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研究生:張依筠
研究生(外文):Yi-Yun Chang
論文名稱:阿拉伯芥Receptor for Activated C-Kinase 1在乾旱逆境下之功能性研究
論文名稱(外文):Functional study of Receptor for Activated C-Kinase 1 (RACK1) in drought stress response in Arabidopsisthaliana
指導教授:張英峯
指導教授(外文):Ying-Feng Chang
口試委員:林讚標謝旭亮鄭秋萍謝明勳
口試委員(外文):Tsan-Piao LinHsu-Liang HsiehChiu-Ping ChengMing-Hsun Hsieh
口試日期:2014-05-01
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:72
中文關鍵詞:RACK1鷹架蛋白氣孔離層酸TCTP乾旱
外文關鍵詞:RACK1scaffold proteinstomataABATCTPdrought
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Receptor for Activated C-Kinase 1 (RACK1)是一個分子量為36-kDa左右的鷹架蛋白質,普遍存在於動物和植物中。在阿拉伯芥中,有三個基因可以轉譯出RACK1蛋白質,分別命名為RACK1A (At1g18080)、RACK1B (At1g48630) 和RACK1C (At3g18130)。阿拉伯芥中的RACK1蛋白質會參與在賀爾蒙反應和植物生長發育的過程中,且會與40S核醣體發生交互作用。目前,RACK1被視為一個多功能性的鷹架蛋白質,在不同的訊息傳遞途徑中扮演重要的角色。除此之外,RACK1在離層酸反應中扮演一個負向調控的角色。然而,RACK1A的生理功能及究竟是與哪些蛋白質發生交互作用,共同參與或調控這些訊息傳遞的過程,都是尚未清楚的部分。本研究利用酵母菌雙雜合篩選的方式,找到了許多有可能與AtRACK1A產生交互作用的蛋白質,其中包括Translationally Controlled Tumor Protein (TCTP),同時進一步以酵母菌雙雜合系統及雙分子螢光互補實驗證實AtTCTP與AtRACK1A是有直接交互作用。藉由分析RACK1A的蛋白質序列,我們也發現到有一段含有10個胺基酸的序列只會存在於雙子葉植物的RACK1中。之後建立AtRACK1A大量表現株及篩選rack1突變株,發現AtRACK1A會參與在根與葉的生長發育中。在模擬乾旱逆境下, AtRACK1A基因表現下降。在rack1a突變株中,被離層酸所調控的乾旱標的基因表現上升。更進一步, rack1a突變株的水分散失性較野生型稍低,經由離層酸處理後,其氣孔關閉程度較高,再者處理乾旱逆境後,其存活率亦比野生型高許多,這些結果皆暗示著AtRACK1A除了正向調節生長發育外,亦可能負向參與調控阿拉伯芥對乾旱逆境的耐受性。

Receptor for Activated C-Kinase 1 (RACK1) is a 36-kDa protein, which is highly conserved in both animals and plants. There are three RACK1 genes in Arabidopsis thaliana, namely RACK1A (At1g18080), RACK1B (At1g48630) and RACK1C (At3g18130). AtRACK1 is involved in multiple hormone responses, developmental processes, and associated with 40S ribosomes in Arabidopsis. Notably, RACK1 is regarded as a versatile scaffold protein which serves as a nexus for multiple signal transduction pathways. Moreover, AtRACK1 plays a negative regulator role under abscisic acid (ABA) responses. However, the specific interactions between AtRACK1 and its binding partners are still unclear. Some proteins and transcription factors that can interact with AtRACK1A were identified by yeast two-hybrid screen, including Translationally Controlled Tumor Protein (TCTP). Furthermore, yeast two-hybrid and bimolecular fluorescence complementation were used to study the interactions between TCTP and RACK1A, and results confirmed that RACK1A interacts with TCTP directly. Protein sequence analysis revealed that a short sequence containing 10 amino acids only exists in dicots. After generating the AtRACK1A overexpression line and isolating the rack1 mutant line, we found AtACK1A participates in roots and rosette leaves development based on phenotype. On the other hand, AtRACK1A expression was down regulated by 20% mannitol treatment. Furthermore, some drought response marker genes that are regulated by ABA are highly expressed in rack1a. After ABA treatment, rack1a showed greater ABA-mediated stomatal closure. We also found that the rack1a showed greatly enhanced tolerance to drought. In summary, these results showed that AtRACK1A not only positively can regulate plant development but also imply that AtRACK1A may negatively participate in the drought stress tolerance.

