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研究生: 劉彥民
Yen-Ming Liu
論文名稱: 阿拉伯芥內CIA2蛋白調節Chaperonin10基因之表現分析
CIA2 regulates expression of Chaperonin10 gene 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: 98
語文別: 中文
論文頁數: 38
英文關鍵詞: cpn10, groES, groEL, CIA2
論文種類: 學術論文
相關次數: 點閱:98下載:1
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  • 植物體內進行光合作用時,葉綠體扮演了非常重要的角色。然而葉綠體內大部分的蛋白質是由細胞核內的基因進行轉錄後,送到細胞質中轉譯產生蛋白質,再藉由葉綠體外膜以及內膜上的轉運蛋白機組(translocon complex)送進葉綠體內。隨後,這些進入葉綠體的蛋白質,必須經由chaperonin 60 (Cpn60)以及chaperonin 10 (Cpn10)所組成的複合體,將其摺疊成成熟的構形,蛋白質才能具有正常的功能。在阿拉伯芥中有兩個同源基因AT2G44650 (Cpn10-II)及AT3G60210 (Cpn10-III)與編譯葉綠體型Cpn10蛋白有關,但是根據我們之前微矩陣以及染色質免疫沉澱分析的研究發現,Cpn10-II表現會受到轉錄因子CIA2的調節,而Cpn10-III則不會明顯地受到CIA2的影響。在此研究中,轉殖植株實驗顯示CIA2不但能調節Cpn10-II基因表現量,並且是造成Cpn10-II有專門組織表現的主因。定量反轉錄聚合酶連鎖反應則顯示Cpn10-II在植株中的表現量的確受CIA2的調節,而且其表現量亦隨植齡的增加而遞減。Cpn10-III基因則是在缺少CIA2轉錄因子的植株中,相對轉錄表現較野生型植株中來得高。這些結果顯示Cpn10-II基因確實會受到CIA2蛋白的調控,而Cpn10-III基因會在Cpn10-II基因表現降低時提高,以彌補Cpn10-II基因的功能。

    Chloroplast plays an important role in plant photosynthesis. However, most chloroplastic proteins are transcribed in nucleus, translated in cytoplasm, and then imported into chloroplasts through translocon complex. Therefore, theses nuclear-encoded chloroplastic proteins have to be folded correctly afterwards by chaperonin complex which are assembled with Chaperonin 60 (Cpn60) and Chaperonin 10 (Cpn10) to maintain their biological functions in chloroplasts. Two chloroplastic Cpn10-encoding genes, AT2G44650 (Cpn10-II) and AT3G60210 (Cpn10-III), are found in Arabidopsis thaliana based on homological search. According to our previous microarray and chromatin immuno-precipatation (ChIP) analyses, we found a transcription factor CIA2 which could directly regulate the expression of Cpn10-II gene but not Cpn10-III. In this study, the results of transgenic studies reveal that CIA2 is responsible for regulating the yield of protein and tissue specificity of Cpn10-II expression. Quantitative RT-PCR analyses also suggest that Cpn10-II expression is up-regulated by CIA2 in an age-dependent manner. In contrast, Cpn10-III seems to have higher expression in cia2 and cia2/Cpn10-II plants. We conclude that Cpn10-II expression is indeed regulated by CIA2, and the Cpn10-II defect can be compensated by Cpn10-III.

