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

張心嚴(Hsin-Yen Chang)

指導教授 ： 孫智雯博士

國立臺灣師範大學/理學院/生命科學研究所/碩士(2008年)

摘要

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。

關鍵字

泛素 ； 阿拉伯芥

並列摘要

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.

並列關鍵字

ubiquitin ； Arabidopsis

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