The Interacting Protein Screening And Biological Functions Of At LecRK-Ⅷ.1 Gene In Arabidopsis Thaliana

In order to cope with the complex and changeable external environment,plants have evolved a complete set of sensing and response mechanisms.Receptor-like kinases(RLKs)play an important role in this process.Lectin receptor-like kinases(Lec RKs)are a class of RLKs that use carbohydrate signaling molecules as potential ligands and contain a lectin receptor domain,a transmembrane domain and an intracellular kinase domain.Lec RKs are widely involved in the regulation of various physiological processes such as plant defense responses,abiotic stress responses,growth and development.In this study,a preliminary study on the Arabidopsis thaliana LecRK-Ⅷ.1 gene is carried out by comprehensively using bioinformatics,genetics and molecular biology.The main results are as follows:(1)Genetic analysis shows that LecRK-Ⅷ.1 is a membrane-localized protein,including extracellular domain,transmembrane domain and intracellular domain.Sequence evolution analysis reveals that there are two members of the eighth subfamily of Lec RKs,LecRK-Ⅷ.1 and Lec RK-VIII.2,with an amino acid sequence similarity of 82%.Tissue-specific analysis shows that LecRK-Ⅷ.1 is highly expressed in the sixth and seventh stages of seed development,suggesting that LecRK-Ⅷ.1 gene may be involved in the regulation of seed development.(2)We obtain T-DNA insertion mutant(lecrk81-1)of the LecRK-Ⅷ.1 gene by the tri-primer PCR.We also gain 35S::LecRK-Ⅷ.1-3x FLAG transgenic lines(OE-1)through Transformation Protocol.In addition,LecRK-Ⅷ.1/ Lec RK-VIII.2 double homozygous deletion mutants and transgenic lines are obtained by hybridization.(3)Under different concentrations of Na Cl,the seed germination rate of OE-1 is similar to that of lecrk81-1,and there is no significant difference between them and wild-type.The seed germination rate of OE-1 is higher than that of the wild type under different concentrations of mannitol treatment,while lecrk81-1 is lower than that of the wild type.It is speculated that the LecRK-Ⅷ.1 gene may be involved in the regulation of exogenous mannitol in the seed germination stage.Mannose and glucose antagonistically regulate seed germination.At the seed germination stage,OE-1 is not sensitive to exogenous mannose response,while there is no significant difference in seed germination rate between lecrk81-1 and wild type.OE-1 is more sensitive to the inhibitory effect of glucose antagonizing mannose.This indicates that the LecRK-Ⅷ.1gene is involved in regulating the response of Arabidopsis to exogenous glucose and mannose.(4)The 35S::LecRK-Ⅷ.1-GFP fusion expression vector is successfully constructed.The transient transformation experiment of tobacco shows that Lec RKVIII.1 is a typical membrane protein.Yeast two-hybrid experiments show that the intracellular kinase domain of LecRK-Ⅷ.1 can form homodimers,and can form heterodimers with the intracellular kinase domain of Lec RK-VIII.2.It is suggested that LecRK-Ⅷ.1 and Lec RK-VIII.2 could play similar roles to regulate plant growth and development.Four proteins interacting with LecRK-Ⅷ.1,MAPKKK4,AGG1,AGG2 and AGG3,are identified by Co-IP/MS and yeast two-hybrid assays.(5)The prokaryotic expression vectors p ET28a-LecRK-Ⅷ.1-LD and p ET28 aLecRK-Ⅷ.1-KD are successfully constructed.The optimal conditions for inducible expression of LecRK-Ⅷ.1-LD target protein are 16 ℃,16 h,0.5 m M IPTG.The LecRK-Ⅷ.1-KD protein may cause body death when expressed in prokaryotic expression strain BL21.