目錄
中文摘要 VII
ABSTRACT VIII
縮寫對照表 X
第一章 前言 1
一、非生物逆境 1
二、乾旱逆境 1
三、離層酸 3
四、RACK1 (Receptor for Activated C-Kinase 1) 蛋白質 4
1. RACK1的起源 4
2. RACK1的分子結構 4
3. RACK1與核醣體的關係 5
4. RACK1的生理功能 5
五、TCTP (Translationally Controlled Tumor Protein) 蛋白質 7
1. TCTP概述 7
2. TCTP的生理功能 8
3. TCTP與逆境的關係 9
六、研究目標 9
第二章 材料與方法 11
一、 植物材料及生長條件 11
二、 重組蛋白質之定點突變 (site-directed mutagenesis) 12
三、 大腸桿菌勝任細胞 (competent cell) 之製備 12
四、 DNA洋菜膠體電泳 13
五、 細菌轉型作用 (tansformation) 13
六、 雙分子雜合互補系統 (BiFC)及蛋白胞內座落位置(subcellular localization) 14
七、 阿拉伯芥原生質體 (protoplast) 的分離和質粒轉殖 14
八、 RNA的抽取及q-PCR分析 15
九、 基因組DNA抽取 16
十、 乾旱逆境耐受性和葉片水分散失性測量 16
十一、 氣孔開合程度測量 17
十二、 酵母菌雙雜合系統 (yeast two-hybrid system) 17
第三章 結果 20
一、RACK1蛋白質序列比對及親緣關係分析 20
二、觀察阿拉伯芥RACK1A在阿拉伯芥原生質體細胞內分布 20
三、培育ATRACK1A和ATRACK1A-S286A大量表現轉殖株以及篩選rack1a突變株 22
四、觀察正常條件下AtRACK1A大量表現轉殖株及rack1a突變株的外表型 22
五、非生物逆境下AtRACK1A的表現量 23
六、乾旱標的基因在各轉殖株的表現量 24
七、AtRACK1A大量表現株及rack1a突變株之乾旱耐受性 24
八、觀察正常條件下AtRACK1A-S286A大量表現株之外表型 26
九、AtRACK1A-S286A大量表現株在高鹽與乾旱逆境的外表型 26
十、以酵母菌雙雜合方法篩選出潛在與阿拉伯芥RACK1A有交互作用的蛋白質 27
十一、驗證阿拉伯RACK1A和TCTP之分子間交互作用 28
十二、觀察阿拉伯芥TCTP在阿拉伯芥原生質體細胞內的分布 29
十三、AtRACK1A大量表現株及rack1a突變株對雷帕黴素 (rapamycin) 的敏感性測試 29
第四章 討論 31
參考文獻 36
圖表 46
附錄 64






























圖目錄
圖1、不同物種的RACK1蛋白質親緣關係分析 46
圖2、各物種的RACK1蛋白質C端之序列比對 47
圖3、AtRACK1A大量表現轉殖株及rack1a突變株之篩選 48
圖4、在正常條件下AtRACK1A大量表現株及rack1a突變株之簇生葉外表型 49
圖5、在正常條件下AtRACK1A大量表現株及rack1a突變株之根部外表型 50
圖6、AtRACK1A大量表現株及rack1a突變株的乾旱標的基因表現量 51
圖7、AtRACK1A大量表現株及rack1a突變株水分散失性試驗 52
圖8、AtRACK1A大量表現株及rack1a突變株的氣孔開閉程度 53
圖9、AtRACK1A大量表現株及rack1a突變株於乾旱逆境處理下之存活率 54
圖10、在正常條件下RACK1A-S286A大量表現株之外表型 55
圖11、在非生物逆境下AtRACK1A-S286A大量表現株之外表型 56
圖12、以雙分子螢光互補系統 (BiFC) 驗證阿拉伯芥TCTP蛋白和RACK1A蛋白具有交互作用 57
圖13、以酵母菌雙雜合系統驗證阿拉伯芥TCTP蛋白質和RACK1A蛋白質具有交互作用 58
圖14、TCTP在阿拉伯芥原生質體中細胞內的分布 59








表目錄
表一、以酵母菌雙雜合系統篩選出潛在與RACK1A有交互作用之蛋白 60
表二、以酵母菌雙雜合系統篩選出潛在與RACK1A有交互作用之轉錄因子 60



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