簡易檢索 / 詳目顯示

研究生: 張心嚴
Hsin-Yen Chang
論文名稱: 阿拉伯芥聚泛素基因UBQ3與UBQ4之表現功能研究
Functional studies of Arabidopsis polyubiquitin genes UBQ3 and UBQ4
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: 52
中文關鍵詞: 泛素阿拉伯芥
英文關鍵詞: ubiquitin, Arabidopsis
論文種類: 學術論文
相關次數: 點閱:124下載:4
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 泛素(ubiquitin)是普遍存在於真核生物體中的小型蛋白質。在不同的生物體中,其序列與結構皆有高度的保守性,是調控許多訊息傳遞功能的重要蛋白質。阿拉伯芥含有許多種類的泛素基因,其中一種為聚泛素基因(polyubiquitin genes)。阿拉伯芥共有五個聚泛素基因,分別為UBQ3、UBQ4、UBQ10、UBQ11與UBQ14。而UBQ3與UBQ4是旁系同源的聚泛素基因,但是在發育過程中卻有不同的基因表現方式。因此本實驗以研究UBQ3與UBQ4的組織表現差異與找尋調控基因表現因子、調控序列為研究目標。從轉殖植物偵測報導基因(GUS)的活性,發現UBQ3與UBQ4在營養器官的表現位置沒有明顯不同,但是在花朵內卻有顯著的表現差異,這代表在生殖生長階段,UBQ3與UBQ4的表現是受不同的途徑調控。另外,更詳細地分析轉殖植物的報導基因表現後,發現內插子序列是調節UBQ3與UBQ4基因表現量最主要的因素:它會增強UBQ3的表現量,卻抑制UBQ4的表現。但序列分析及電泳膠遲緩實驗亦證明UBQ3及UBQ4的內插子序列仍共享二條順式作用序列。最後藉由給予阿拉伯芥不同光線與溫度的環境刺激,發現只有UBQ3的基因表現量會因紅光、藍光、黑暗與紫外線的刺激而增加,而UBQ4的基因表現量並不受光線或溫度的刺激而改變,表示UBQ3的表現會受光線所調控,UBQ4則否。綜合以上結果,在不同發育時期或在不同環境因子下生長,影響UBQ3與UBQ4基因表現差異的主要因素是轉錄調節。

    Ubiquitin is a small protein existing in all eukaryotes with a conserved sequence and structure. It has been found to play important roles in controlling cellular signal transduction. In Arabidopsis, ubiquitins are primarily encoded by five polyubiquitin genes, including UBQ3, UBQ4, UBQ10, UBQ11, and UBQ14. UBQ3 and UBQ4 are the paralogous polyubiquitin genes, however, they express differentially through various developmental stages. Thus, in this study, we first investigated the spatial expression profile of UBQ3 and UBQ4, and the regulatory mechanisms of gene expression. GUS activity assay in transgenic plants revealed that UBQ3 and UBQ4 were expressed at the same locations in vegetative tissues, but the expression patterns varied in reproductive tissues. Therefore, we suggest that UBQ3 and UBQ4 are differentially expressed in reproductive tissues due to different regulatory pathways. Further studies indicated that intron sequence play an important role in regulating the expression of UBQ3 and UBQ4 in planta: it enhances the transcript level of UBQ3, but represses that of UBQ4. Nevertheless, sequence analyses and EMSA experiments revealed that intron sequences of UBQ3 and UBQ4 still share two conserved cis-acting elements. Finally, Arabidopsis seedlings were treated under different light wavelength and temperature conditions. We found that UBQ3 increases transcript levels in dark, red light, blue light, and ultraviolet rays, but UBQ4 remains similar transcript levels under these treatments. In conclusion, the differential expression of UBQ3 and UBQ4 is predominantly regulated at the transcriptional levels during reproductive development and under various environmental factors, such as light and temperature.

