Proteinic Study On The OsSF3b3of A Rice Lesion Mimic Phynotype Regulator

Plants lesion mimic refers to a kind of mutant which spontaneously cause cell death without pathogens invasion, it is often caused by mutation of disease-resistant or apoptosis-related gene, leading to the activation of plant defense system, disorder of normal physiological metabolism as well as the early senescence and death of cell. Rice lesion mimic mutant spl5can spontaneously cause the death of blade cell, moreover, the plant’s resistance to rice blast and bacterial leaf blight has increased significantly. In previous research, by map-based-cloning technology, we cloned the genes that control rice sp15lesion mimic Necrosis phenotype, and found that the protein encoded by SPL5and one domain of human’s splice factor3(SF3) are homologous. Because human’s SF3factor is a protein complex polymer, we speculate that interaction protein of SPL5(namely OsSF3b3) may likely exist in rice, these proteins together participate in the regulation of such lesions, therefore this study screened and analyzed OsSF3b3’s interaction protein. First, by homology-based cloning, from the rice we cloned subunits OsSF3b1, OsSF3b2, OsSF3b4and OsSF3b5, which are homologous with human’s SF3protein complex, and through yeast two-hybrid technology, we found that OsSF3b3’s structural domain CPSF_A interacts with OsSF3b2and OsSF3b5respectively. To further analyze OsSF3b3’s interaction protein, we built the prokaryotic expression vector of OsSF3b3:His fusion, after IPTG induction in BL21bacterial strain, by electrophoresis the extracted protein gained a stripe with size similar to that of OsSF3b3. At the same time, we built the over-expression vector of OsSF3b3:GST fusion, which is mediated by agrobacterium and transformed to the callus of Nipponbare, obtaining transgenic plant, and through PCR detection of Carrier specific sequence and RT-PCR analysis of OsSF3b3gene expression, screening for high-expressed transgenic Strain. These results laid a solid foundation for the analysis of OsSF3b3interaction protein by Pull down test.