阿拉伯芥蛋白激酶AtYak1之功能探討

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吳怡蓁(Yi-Chen Wu) 查詢紙本館藏

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論文名稱

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

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摘要(中)

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

摘要(英)

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.

關鍵字(中)

★ 光訊息傳導
★ 蛋白激酶

關鍵字(英)

★ AtYak1
★ light signal transduction
★ DYRK protein kinase

論文目次

中文摘要·······································································································i
英文摘要······································································································ii
目錄············································································································ iii
圖表目錄······································································································v
縮寫對照表·································································································vi
前言··············································································································1
一、植物光訊息傳導（light signal transduction）及光型態形成
( photomorphogenesis） ··········································································· 1
二、阿拉伯芥受紅光/遠紅光調控之基因························································ 2
三、LWD1（light-regulated WD repeat 1）基因·············································· 5
1.LWD1 基因簡介······················································································· 5
2.WD-repeat 蛋白質結構與功能································································ 6
四、AtYak1（Arabidopsis thaliana Yak1-related protein kinase）基因··········· 8
1. AtYak1 同源基因與結構的簡介····························································· 8
2. AtYak1 同源基因的表現與功能····························································· 9
五、研究目的····································································································· 11
材料與方法····································································································12
一、酵母菌雙雜合反應····················································································· 12
1). pACT2(AD)-LWD1 及pAS2(BD)-AtYak1 質體之建構······················ 12
2). 酵母菌細胞轉型··················································································· 16
3). β-galactosidase 活性分析······································································· 18
二、AtYak1 基因功能研究分析········································································ 19
1). AtYak1 基因在細胞中的表現位置························································ 19
2). AtYak1 基因在各組織之基因表現分析················································ 23
iv
三、AtYak1 突變株之篩選、性狀觀察與分析················································ 27
1). 阿拉伯芥突變株種植與生長······························································· 27
2). 阿拉伯芥AtYak1 T-DNA 嵌入突變株之篩選···································· 27
3). T-DNA 嵌入數目之確認-南方墨漬法·················································· 29
4). 大量表現AtYak1 基因阿拉伯芥轉殖株之獲得·································· 37
5). 大量表現AtYak1 轉殖株與AtYak1 T-DNA 插入
突變株之基因表現··············································································· 41
結果與討論····································································································42
一、LWD1 與AtYak1 交互作用之確認··························································· 42
二、AtYak1 基因功能推測················································································ 44
1). AtYak1 在細胞中位置的定位（subcellular localization） ·························· 44
2). AtYak1 於植物組織之表現量分析（expression pattern）·························· 45
三、AtYak1 突變株之性狀觀察與分析···························································· 46
1). 阿拉伯芥AtYak1 T-DNA 嵌入突變株同型合子的獲得···························· 46
2). 阿拉伯芥突變株中T-DNA 嵌入數目之確認············································· 47
3). 利用RT-PCR 對AtYak1 於突變株之基因表現分析·································· 48
4). T-DNA 嵌入AtYak1 基因突變株表型的觀察············································· 49
5). 利用Real time RT-PCR 確認AtYak1 基因於轉殖株中大量表現·············· 50
未來展望········································································································54
參考文獻········································································································55
表····················································································································62
圖····················································································································63
附圖················································································································79
附錄················································································································84

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指導教授

吳素幸、吳少傑
(Shu-Hsing Wu、Shaw-Jye Wu)

審核日期

2004-7-14

推文