    中文摘要……………………………………………………………………… 1 英文摘要……………………………………………………………………… 2 縮寫表………………………………………………………………………… 3 緒論…………………………………………………………………………… 4 材料與方法…………………………………………………………………… 8 實驗結果……………………………………………………………………… 13 分析與討論…………………………………………………………………… 18 參考文獻……………………………………………………………………… 21 表格與圖片…………………………………………………………………… 27 表一 專一引子對序列資料……………………………………………… 27 表二 UBQ3與UBQ4轉殖株之GUS定量結果…………………………… 28 表三 阿拉伯芥聚泛素基因序列之相似片段數目……………………… 29 表四 UBQ3與UBQ4內插子之相異序列………………………………… 30 表五 UBQ3與UBQ4內插子序列之強化訊息分析……………………… 31 表六 聚泛素基因表現之微陣列資料分析……………………………… 32 圖一 泛素化作用機制…………………………………………………… 33 圖二 阿拉伯芥聚泛素基因與泛素延伸基因之結構…………………… 34 圖三 UBQ3啟動子連接不同組合五端未轉譯區域/內插子之構築序列… 35 圖四 UBQ4啟動子連接不同組合五端未轉譯區域/內插子之構築序列… 36 圖五 比對阿拉伯芥聚泛素基因之五端未轉譯區域序列……………… 37 圖六 內插子調控序列UIE1之序列資訊、相似度與位置……………… 38 圖七 內插子調控序列UIE2之序列資訊、相似度與位置……………… 39 圖八 內插子調控序列UIE3之序列資訊、相似度與位置……………… 40 圖九 內插子序列與總蛋白質之交互作用結果………………………… 41 圖十 UIE1之總蛋白質競爭實驗結果…………………………………… 42 圖十一 UIE2之總蛋白質競爭實驗結果………………………………… 43 圖十二 UIE3之總蛋白質競爭實驗結果………………………………… 44 圖十三 溫度對阿拉伯芥UBQ3和UBQ4基因表現的影響……………… 45 圖十四 藍光與紅光對UBQ3和UBQ4基因表現的影響………………… 46 圖十五 黑暗處理對UBQ3和UBQ4基因表現的影響………………… 47 圖十六 紫外線(UV-B)處理對UBQ3和UBQ4基因表現的影響………… 48 附錄一 阿拉伯芥泛素基因家族……………………………………… 49 附錄二 14天齡含U3.1、U3.2或U3.3序列的不同品系轉殖株之GUS染色結果 50 附錄三 14天齡含U4.1、U4.4或U4.5序列的不同品系轉殖株之GUS染色結果 51 附錄四 14天齡含U4.2、U4.3或U4.6序列的不同品系轉殖株之GUS染色結果 52

