The Molecular Genetical Research Of OsSCPD1 In Drought Tolerance Regulation In Rice

The serine carboxypeptidases belong to a large family.Studies have found that serine carboxypeptidases are involved in many important biochemical reactions,including protein folding,herbicide conjugation,secondary metabolite biosynthesis and so on.Serine caboxypeptidases play an important role in cellular regulation of plant,synthesis of plant secondary metabolites and the regulatory role in stress tolerance.The previous studies found that drought tolerance in rice OsSCPD1 mutant mutants was lower than the wild type,whereas drought tolerance in transgenic rice plants was enhanced.After defining this phenotype,the fluorescent label vector of rice OsSCPD1 was constructed and the OsSCPD1 was located on the cell membrane by fluorescence microscopy.At the same time,the yeast two-hybrid vector of OsSCPD1 and the cDNA library of drought stress in rice were constructed.The nine proteins interacting with OsSCPD1 under drought stress were screened by yeast two-hybrid system,they wereOsHSP40?Ospoll-L?Os21980?OsMADS1?Osp59-Y1?OsUPF0195-L?OsRPL14E?OsS59-Land OsCaLB-1.The interaction between two proteins?OsUPF0195-L,OsHSP40?and OsSCPD1 protein was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation analysis.At the same time,the functional domain of OsSCPD1 and protein interaction was identified as proteolytic enzyme peptidase S10.Further studies showed that OsSCPD1interacted with OsHSP40 on the cell membrane and was regulated by Mg²⁺.This study hypothesized that OsHSP40,located in the cytoplasm and OsSCPD1 located on the cell membrane,were interacted on the cell membrane.The OsHSP40 to be migrated to the cell membrane and the frist polypeptide segment of the J domain on the N-terminal DnaJ protein was excised by OsSCPD1.So as to complete the modification of OsHSP40 protein,which was involved in the regulation of drought tolerance of rice plants.The results of iTRAQ analysis showed that the most significant accumulation of differentially expressed proteins was amino acid metabolism and electron signal transport chain associated protein,KEGG Pathway functional enrichment analysis revealed that differential proteins were associated with carbon metabolism,glycolysis/gluconeogenesis and amino acid biosynthesis and unsaturated fatty acid metabolic pathways.In addition,Overexpression of OsUPF0195-L and OsHSP40 genes and CRISPR/Cas9 mutant plants were obtained by transgenic technology to further verify that OsSCPD1 interaction genes participate in the drought response of rice.The drought tolerance was significantly reduced in the OsHSP40-Cas transgenic plants,but was enhanced in the OsHSP40 overexpression and the wild type plants.From the above results,the mechanism of OsSCPD1involved in drought tolerance of rice by protein level was elucidated and theoretical and material basis for the development of new materials for drought tolerance were provided.At the same time,the discovery of some drought-related proteins would provide important information to further enrich and improve the drought stress signal pathway and network.