Functional Analysis Of Rice OsRLCK91 In Grain Development And Osmotic Stress Response In Rice

Receptor-like cytoplasmic kinases(RLCKs)are a special class of receptor-like kinases(RLKs).Compared with receptor-like kinases,typical receptor-like cytoplasmic kinases has no extracellular receptor domain and transmembrane domain,only the cytoplasmic kinase domain.Receptor-like cytoplasmic kinases are mainly localized in the cytoplasm,but some RLCKs can be localized into the cell membrane by amino-terminal palmitoylation or myristoylation.According to reports,receptor-like cytoplasmic kinases are involved in signal transduction pathways such as plant growth and development,immunity,and abiotic stress.Studies have shown that there are 379 members of the receptor-like cytoplasmic kinase in rice,which are divided into 58 subfamilies.However,the function of these receptor-like cytoplasmic kinase genes in rice are largely unknown in the process of growth,development and stress response.In this work,we mainly analyze the gene function of OsRLCK91 and screen the OsRLCK91 interacting proteins through yeast two-hybrid experiment.The results obtained are as follows:1.GUS staining assays of OsRLCK91pro: GUS transgenic plants showed that OsRLCK91 is mainly expressed in the base of seedlings,stem nodes,mature leaves,young spikes,anthers,stigmas and developing seeds.2.Through genetic transformation of rice,we identified some homozygous osrlck91 gene knockout mutants by CRISPR/cas9 technique.To analyze the phenotype of this mutants,it was found that the grain width of the osrlck91 mutant was significantly narrower than wild type,indicating that OsRLCK91 play an important role in the regulation of grain development.3.Using OsRLCK91 as bait to screen rice cDNA library through yeast two-hybrid system,we found OsMAPK4,OsAHP1,OsAHP2 interacted with OsRLCK91 in yeast cells.In addition,we also found that OsMKK3,OsMKK10-2,OsGEF7 A interacted with OsRLCK91.As a homologous gene of OsRLCK91,OsRLCK197 also interacts with OsAHP1.4.Under 150 mM NaCl treatment,the expression levels of OsRLCK91,OsMAPK4,and two homologous genes OsRLCK197 and OsRLCK305 were up-regulated by q RT-PCR analysis.5.Under the treatment of 200 mM mannitol,the shoot length of OsRLCK91 overexpression lines was longer than wild type,and the shoot fresh weight and root fresh weight are increased significantly than wild type,indicating that OsRLCK91 overexpression lines were resistant to osmotic stress.6.Using CRISPR/cas9 gene knockout technology,we have also constructed OsMAPK4,OsRLCK197,OsRLCK305 knockout vectors and OsRLCK197,OsRLCK305 double knockout vectors and OsRLCK91,OsRLCK197,OsRLCK305 triple knockout vectors.Through genetic transformation we have obtained some homozygous mutants.In summary,OsRLCK91 is specifically expressed in rice tissues,such as young panicle,anthers and stigmas.OsRLCK91 is up-regulated under salt stress.OsMAPK4,OsAHP1,OsAHP2,OsMKK3,OsMKK10-2,OsGEF7 A interacted with OsRLCK91 in yeast cell.OsRLCK91 plays an important role in grain development and osmotic stress.In addition,OsMAPK4,OsRLCK197,and OsRLCK305 are up-regulated under salt stress.They may have the similar function as OsRLCK91 and play a similar role in salt stress.