Studies on Rice Sucrose Synthase 1:Sugar-Modulated Gene Expression and Subcellular Localization
Date Issued
2011
Date
2011
Author(s)
Chang, Jui-Che
Abstract
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.
Subjects
electrophoresis mobility shift assay
promoter
sucrose-induced
immunoprecipitation
onion epidermal cells
protoplasts of rice cells
Type
thesis
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