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研究生: 陳怡彰
Yi-Jhang Chen
論文名稱: 阿拉伯芥atToc33與atToc34基因之功能性研究
Functional studies of translocon genes atToc33 & atToc34 in Arabidopsis thaliana
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: 2009
畢業學年度: 97
語文別: 中文
論文頁數: 35
中文關鍵詞: atToc33atToc34
英文關鍵詞: atToc33, atToc34
論文種類: 學術論文
相關次數: 點閱:104下載:1
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  • 葉綠體是植物行光合作用的重要胞器,在遠古時代由藍綠菌與真核細胞內共生而來。然而在演化過程中,超過90%的葉綠體蛋白質基因已經轉移至真核細胞的細胞核內。這些基因產物必須在細胞核內轉錄、在細胞質內轉譯及修飾後再送到葉綠體內。位於葉綠體膜上的轉運蛋白機組(translocon complex)負責辨識及運輸這些蛋白質,其中包含位在外膜上的Toc和位在內膜上的Tic兩大複合體。在阿拉伯芥中已知的轉運機組成員裡,atToc33和atToc34是豌豆Toc34的兩個同源蛋白,在運輸蛋白質進入葉綠體的過程中,扮演最初辨識蛋白質的角色。轉運機組的成員其基因必須被適當的調節,否則這些轉運機組將無法準確的組裝以執行適當的功能。為了了解atToc33和atToc34基因表現的調節的機制,atToc33和atToc34基因的啟動子序列接上GUS報導基因後送入野生型阿拉伯芥,結果顯示這兩個基因5’端非轉譯區內第一內插子對於促進基因表現有顯著的影響。此外,5’端啟動子刪減及膠體電泳位移分析實驗亦找出數個可能調節atToc33基因表現的啟動子區域。

    Chloroplast, originated from endosymbiotic cyanobacteria in the ancient times, is one of the most important organelles in plants for it’s function of photosynthesis. During the evolution of plant cells, more than ninety percent of the plastid genes were gradually transferred to the host nuclear genome. However, these genes have to be transcribed in nucleus, translated and post-translationally modified in cytoplasm, and imported to plastids accurately. The translocons on the outer envelope and inner envelope membrane of chloroplast play key roles on machinery of protein import into the chloroplast. They compose two complexes: Toc complex (Translocon at the Outer envelope membrane of the Chloroplast) and Tic complex (Translocon at the Inner envelope membrane of the Chloroplast). Among the Toc and Tic components identified in Arabidopsis thaliana, atToc33 and atToc34 are homologous to pea psToc34 and play important roles of pre-protein recognition. Evidently, the expression of translocon genes have to be regulated properly, or their gene products will not be accurately integrated to their destination and proceed their mission. In order to reveal the regulatory mechanism of atToc33 and atToc34 gene expression, transgenes containing the promoter sequences of atToc33 and atToc34 gene and GUS coding sequence were transferred into wild-type Arabidopsis. The 1st intron in the 5’UTR of both gene have obvious effect on the regulation of gene expression. Furthermore, 5’ promoter deletion and EMSA experiments reveal several putative regulation regions of promoter sequence to modulate atToc33 expression.

    一、緒論 1 二、材料與方法 1.植物材料 3 2.AtToc33和atToc34基因promoter及5’UTR序列的選殖 3 3.聚合酶連鎖反應(polymerase chain reaction, PCR) 3 4.轉殖質體製備 4 5.大腸桿菌(DH5α)勝任細胞轉型(transformation) 4 6.大腸桿菌轉殖質體萃取 5 7.農桿菌(Agrobacteria GV3101)轉型 5 8.花序沾黏法(floral dipping)轉殖阿拉伯芥 5 9.GUS定性染色 6 10.GUS定量 6 11.基因槍轉殖 6 12.LUC (luciferase)定量 7 13.順式作用元素(cis-acting element)序列分析: 7 14.阿拉伯芥DNA萃取 8 15.阿拉伯芥蛋白質萃取 8 16.分離植物細胞核 8 17.植物細胞核蛋白萃取 9 18.以Biotin-N11-CTP標記順式作用元素寡核苷酸 10 19.EMSA (Electrophoretic mobility shift assay)分析 10 三、結果 1.atToc33及atToc34在阿拉伯芥中的表現 12 2.5’UTR對atToc33及atToc34基因表現的影響 13 3.atToc33啟動子基因調節區域 14 4.atToc33順式作用元素之分析 14 四、討論 16 五、參考文獻 18 六、表格 21 七、圖 24 八、附錄 35

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