阿拉伯芥蛋白激?AtYak1之功能探討; The studies of AtYak1, a novel DYRK family member in Arabidopsis thaliana

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题名:	阿拉伯芥蛋白激?AtYak1之功能探討;The studies of AtYak1, a novel DYRK family member in Arabidopsis thaliana
作者:	吳怡蓁;Yi-Chen Wu
贡献者:	生命科學研究所
关键词:	蛋白激酶;AtYak1;light signal transduction;DYRK protein kinase
日期:	2004-07-08
上传时间:	2009-09-22 10:18:13 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	植物對於周遭自然環境狀態的改變非常敏感，因此必須藉由不同的反應機制來適應環境的變動；其中，光對植物的影響最為重要，它不僅是能量的來源，同時也是植物生長與發育過程中不可或缺的因子之一。植物一方面透過光接受器接收或偵測外在光源，另一方面則在光訊息傳遞路徑過程中藉由許多基因的表現與調節，進而對光產生多重層次的詮釋及反應。經由DNA 微矩陣技術，在光照對阿拉伯芥基因表現影響的初步研究中，鑑定到許多在光訊息傳遞中受調控的基因；在這群基因中，先針對一個具有WD-repeat motif 的蛋白質做測試，由於此基因的表現量隨光照時間增加而增加，而於暗處理時，又明顯造成此基因表現量降低，因此顯示其受光調節，故將此基因命名為LWD1。如同其他具有WD-repeat motif 的蛋白質，WD-repeat motif 提供了穩定各個蛋白質間交互作用的平台，故假設LWD1 也藉由和其他不同蛋白質間的交互作用，進而調節植物體內一些訊號的傳遞。利用酵母菌雙雜合技術可發現， AtYak1 是其中一個表現與LWD1 有交互作用之蛋白質。AtYak1 也是第一個在植物中找到的DYRKs （dual specificity Yak1-related protein kinase）。之前研究顯示DYRKs 可調節細胞的生長與發育，為了更進一步了解AtYak1 是如何在光訊息路徑中調控植物生長和發育，本論文將針對AtYak1 的生物功能來作探討，透過AtYak1 在植物細胞中表現的位置、不同組織之表現量及突變株性狀之觀察與分析，進而推測其生理功能及意義。結果顯示，當AtYak1 與DNA binding domain 融合單獨送入酵母菌時即啟動β-galactosidase 活性，因此我們推測AtYak1 可能具有轉錄作用活化者的功能。我們也可發現AtYak1 分佈於細胞質及細胞核內，由此我們認為在植物中之AtYak1 可能也和其他真核細胞之 DYRKs 功能類似，而且會依據外在環境因素或細胞本身之生理狀態而存在細胞中不同的位置以執行不同之功能。在觀察AtYak1 在組織表現量方面，結果顯示，其於葉片、花、果實、根部均有表現，其中以根部表現量最多。另外，藉由突變株的觀察，我們發現AtYak1 突變株在長/ 短日照中生長葉片的型態和發育均和野生型不同，植株也較小，尤其以短日照最為明顯，故推測AtYak1 可能也參與在阿拉伯芥葉片型態生長和發育路徑中 Light provides an important environmental cue regulating plant growth and development. Light perception is mediated by the action of several photoreceptors. The signal transduction and light responses in higher plants involve the regulation of differential gene expression. Considerable amount of efforts have been put in the studies of light-mediated signaling pathway in plants. DNA microarray technology serves as a powerful approach in addition to the biochemical and genetic methods in identifying novel molecular components in light signaling pathway. After analyzing microarray, we choose a gene encoding WD-containing protein, termed LWD1, which is clearly up-regulated in response to light treatment and is dramatically down-regulated in dark-treated Arabidopsis leaves. Similar to other WD-containing proteins, LWD1 could function in interacting or coordinating multiple protein partners responsible for proper signal transduction. Via yeast two-hybrid, preliminary data reveal strong protein-protein interaction between LWD1 and AtYak1. AtYak1 is the first DYRK（dual specificity Yak1-related protein kinase）family member identified in plants and exists as a single copy gene in Arabidopsis. In this thesis, I will report the initial efforts in characterizing the biological properties and functions of AtYak1. Results suggest that AtYak1 is a putative transcriptional activator. Subcellular localization of AtYak1 is analyzed by generating a AtYak1-GFP translational fusion protein for protoplast transient assay. AtYak1-GFP is observed both in the cytoplasm and in the nucleus. Real time RT-PCR was employed to study the expression pattern of AtYAK1 in various tissues of Arabidopsis, including rosette leaves, flowers, silique, and roots. Results show that AtYak1 is expressed ubiquitously but predominantly in roots. Furthermore, to gain more insights on the biological functions of AtYak1, we obtained AtYak1 T-DNA insertion lines and transgenic Arabidopsis overexpressing AtYak1. Phenotype comparison between wild type and AtYak1 mutant Arabidopsis indicate that AtYak1 may involve in leaf differentiation and development.
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