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Localization and expression analysis of U-box-containing E3 ligase OsUPS result from phosphate deficiency in rice
Rice is a staple food crop in the world. A number of agronomically important traits including enhancement of stress tolerance, quality improvement, and nutrition value increases have been introduced to rice. In this study, an Oryza sativa cDNA containing a U-box motif was cloned; its deduced amino acid sequence was compared to that of other U-box genes and indicated that encodes a U-box-containing E3 ligase. E3 ligases are structurally divided into three groups. We isolated the OsUPS gene from rice (Oryza sativa). The OsUPS protein has domain which is a single~70-amino acid region of the protein and GKL domain containing conserved Glycine, Lysine/ Araginine residues and leucine-rich feature. A full-length expression of OsUPS was up-regulated in the rice plant and in cell culture in the absence of phosphate. To express the OsUPS cDNA, it was inserted into the pGEX-2T vector. And the gene was expressed in E.coli strain BL21 (DE3). Induced after 3h of IPTG treatment and was isolated by affinity chromatography. Using the GUS reporter genes regulated by the OsUPS promoter, we have carried out the analysis of transcriptional and spatial regulation of gene expression. To investigate the function of these genes, the CaMV 35S promoter-driven these genes were introduced into Arabidopsis and rice via Agrobacterium tumefaciens-mediated gene transformation. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (Pi). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the Pi signaling pathway through the ubiquitin-26S proteasome system.