    縮寫表 3 中文摘要 4 英文摘要 5 序論 6 材料與方法 8 結果 14 討論 18 參考文獻 21 表一、Cpn10-II、Cpn10-II以及CIA2專一引子序列資料 24 表二、不同品系的同型合子轉殖植株 25 圖一、AT2G44650 (Cpn10-II)以及AT3G60210 (Cpn10-III)的基因結構示意圖 26 圖二、雙突變植株的分析 27 圖三、4週齡大的WT、cia2、cpn10-II、cia2/ cpn10-II-3-4的外表型 28 圖四、Cpn10-II及Cpn10-III在WT、30-2、cpn10-II、cia2/cpn10-II植株中的表現差異 29 圖五、含有Cpn10-II及Cpn10-III上游序列的報導基因質體示意圖 30 圖六、同型合子之T4轉殖阿拉柏芥轉殖植株GUS染色結果 31 圖七、轉殖植物的GUS活性分析 32 圖八、Cpn10-II及Cpn10-III在不同年齡WT、cia2、cia2/cpn10-II植株內的表現 33 圖九、Cpn10-II以及Cpn10-III在不同植物組織的表現 34 圖十、構築Cpn10-II、以及Cpn10-III基因啟動子刪減實驗的質體 35 圖十一、Cpn10-II啟動子刪減的報導基因定量分析 36 圖十二、CIA2轉錄蛋白調節Cpn10-II基因表現之可能機制 37 附錄一、GroEL-GroES複合體的結構示意圖 38

    Clough, S.J., Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The plant journal. 16:735-743.
    Martin, J. and Hartl, F. U. (1997). Molecular Chaperones in the Cytosol: From Nascent Chain to Folded Protein. Science. 295:1852-1858.
    Koumoto, Y., Shimada, T., Tsugeki, R. (1996). Chloroplasts have a novel Cpn10 in addition to Cpn20 as co-chaperonins in Arabidopsis thaliana. The plant Journal. 10: 1119-1125.
    Kouoto, Y., Shimada, T. (1999). Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana. The plant Journal. 17:467-477.
    Kouoto, Y., Shimada, T., Kondo, M. (2001). Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant. The Journal of Biological Chemistry. 276:29688-29694.
    Koumoto, Y. (2001). Chloroplasts Have a Novel Cpn10 in Addition to Cpn20 as Co-chaperonins in Arabidopsis thaliana. The Journal of Biological Chemistry. 2:29688-29694.
    Nakamichi, N., Kita, M., Ito, S., Sato, E., Yamashino, T., Mizuno, T. (2005). The Arabidopsis pseudo-response regulators, PRR5 and PRR7, coordinately play essential roles for circadian clock function. Plant Cell Physiol, 46:609-619.
    Putterill, J., Robson, F., Lee, K., Simon, R., and Coupland, G. (1995). The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc-finger transcription factors. Cell. 80:847–857.
    Rybicki. E.P. ., M.B. von Wechmar. (1982). Enzyme-assisted immune detection of plant virus proteins electroblotted onto nitrocellulose paper. Journal of Virological Methods. 5: 267-278.
    Strayer, C., et al (2000). Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science. 289:768-771.
    Sun, CW., Chen, L.J., Lin, L.C., and Li, H.M. (2001). Leaf-specific upregulation of chloroplast translocon genes by a CCT Motif–containing protein, CIA2. The Plant Cell. 13:2053-61.
    Sun CW., Huang YC., and Chang HY. (2009). CIA2 coordinately up-regulates protein import and synthesis in leaf chloroplasts. Plant Physiology. 150:879-888.
    Soll, J. and Schleiff, E. (2004). Protein import into chloroplasts. Nature reviews molecular cell Biology. 5:198-208.
    Salome, P.A., To, J.P.C., Kieber, J.J., and McClung, C.R. (2006). Arabidopsis response regulators ARR3 and ARR4 play cytokinine independent roles in the control of circadian period. Plant Cell. 18:55-69.
    Salbi, HR. (2008). Chaperone machines in actions. Curr. Opin. Struct. Biol. 18:35-42.
    Todd, M.J. (1994). Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding. Science. 265:659-666.
    Viitanen, P., Schmidt, M. (1995). Functional characterization of the higher plant chloroplast chaperonins. The Journal of Biological Chemistry. 270:18158-18164.
    Wessiman, J.S., et al (1995). Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES. Cell. 83:577-587.
    Yang, T., Poovaiah, W.B. (2000). Arabidopsis Chloroplast Chaperonin 10 is a Calmodulin-Binding Protein. Biochemical and Biophysical Research Communications. 275:601–607.

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