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研究生: 黃豔喬
Yen-Chiao Huang
論文名稱: 阿拉伯芥CIA2蛋白調節下游基因表現之分析
The regulatory expression of CIA2 downstream genes in Arabidopsis
指導教授: 孫智雯
Sun, Chih-Wen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 40
中文關鍵詞: 轉運蛋白機組葉綠體核糖體蛋白基因染色質免疫沈澱實驗膠體電泳位移分析
論文種類: 學術論文
相關次數: 點閱:207下載:8
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  • 在植物演化過程中,葉綠體將大多數基因已經轉送至植物細胞核內。因此葉綠體發育及執行正常功能皆受到細胞核基因調控。之前研究發現CIA2是一個轉錄因子,負責調節轉運蛋白機組基因atToc75和atToc33的表現,及調控葉綠體輸入蛋白質的效率。並由阿拉伯芥基因微矩陣晶片調查發現CIA2調節葉綠體分子伴侶蛋白CPN10以及葉綠體核糖體蛋白基因表現。為了要了解CIA2是否在植物細胞內直接調節這些下游基因表現,我們利用染色質免疫沈澱實驗來探討CIA2與否直接鍵結在下游基因啟動子上。實驗結果顯示,CIA2直接參與調控atToc33、atToc75-III、CPN10,及部分葉綠體核糖體蛋白RPL18、RPS6、及RPL15基因。而我們也利用生物資訊工具調查CIA2下游atToc33、atToc75-III、CPN10及葉綠體核糖體蛋白基因啟動子,結果發現兩個未知的順式作用元素CIARE1及CIARE2。由膠體電泳位移分析發現CIARE1 (CIA2-regulatory element)及CIARE2皆會與蛋白質結合。因此,染色質免疫沈澱實驗及膠體電泳位移分析結果進一步釐清CIA2參與調控下游基因的過程,並且幫助了解CIA2參與調控葉綠體發育相關之基因表現的途徑。

    目錄 一、縮寫表 I 二、中文摘要 III 三、英文摘要 IV 四、序論 1 五、研究材料與方法 1. 植物材料 5 2. 染色質免疫沈澱法 5 3. 總蛋白質萃取 8 4. 細胞核蛋白質萃取 9 5. 蛋白質濃度測定 10 6. 利用生物資訊軟體預測CIA2下游基因啟動子共有的順式作用元素 10 7. 膠體電泳位移分析探討順式作用元素與蛋白質的結合 10 六、結果 1. CIA2在植物細胞內與atToc33、atToc75-III、CPN10,及部分葉綠體核糖體蛋白RPL18、RPS6、及RPL15基因啟動子交互作用 13 2. CIA2參與調控atToc75-III基因表現 14 3. CIA2參與調控atToc33基因表現 14 4. CIARE1及CIARE2為CIA2下游基因啟動子上的保守序列 15 5. CIARE1及CIARE2具有蛋白質結合能力 16 6. CIARE1-F、CIARE2-F、及CIARE2-R與蛋白質結合具專一性 16 七、討論 18 八、參考文獻 21 九、圖表 圖一、野生型、cia2突變株、c-Myc:CIA2 (cia2)轉殖株外表型比較 26 圖二、CIA2在植物細胞內與atToc33、atToc75-III及CPN10啟動子結合 27 圖三、CIA2在植物細胞內與葉綠體核糖體蛋白基因RPL18、RPS6、RPL15啟動子交互作用 28 圖四、CIA2在植物細胞內調控atToc75-III基因表現 29 圖五、CIA2在植物細胞內調控atToc33基因表現 30 圖六、CIARE1順式作用元素 31 圖七、CIARE2順式作用元素 32 圖八、CIARE1及CIARE2順式作用元素位於啟動子上相對位置 33 圖九、EMSA分析CIARE1與總蛋白質交互作用 34 圖十、EMSA分析CIARE2與總蛋白質交互作用 35 圖十一、標記與未標記CIARE1-F核苷酸與定量蛋白質進行競爭 36 圖十二、標記與未標記CIARE2-F核苷酸與定量蛋白質進行競爭 37 圖十三、標記與未標記CIARE2-R核苷酸與定量蛋白質進行競爭 38 表一、CIA2調節16個葉綠體核糖體蛋白基因表現 39 表二、染色質免疫沈澱實驗中使用的引子序列 40

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