將使用晶片分析使用AtWRKY25 基因的過度表達植株在鹽壓力或離層酸處理下揭示下游受調控之基因。WRKY25之轉錄活性分析也將在 阿拉伯芥之原生質體中進行。我們將利用共免疫沉降方式驗證AtWRKY25與14-3-3之交互作用。我們將 利用激&#37238;分析搭配質譜分析找到AtWRKY25之磷酸化位點。為了找到14-3-3結合位點，AtWRKY25之磷酸 化位點將被點突變成麩胺酸並觀察14-3-3結合是否受影響。此外，AtWRKY25過度表達植株中AtWRKY25 之磷酸化位點及14-3-3結合位點將被點突變成麩胺酸、天門冬胺酸或麩胺酸並觀察外表型。我們希望 這項研究將導致更好地瞭解WRKY25基因的作用機轉，並希望最終可以應用於農作物(如水稻)改善鹽逆 境抗性和提高作物產量。<br> Abstract: WRKY is a plant specific transcription factor which can bind to W-box of target gene promoter for transcriptional regulation. In previous studies, AtWRKY was found to be involved in salt or osmotic stress response and tolerance, and abscisic acid (ABA) signaling pathway. In our preliminary results, we found that overexpression line of a type I WRKY AtWRKY25 is salt-tolerant. In addition, lateral root development is impaired. Moreover, WRKY25 interacts with 14-3-3 protein. We hypothesize that binding of 14-3-3 affects salt stress and ABA response, and regulates lateral root development. In this proposed project we will use Arabidopsis thaliana as our model system. The ultimate goal of this project is to elucidate the function and mechanism of 14-3-3 interacting AtWRKYs in salt stress response, and ABA signal transduction pathway. In particular, we will investigate the biological function and mechanism of 14-3-3 binding to WRKY25. Overexpression line of WRKY25 driven by 35S promoter will be generated and subjected to ABA treatment for sensitivity test. ABA content will also be measured to test whether WRKY25 affects ABA accumulation. As for lateral root development, auxin level will be measured by GC-MS. DR5-GUS line will be crossed and test for auxin distribution. In order to identify downstream target genes, microarray analysis will be introduced to reveal up-regulated and down-regulated genes using WRKY25 over-expression line. Transactivation assay will be performed to determine the transcriptional activity of WRKY25. On the other hand, 14-3-3 interaction will be validated by co-immunoprecipitation. WRKY25 in vitro phosphorylation site will be mapped using kinase assay followed by mass spectrometry analysis. To identify 14-3-3 binding site, phosphorylation site of WRKY25 will be point-mutated to alanine and test if the 14-3-3 interaction is abolished. Finally, we will generate overexpression line expressing WRKY25 with phosphorylation and 14-3-3 sites point-mutated to alanine or apartate/glutamate (phosphomimic). We will determine if the site contributes to the salt-tolerance and lateral root phenotype. We hope that this study will eventually be applied to engineering crops against salt stress and improving crop yield.14-3-3WRKY25磷酸化鹽根離層酸14-3-3WRKY25phosphorylationsaltrootABA