| 摘要: | 生物遇到高溫逆境時皆會誘導合成熱休克蛋白質，誘導機制為細胞內因高溫造成蛋白質變性，這些變性蛋白質可致活熱休克因子﹙Heat shock factor; HSF﹚及促使熱休克因子與熱休克元素﹙Heat shock element; HSE﹚結合。除熱休克外，許多因子亦可誘導熱休克蛋白質合成，如胺基酸類似物，砷、鎘及離層酸皆可在常溫下誘導熱休克蛋白質合成。水稻中第一族小分子量熱休克蛋白質–Oshsp17.3 可在常溫下被proline 的類似物 azetidine﹙Aze﹚誘導合成，經利用啟動子5’端刪除法與暫時表現系統研究確認出一段 Aze 反應元素﹙Azetidine responsive element；AZRE﹚，位於Oshsp17.3 啟動子-186 至-176 核?酸位置。進一步分析AZRE 顯示AZRE 無法使非Aze 誘導型啟動子對Aze 產生明顯反應﹙約增加2~3 倍﹚；但若刪除AZRE 則會使Oshsp17.3 啟動子完全失去對Aze 反應性，另刪除AZRE 亦會使Oshsp17.3 的熱休克反應降低90％，相同結果亦見於阿拉伯芥的相關轉殖株。因此AZRE 會影響細胞內的Aze 反應與熱休克反應。利用yeast one–hybrid selection 技術篩選與AZRE 結合之蛋白質﹙ AZRE binding protein; AZREBP﹚，已篩得17 個可能基因，正利用electrophoretic mobility shift assay 確認中。本計劃預計三年完成，主要目標是研究第一族小分子量熱休克蛋白質受熱及Aze 誘導的調控機制。主要工作包括AZRE 與AZREBP 的特性分析、HSFs 在熱及Aze 逆境下的差異性表現和AZRE 如何影響HSFs 與HSE 的結合。首先預計分析熱及Aze 逆境對AZREBP 在細胞內表現位置與表現模式的影響。另方面改用取代方式改變Oshsp17.3 啟動子序列，以暫時性表現系統分析除AZRE 外是否尚有其它elements 參與水稻Aze 反應途徑中。上述結果有助於我們了解Aze 啟動第一族小分子量熱休克蛋白質的作用機制。接著預計建立過度表現與抑制AZREBP 基因的水稻轉殖株，分析轉植株外部型態及對逆境的抗性與生理反應。此部分結果可幫助我們探討AZREBP 的生理功能。最後我們將全面蒐尋所有水稻HSFs，利用Northern–blot 與RT–PCR 方式分析各個HSF 在熱及Aze 逆境下的表現模式，藉此分析HSFs 對不同群的小分子量熱休克蛋白質是否有差異性調控機制存在。 All organisms respond to heat shock (HS) stress by the predominant synthesis of heat shock proteins (HSPs). The transcription of HSP genes is triggered by thermal misfolding of cellular proteins, which leads to the activation of HS factor (HSF) binding to HS elements (HSEs). Other than HS, many agents such as amino acid analogs, arsenite, cadmium and abscisic acid activate HSP expression under normal temperatures. In rice, a class I small heat shock protein gene (sHSP-CI), Oshsp17.3 is selectively induced by azetidine (a proline analog; Aze). Using 5’ promoter deletion analysis of transient expression by rice coleoptile, a motif of GTCCTGGAC located between nucleotides -186 and -178 relative to the transcription initiation site of Oshsp17.3 was identified. By addition of the putative Aze responsive element (AZRE) to non-Aze-inducible promoters (Oshsp16.9A), we cannot observe significant Aze inducibility (2- to 3-fold). In contrast, by deletion of this putative AZRE inducibility of Oshsp17.3 were totally abolished by Aze and also greatly reduced by HS (90% loss). The requirement of this AZRE for Aze and HS inducibility was further confirmed in transgenic Arabidosis. Thus, the putative AZRE is essential for response to Aze and to certain extent required for HS inducibility. Conducting yeast one-hybrid selection techniques, we screened 17 possible AZRE binding proteins (AZREBPs). Now we are working on identifying the trans-acting factor by electrophoretic mobility shift assay. In this three-year project, our major goal is to study differential regulatory mechanism of sHSP-CIs induction by HS and Aze. The major works are focused on characterizing AZRE as well as AZREBPs, differential expression pattern of HSFs in response to HS and Aze treatment, and the effect of AZRE on the binding of HSFs to HSEs. Firstly, we plan to analyze cellular localization and expression pattern of AZREBP during HS and Aze treatment. Besides, to define more elements interacted with AZRE and HSE on Oshsp18.0 promoter region, we make constructs with an approximately 50-bp serial replacement from the 5’-end of the Oshsp17.3 for transient expression assay. The data obtained from here can provide us more information for the signaling pathway of Aze on activation of sHSP-CIs. Secondly, we plan to introduce AZREBP gene into rice to express AZREBP constitutively and use RNA interference techniques to knock down the transcription level of AZREBP in rice at any conditions including normal temperature, HS, and Aze treatment. Assay for phenotype and tolerance to stresses of these transgenic rice plants is being conducted to find the physiological function of AZREBP. Finally, we will mine all HSF genes from rice genome by bioinformatics tools and detect the expression level under HS and Aze treatment using Northern-blot and RT-PCR analysis. Besides, the interaction between HSFs and AZRE is also studied. The results will be helpful for understanding the differential regulatory mechanism of sHSPs. 研究期間 : 9808 ~ 9907 |
