Molecular Cloning Of A Rice Sphingosine-1-phosphate Phosphatase Gene, OsSPP1

Sphingolipids are ubiquitous, essential components of eukaryotic membranes and function as the dynamically signal factors between cells and the outer surroundings. In mammals, it has been demonstrated that some sphingolipid metabolites, ceramide(Cer), sphingosine(Sph), and sphingosine-1-phosphate (S1P) play important roles in the regulation of cell proliferation, survival, and cell death. S1P level in cells is regulated through its formation from sphingosine by the activity of sphingosine kinase (SPK) and to a lesser extent by its degradation by S1P lyase and S1P phosphatase activities. To elucidate the potential function of S1P in plant responses to abiotic and biotic stresses, we cloned and identified a rice SPP gene, OsSPPl.The cDNA of OsSPPl was amplified by PCR using phage DNA prepared from a rice cDNA library as template. The full-length cDNA of the OsSPPl is 1667 bp with a predicted open reading frame of 1248 bp in length, which predicted to encode a putative SPP protein with 415 amino acid residues with a predicted molecular mass of 45.6kDa and pI of 8.7. The OsSPPl gene was located on chromosome 3 of the rice genomes and is composed of 8 exons and 7 introns. Alignment of OsSPPl with other SPPs from human, mouse, yeast, fungi and plants showed that OsSPPl has a conserved PAP2_SPPasel domain.To study the biochemical activity and subcellular localization of OsSPPl, we cloned the coding sequence of OsSPPl into a protein expression vector pRSET-A and obtained recombinant plasmid pRSETa-OsSPP1. Attempts to purify recombinant OsSPPl protein from E. coli failed. Optimization of the induction and purification conditions for OsSPPl is in progress. Meanwhile, we also cloned into a vector used for subcellular localization (GFP3G) and obtained recombinant plasmid GFP3G-OsSPP1. Analysis of the cellular localization of OsSPPl using GFP3G-OsSPP1 plasmid is also in progress.