Molecular Cloning And Functional Analysis Of A Rice Sphingosine Kinase Gene OsSPK1

Sphingolipids are ubiquitous constituents of membrane systems in all eukaryotic organims. There is now substantial evidence that sphingolipids and their metabolic products are involved in the regulation of cell growth, differentiation, survival, proliferation and programmed cell death via their participation as second messengers in specific signal-transduction pathways. As a novel class of sphingolipid metabolites, sphingisine-1-phosphate (SPP) functions as a second messenger and as ligands for cell surface receptors. Cellular levels of SPP are largely mediated by its formation from sphingosine by the activity of sphingosine kinase (SPK) and to a lesser extent by its degradation by SPP lyase and SPP phosphatase activities. In order to elucidate the biological function of SPP in plant responses to abiotic and biotic stresses, we cloned and identified a rice SPK gene, OsSPKl, and studied its function in disease resistance response by overexpressing in transgenic tobacco.The cDNA of OsSPKl was amplified by PCR using phage DNA prepared from a rice cDNA library as template. The full-length cDNA of the OsSPKl is 2910 bp with a predicted open reading frame of 2247 bp in length, which predicted to encode a putative SPK protein with 757 amino acid residues with a predicted molecular mass of 83.27 kDa and pI of 7.4. The OsSPKl gene was located on chromosome 10 of the rice genomes and is composed of 8 exons and 7 introns. Alignment of OsSPKl with other SPKs from human, mouse, yeast, Caenorhabditis elegans and plants showed that OsSPKl has a conserved diacylglycerol kinase catalytic domain of ~125 amino acid residues in length. Also, OsSPKl contains an extra conserved C1-C4 regions, including GXGX motif in the C1 domain, which may be involved in ATP binding.To better understand the function of OsSPKl in disease resistance response, we cloned the OsSPKl coding region into a plant binary vector, CHF3pp2p212, under control of the CaMV 35S promoter and transformed tobacco leaf discs by Agrobacterium-mediated transformation. A total of fourteen independent OsSPKl transgneic tobacco lines were obtained and confirmed by PCR detection. RT-PCR analysis indicated that OsSPKl gene was transcribed in the transgenic tobacco plants. We evaluated disease resistance of the transgenic lines and found that the transgenic plants showed enhanced disease resistance against Phytophthora parasitica var nicotianae and Phytophthora parasitica var nicotianae. Our preliminary results indicate that OsSPKl-mediated SPP signaling may play a role in disease resistance response in plants.