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研究生:鄧伊珊
研究生(外文):Yi-Shan Teng
論文名稱:葉綠體內膜運輸機組蛋白CIA5的功能與特性分析
論文名稱(外文):Characterization of Chloroplast Inner Envelope Membrane Protein,CIA5(Chloroplast-Import-Apparatus)
指導教授:李秀敏李秀敏引用關係
指導教授(外文):Hsou-min Li
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:2004
畢業學年度:92
語文別:中文
論文頁數:73
中文關鍵詞:內膜運輸機組蛋白葉綠體
外文關鍵詞:chloropolastCIA5
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中文摘要

大部分葉綠體蛋白質是由細胞核基因所轉錄,於細胞質中合成後送入葉綠體中。由細胞核基因轉錄轉譯而來的葉綠體前驅蛋白質(precursor proteins)在N端具有導引訊息(transit peptide),主要會經由葉綠體內膜及外膜上的運輸機組(translocon complex)一連串的作用輸入葉綠體中。運輸機組成員中位於葉綠體外膜者稱為Toc (translocons at the outer envelope membrane of chloroplasts)蛋白,位於內膜者稱為Tic (translocons at the inner envelope membrane of chloroplasts)蛋白。利用標的基因(marker gene)篩選蛋白質輸入葉綠體機制發生缺失突變株的方法,我們得到一個阿拉伯芥突變株cia5 (chloroplast import apparatus 5)。經由實驗結果顯示這個蛋白質為一葉綠體內膜蛋白質並具有四個transmembrane domains,且N端和C端朝向葉綠體基質(stroma)。在cia5 突變株中,可以觀察到葉綠體前驅蛋白質會累積在葉綠體外,無法輸入葉綠體中,並且內膜運輸機組蛋白的含量,也會有下降的情形。利用免疫共沉澱法(coimmunoprecipitation),發現CIA5為新發現的葉綠體內膜運輸機組蛋白。將CIA5的胺基酸序列透過電腦程式分析及比較,顯示CIA5在許多植物種類中都有同源蛋白質,且保守度相當高,它可能具有膜通道(membrane permease)的功能。
Abstract

Most chloroplast proteins are encoded by the nucleus, synthesized in the cytosol and imported into chloroplasts. These nucleus-encoded proteins are synthesized as precousors with N-terminal transit peptides, and are transported into chloroplasts by a set of translocon complex located in the chloroplast outer and inner envelope membranes. Translocon components are termed Toc (translocons at the outer envelope membrane of chloroplasts) and Tic (translocons at the inner envelope membrane of chloroplasts) proteins. We have isolated an Arabidopsis mutant, cia5 (chloroplast import apparatus 5), which is defective in chloroplast protein import. CIA5 is predicted to be a membrane premease-like protein, and is extremely conserved in many plant species. Here I show that CIA5 is a chloroplast inner envelope membrane protein containing four transmembrane domains and both its N and C termini face the stroma. I also found that loss of CIA5 resulted in defects in chloroplast protein import, and in reduced amount of inner membrane translocon proteins. Coimmunoprecipiptation studies suggest that CIA5 is a new Tic component.
目錄

一、縮寫表……………………………………………………………………Ⅰ
二、中文摘要…………………………………………………………………Ⅵ
三、英文摘要…………………………………………………………………Ⅴ
四、前言………………………………………………………………………1
五、材料與方法
1. 植物材料……………………………………………………………………7
2. 阿拉伯芥葉綠體製備………………………………………………………8
3. 豌豆葉綠體製備…………………………………………………………...10
4. 以硫酸十二脂鈉聚丙烯醯胺膠片進行電泳分析(SDS-PAGE)…...……...10
5. 活體外轉錄(In vitro transcription)…………………………………………11
6. 活體外轉譯(In vitro translation)……………………………………………12
7. 蛋白質濃度測定……………………………………………………………12
8. 抽取阿拉伯芥植物組織蛋白質……………………………………………13
9. 西方點漬法(Western bolt)………………………………………………….13
10. 抽取少量質體DNA………………………………………………………..15
11. 構築CIA5部分片段刪除載體(delection constructs)……………………...15
12. 葉綠體蛋白質定位(localization)…..……………………………………….18
13. 銀染(silver stain)……………………………………………………………19
14. 免疫共沉澱法(co-immunoprecipitation)…………………………………...20
15. CIA5於葉綠體內膜方向性(topology)之分析……………………………..21
16. CIA5 RNA輸入內質網囊泡(microsome)之分析………………………..24
17. 利用非洲爪蟾(Xenopus laevis)卵做為表現系統……………….………..25
18. 以螢光標示抗體染色觀察蛙卵表現情形………………………………..27
六、結果
1. CIA5位於葉綠體內膜……………………………….……………………29
2. cia5突變株會影響葉綠體的蛋白質運輸能力…………………………...30
3. cia5突變株中內膜運輸機組成員的含量會下降………………………...31
4. CIA5為新的內膜機組成員………………………………………….........32.
5. CIA5具有四個transmembrane domains且N端與C端皆朝向基質…..33
6. CIA5 homolog--synCIA5可以表現在內質網囊泡(microsome)上………36.
7. 經由螢光標定法觀察到synCIA5-cMyc可以成左穛{在非洲爪蟾卵母細胞細胞膜上………………………………………………………………...39
8. 由免疫共沉澱法預期找出新的與CIA5在一起的內膜機組蛋白………39.
七、討論………………………………………………………………………41
八、參考文獻…………………………………………………………………46
九、圖表
圖一、前驅蛋白質輸入葉綠體示意圖……………………………………….50
圖二、篩選葉綠體蛋白質輸入葉綠體機制發生缺失突變株之示意圖…….51
圖三、cia突變株外表型……………………………………………………....52
圖四、CIA5之染色體定位圖譜………………………………………………53
圖五、cia5、ppi2、tic20突變株與同時期的野生型外表型之比較.….........54
圖六、CIA5與其他物種CIA5 homolog序列比對…………………………..55
圖七、CIA5為葉綠體蛋白質……………………………………………….56
圖八、CIA5表現於葉綠體膜上……………………………………………..57
圖九、CIA5位於葉綠體內膜………………………………………………..58
圖十、cia5突變株會影響葉綠體蛋白質的運輸能力………………………59
圖十一、cia5突變株會影響蛋白質進入葉綠體中…………………………60
圖十二、cia5突變株中內膜運輸機組成員含量會下降……………………61
圖十三、ppi2突變株中內外膜運輸機組成員含量都會下降………………62
圖十四、tic20突變株中內外膜運輸機組成員含量都會下降……………...63
圖十五、CIA5為新的內膜機組成員………………………………………..64
圖十六、內膜囊泡形成示意圖………………………………………………65
圖十七、Pro35S:CIA5-cMyc轉植株CIA5於阿拉伯芥葉綠體囊泡的
方向性………………………………………………………………66
圖十八、不同長度的CIA5基因缺失片段示意圖………………………….67
圖十九、CIA5基因缺失片段於豌豆葉綠體內膜上的方向性…..................68
圖二十、Pro35S:CIA5-cMyc於阿拉伯芥葉綠體內膜上的方向性…...........69
圖二十一、CIA5基因缺失片段於豌豆葉綠體內膜囊泡上的方向性……..70
圖二十二、SynCIA5-cMyc可以成打﹞J內質網囊泡膜上………………...71
圖二十三、SynCIA5-cMyc可以成左穛{於蛙卵細胞膜上………………...72
圖二十四、以免疫共沉澱法找出新的與CIA5在一起的內膜蛋白質……..73
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