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系統識別號 U0026-0812200910212834
論文名稱(中文) 蛋白質酪胺酸磷酸化在阿拉伯芥植物根部發育之角色
論文名稱(英文) The Role of Protein Tyrosine Phosphorylation in Arabidopsis Root Development
校院名稱 成功大學
系所名稱(中) 生物學系碩博士班
系所名稱(英) Department of Biology
學年度 90
學期 2
出版年 91
研究生(中文) 林玉梅
研究生(英文) Yu-Mei Lin
電子信箱 lomaykimo@yahoo.com.tw
學號 l5689118
學位類別 碩士
語文別 中文
論文頁數 54頁
口試委員 指導教授-黃浩仁
指導教授-黃定鼎
口試委員-吳文鑾
召集委員-張文粲
中文關鍵字   器官發育  阿拉伯芥  蛋白質酪胺酸磷酸化 
英文關鍵字 Root  Organogenesis  Arabidopsis  Protein Tyrosine Phosphorylation 
學科別分類
中文摘要 生物體常藉由磷酸化或去磷酸化蛋白質分子調控其活性,進而影響細胞生理反應。這些磷酸化或去磷酸化生化反應需藉由蛋白質激(protein kinases)或蛋白質去磷酸(protein phosphatases)進行催化。蛋白質上可以被磷酸化與去磷酸化的胺基酸主要為絲胺酸(serine)、酥胺酸(threonine)、酪胺酸(tyrosine)。其中,蛋白質酪胺酸磷酸化(protein tyrosine phosphorylation)已在動物中證實可以經由訊息傳遞系統調控細胞生長發育與分化;然而,在植物的發育過程所扮演的角色卻不清楚。

本研究藉由培養阿拉伯芥(Arabidopsis thaliana)下胚軸(hypocotyls)培養在CIM培養基(callus-inducing medium)誘導中柱細胞(stele)去分化形成癒傷組織(callus),並進一步移至BM (B5 medium)誘導不定根的發育。我們以抗體偵測酪胺酸磷酸化蛋白質在植物根部器官分化時的表現;我們亦利用溫度敏感突變株srd2 (在22℃可被誘導成癒傷組織,在28℃則否)欲證明酪胺酸磷酸化蛋白質的改變並非因為外加荷爾蒙的刺激。接著,再利用各種外加蛋白質激抑制劑及去磷酸抑制劑了解蛋白質磷酸化(主要為蛋白質酪胺酸磷酸化)是否調控植物發育。

結果顯示,酪胺酸磷酸化蛋白質會在阿拉伯芥下胚軸誘導癒傷組織時大量表現;而且,培養在CIM培養基的srd2突變株在22℃時有大量的酪胺酸磷酸化蛋白質,28℃則否。將野生型與srd2突變株之下胚軸培養於CIM培養基一天時,皆有酪胺酸磷酸化蛋白質表現量上升;但由野生型之下胚軸培養於BM及CIM培養基中一天,亦可見酪胺酸磷酸化蛋白質表現量上升,故推論培養早期所造成的酪胺酸磷酸化蛋白質表現量上升可能為植株受傷所誘導,而非癒傷組織發育所必需。在抑制劑的處理實驗結果,我們發現蛋白質激抑制劑staurosporine及蛋白質去磷酸抑制劑OA、cantharidin、sodium orthovanadate和PAO可抑制癒傷組織的形成,而且sodium orthovanadate亦會抑制酪胺酸磷酸化蛋白質的表現量;在不定根的分化過程中發現癒傷組織生成所必需的酪胺酸磷酸化蛋白質表現會隨著時間減少,且不定根的分化可被OA、cantharidin及sodium orthovanadate抑制。而staurosporine和NH4-molybdate則部分抑制。綜合本研究結果,我們推論酪胺酸磷酸化蛋白質可能參與根部發育過程。

英文摘要 Protein phosphorylation and dephosphorylation are involved in the regulation of many eukaryotic intracellular processes. Protein tyrosine phosphorylation catalyzed by protein tyrosine kinase is important in the control of fundamental cellular activities. In animal system, protein tyrosine phosphorylation and dephosphorylation play a central role in a variety of signal transduction pathway regulating cell growth, development and differentiation. Though there were increasing evidences to the importance of reversible protein phosphorylation in cell responses to external stimuli in plants, data related to the involvement of protein tyrosine phosphorylation in plant growth and development was limited.

Rooting initiation involves cell divisions of induced cells followed by the formation of a root meristem. In Arabidopsis, root organogenesis in vitro can be induced from hypocotyls by a two step culture method. Hypocotyls are pre-cultured on callus-inducing medium and then transferred onto root-inducing medium for root re-differentiation.

We have investigated the possible role of tyrosine phosphorylation in this process with hypocotyls explants of Arabidopsis thaliana. Phytohormone-stimulated cell cycle reactivation in hypocotyls was accompanied by tyrosine phosphorylation of several proteins. Such regulation of the tyrosine phosphorylation in these proteins was not observed in a callus-formation mutant, srd2, a result which suggested that the induction of tyrosine phosphorylation occurs as a specific event in the cell cycle re-entry. Additionally, treatment of hypocotyls with a protein kinase inhibitor, staurosporine, and tyrosine phosphatase inhibitors, sodium orthovanadate and PAO, prevented the induction of cell division by phytohormones.

To investigate molecular mechanisms controlling root development, we examined the possible role of protein tyrosine phosphorylation during root organogenesis. The disappearance of three tyrosine-phosphorylated proteins at 63, 70 and 80 kDa correlated with the induction of root organogenesis was detected. We also found that tyrosine phosphatase inhibitors, V and NH4-molybdate, were able to inhibit root re-differentiation. This study highlighted that the role of protein tyrosine phosphotase(s) may play an important regulatory role in root development.

論文目次 致謝…………………………………………………………3
中文摘要……………………………………………………4
英文摘要……………………………………………………6
目錄…………………………………………………………8
圖目錄………………………………………………………9
表目錄………………………………………………………10
縮寫對照表…………………………………………………11
前人研究……………………………………………………12
一、側根發育的調控機制…………………………………12
二、蛋白質激(protein kinases)…….……………13
三、蛋白質去磷酸(phosphoprotein phosphatase;PPases)……………………………………………………17
材料方法……………………………………………………21
結果…………………………………………………………24
一、植物荷爾蒙刺激之下胚軸誘導癒傷組織生成………24
二、酪胺酸磷酸化蛋白質在植物荷爾蒙刺激下胚軸誘導癒傷組織的表現………………………………………………24
三、植物荷爾蒙刺激下胚軸誘導癒傷組織的調控途徑…25
四、移除植物荷爾蒙刺激下胚軸癒傷組織在分化成不定根……………………………………………………………26
五、不定根分化時,酪胺酸磷酸化蛋白質的表現………26
六、不定根分化時可能的調控途徑………………………26
討論…………………………………………………………28
一、荷爾蒙在癒傷組織形成及根部器官發育中相互作用28
二、酪胺酸磷酸化蛋白質在癒傷組織形成及根部器官發育的表現………………………………………………………29
三、調控癒傷組織形成及根部器官發育之訊息傳遞途徑30
參考文獻……………………………………………………33
圖……………………………………………………………Ⅰ
表……………………………………………………………Ⅸ
自述………………………………………………………ⅩⅣ
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