    1. Bachmair, A., Novatchkova, M., Potuschak, T., and Eisenhaber, F. (2001). Ubiquitylation in plants: a post-genomic look at a post-translational modification. TRENDS in Plant Science. 6, 463-470.
    2. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., and Elledge, S.J. (1996). Skp1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 86: 263-274.
    3. Bailey, T.L., and Gribskov, M. (1998). Methods and statistics for combining motif match scores. Journal of computational biology. 5: 211-221.
    4. Brosché, M., Schuler, M.A., Kalbina, I., Connor, L., and Strid, Å. (2002). Gene regulation by low level UV-B radiation: identification by DNA array analysis. Photochemical and photobiological sciences. 1: 656-664.
    5. Burke, T.J., Callis, J., and Vierstra, R.D. (1988). Characterization of a polyubiquitin gene from Arabidopsis thaliana. Molecular and general genetics. 213:435-443.
    6. Callis, J., Raasch, J.A., and Vierstra, R.D. (1990). Ubiquitin extension proteins of Arabidopsis thaliana. Structure, localization, and expression of their promoters in transgenic tobacco. Journal of biological chemistry. 265: 12486-12493.
    7. Callis, J., Carpenter, T., Sun, C.W., and Vierstrat, R.D. (1995). Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia. Genetics. 139: 921-939.
    8. Carter, C., Graham, R.A., and Thornburg, R.W. (1998). Arabidopsis thaliana contains a large family of germin-like proteins: characterization of cDNA and genomic sequences encoding 12 unique family members. Plant molecular biology. 38: 929-943.
    9. Catic, A., and Ploegh, H.L. (2005). Ubiquitin - conserved protein or selfish gene? Trends in biochemical sciences. 30: 600-604.
    10. Chae, E., Tan, Q.K.G., Hill, T.A., and Irish, V.F. (2008). An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development. Development. 135: 1235-1245.
    11. Chau, V., Tobias, J.W., Bachmair, A., Marriott, D., Ecker, D.J., Gonda, D.K., and Varshavsky, A. (1989). A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 243: 1576-1583.
    12. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical biochemistry. 162: 156-159.
    13. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The plant journal. 16: 735-743.
    14. Crooks, G.E., Hon, G., Chandonia, J.M., and Brenner, S.E. (2004). WebLogo: a sequence logo generator. Genome research.14: 1188-1190.
    15. Fan, W.H., and Dong, X.N. (2002). In vivo interaction between NPR1 and transcription factor TGA2 leads tosalicylic acid–mediated gene activation in Arabidopsis. The plant cell. 14:1377-1389.
    16. Finley, D., Özkaynak, E., and Varshavsky, A. (1987). The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell. 48: 1035-1046.
    17. Finley, D., and Chau, V. (1991). Ubiqitination. Annual review of cell biology. 7: 25-69.
    18. Finley, D., Sadis, S., Monia, B.P., Boucher, P., Ecker, D.J., Crooke, S.T., and Chau, V. (1994). Inhibition of proteolysis and cell cycle progression in a multiubiquitination- deficient yeast mutant. Molecular and cell biology. 14: 5501-5509.
    19. Fisk, H.A., and Yaffe, M.P. (1999). A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae. Journal of cell biology. 145: 1199-208.
    20. Galan, J.M., and Haguenauer-Tsapis, R. (1997). Ubiquitin Lys63 is involved in ubiquitination and endocytosis of a yeast plasma membrane protein. The EMBO journal. 16: 5847-54.
    21. Glickman, M.H., and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiological reviews. 82: 373-428.
    22. Goldstein, G., Scheid, M., Hammerling, U., Schlesinger, D.H., Niall, H.D., and Boyse, E.A. (1974). Isolation of a polypeptide that has lymphocyte- differentiating properties and is probably represented universally in living cells. Proceedings of the national academy of sciences of the United States of America. 72: 11-15.
    23. Gray, W.M., del Pozo, J.C., Walker, L., Hobbie, L., Risseeuw, E., Banks, T., Crosby, W.L., Yang, M., Ma, H., and Estelle, M. (1999). Identification of an SCF ubiquitin ligase complex required for auxin response in Arabidopsis thaliana. Genes and development. 13: 1678-1691.
    24. Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system: resolution, affinity purification and role in protein breakdown. The journal of biological chemistry. 258: 8206-8214.
    25. Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984). ATP-dependent degradation of ubiquitin-protein conjugates. Proceedings of the national academy of sciences. 81: 1619-1623.
    26. Hershko, A., and Ciechanover, A. (1992). The ubiquitin system. Annual review of clinical biochemistry. 61: 761-807.
    27. Hochstrasser, M. (2006). Lingering mysteries of Ubiquitin-Chain assembly. Cell. 124: 27-34.
    28. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G., and Jentsch, S. (2002). RAD6- dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 419: 135-41.
    29. Hofmann, K., and Bucher, P. (1998). The PCI domain: a common theme in three multi-protein complexes. Trends in Biochemical Sciences. 23: 204-205.
    30. Imaizumi, T., Tran, H.G., Swartz, T.E., Briggs, W.R., and Kay, S.A. (2003). FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis. Nature. 426: 302-306.
    31. Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS-fusions: β-glucuronidase as a sensitive and versatile fusion marker in higher plants. The EMBO journal. 6: 3901-3907.
    32. Lai, Z., Ma, W., Han, B., Liang, L., Zhang, Y., Hong, G., and Xue, Y. (2002). An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum. Plant molecular biology. 50: 29-42.
    33. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia plantarum. 15: 473-497.
    34. Ni, W., Xie, D., Hobbie, L., Feng, B., Zhao, D., Akkara, J., and Ma, H. (2004). Regulation of flower development in Arabidopsis by SCF complexes. Plant physiology. 134: 1574-1585.
    35. Norris, S.R., Meyer, S.E., and Callis, J. (1993). The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant molecular biology. 21: 895-906.
    36. Osterlund, M.T., Hardtke, C.S., Wei, N., and Deng, X.W. (2000). Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature. 405: 462-466.
    37. Özkaynak, E., Finley, D., and Varshavsky, A. (1984). The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein. Nature. 312: 663-666.
    38. Patton, E.E., Willems, A.R., and Tyers, M. (1998). Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. Trends in genetics. 14: 236-243.
    39. del Pozo, J.C., Timpte, C., Tan, S., Callis, J., and Estelle, M. (1998). The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science. 280: 1760-1763.
    40. Rose, A.B., Elfersi, T., Parra, G., and Korf, I. (2008). Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. The plant cell. 20: 543-551.
    41. Schlessinger, D.H., Goldstein, G., and Niall, H.D. (1975). The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells. Biochemistry. 14: 2214-2218.
    42. Schwechheimer, C., Serino, G., Callis, J., Crosby, W.L., Lyapina, S., Deshaies, R.J., Gray, W.M., Estelle, M., and Deng, X.W. (2001). Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Science. 292: 1379- 1382.
    43. Shanklin, J., Jabben, M., and Vierstra, R.D. (1987). Red light-induced formation of ubiquitin-phytochrome conjugates: identification of possible intermediates of phytochrome degradation. The proceedings of the national academy of sciences. 84: 359-363.
    44. Sun, C.W., and Callis, J. (1997). Independnet modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes. The plant journal. 11: 1017-1027.
    45. Tornero, P., Merritt, P., Sadanandom, A., Shirasu, K., Innes, R.W., and Dangl, J.L. (2002). RAR1 and NDR1 contribute quantitatively to disease resistance in Arabidopsis, and their relative contributions are dependent on the R gene assayed. The Plant Cell. 14: 1005-1015.
    46. Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D., and Xie, D. (2002). The SCFCOI1 ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. The plant cell. 14: 1919-1935.
    47. Yin, X.J., Volk, S., Ljung, K., Mehlmer, N., Dolezal, K., Ditengou, F., Hanano, S., Davis, S.J., Schmelzer, E., Sandberg, G., Teige, M., Palme, K., Pickart, C. and Bachmaira, A. (2007). Ubiquitin lysine 63 chain-forming ligases regulate apical dominance in Arabidopsis. The plant cell. 19: 1898-1911.
    48. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004). GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant physiology. 136: 2621-2632.

    QR CODE