水稻蔗糖合成酶 RSuS1 之研究：受糖調控之基因表現與細胞內定位

Abstract

蔗糖合成酶催化蔗糖與 UDP 形成果糖與 UDPG 的可逆反應。水稻中有至少有六種蔗糖合成酶基因，其中 RSus1 的表現在轉錄層次上會受蔗糖誘導。為了瞭解 RSus1 基因表現受蔗糖調控的機制，本研究以水稻懸浮培養細胞 (Oryza sativa L. cv. Tainung 67) 為材料，探討細胞以蔗糖或甘露醇處理後所抽取得之核蛋白質，是否與 RSus1 基因 5’ 端的調控區域之間有交互作用。由電泳遲滯分析的結果得知，主要的蛋白質結合區域位於 RSus1 之啟動子轉錄起始點上游 -1045 ~ -1007 (命名為 A-3-2)。A-3-2 序列中富含 purine，且有四個 GCGGCG elements。我們從水稻懸浮培養細胞中，純化出一個可直接與 A-3-2 專一結合的蛋白質。其屬於 purine-rich DNA 結合蛋白質，將之命名為 OsPurα。OsPurα 與阿拉伯芥 Purα-1 之蛋白質序列同質性達 73%，且其預測之蛋白質結構與果蠅 Purα 相似。利用大腸桿菌表現重組 OsPurα 蛋白質後，進一步以膠體電泳遲滯分析，結果顯示重組 OsPurα 蛋白質可與 DNA 片段 A-3-2 產生交互作用。此外，將兩重覆之 A-3-2 片段接於 CaMV 35S minimal 啟動子前端，並對水稻懸浮培養細胞進行轉形。結果顯示，細胞在蔗糖誘存在下，OsPurα 可增強報導基因 GUS 之表現。以蔗糖培養的細胞中，OsPurα 與 A-3-2 間有較佳的結合；然而，OsPurα mRNA 之累積量卻不受蔗糖存在與否而影響。由這部份的研究結果得知，OsPurα 參與 RSus1 基因受蔗糖誘導的調控，但可能還需有其他蛋白質共同參與。 本論文第二部份的研究是探討 RSuS1 蛋白質在細胞中的定位。以西方轉印分析法及免疫共沉澱法，偵測出細胞質與細胞核中皆存在 RSuS。以洋蔥表皮細胞與水稻原生質體進行 sGFP-RSuS 融合蛋白質短暫表現分析，以及對水稻原生質體進行免疫定位的實驗結果，進一步證實 RSuS1 可以進入細胞核中。此外，我們發現磷酸化修飾，並不會影響 RSuS 於細胞質或細胞核的分布。另一方面，利用 DNA 親和層析法純化 A-3-2 結合蛋白質時，發現 RSuS 可能存於其中。因此，推測 RSuS 可能參與調控 RSus1 基因表現，但仍需進一步的研究。

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. The enzyme is encoded by at least six differentially expressed genes in rice. Among the six genes, the RSus1 gene is transcriptionally induced by sucrose. To gain insight into the mechanisms underlying the sucrose-mediated regulation of RSus1 expression, the interactions between the 5’ regulatory region of RSus1 and the nuclear proteins, which were isolated from suspension-cultured cells of rice (Oryza sativa L. cv. Tainung 67) under sucrose and mannitol treatments, were investigated. The results of electrophoresis mobility shift assay suggested that the major protein-binding region was between -1045 bp and -1007 bp upstream the transcription initiation site of RSus1. This region, designated A-3-2, was rich in purine bases and contained four GCGGCG–repeat elements. A protein that specifically and directly interacted with A-3-2 was isolated from the suspension-cultured cells of rice and was subsequently identified as a purine-rich DNA binding protein. The amino acid sequence of this protein, OsPurα, exhibited 73% identity with the Arabidopsis Purα-1 protein, and its modeled structure resembled the structure of Purα in Drosophila. Recombinant OsPurα expressed and purified from E. coli was demonstrated to have DNA-binding activity and to interact with A-3-2 specifically. Moreover, OsPurα was able to enhance sucrose-induced expression of the β-glucuronidase (GUS) reporter gene, which was transcriptionally fused to two copies of a DNA fragment containing A-3-2 and the CaMV 35S minimal promoter, in vivo. The level of OsPurα bound to A-3-2 was higher in cells cultured in the presence of sucrose; however, the level of OsPurα mRNA in cells was not affected by sucrose. The results of this study demonstrate that OsPurα participates in the regulation of RSus1 expression in response to sucrose; nevertheless, it may require other partner proteins for full function In the second part of this study, I investigated the subcellular localization of RSuS proteins. The proteins were detected in both the cytoplasm and the nucleus by using immunoprecipitation and western analysis. The results of transient expression of sGFP-RSuS1 fusion protein in the onion epidermal cells and in the protoplasts of rice cells, and immunolocalization analysis of RSuS proteins in rice cells further confirmed the presence of RSuS proteins in nuclei. Moreoer, phosphorylation of RSuS proteins did not affect their distributions in cytosol and nuclei. Presence of RSuS in the fraction of A-3-2-binding proteins, which were purified by DNA-affinity chromatography, suggested that RSuS may participate in the modulation of RSus1 gene expression; however, it requires further investigation.