Screening And Functional Verification Of Protein Interacting With The AtPHB6

Salt-alkali is a kind of abiotic stress which exists widely in nature.Scholars have done a lot of research work on the mechanism of plant salt-alkali tolerance.The PHB gene was mainly used to study its anti-cell proliferation and anti-tumor effects,but there are few studies related to salt-alkali stress.Through in-depth study of the molecular mechanism of this gene,it is important for plant salt-alkali tolerance molecular breeding and full utilization of salt-alkali land resources in China.In this study,qRT-PCR technique was used to find that the AtPHB6 gene of Arabidopsis thaliana responded to salt-alkali stress.The stress resistance of wild type,overexpressed AtPHB6 and mutant A.thaliana strains to salt-alkali stress was observed by measuring root length and fresh weight.The interaction protein with AtPHB6 protein was screened by yeast two-hybrid system.Pull-down and bimolecule fluorescence complementarity assay(BiFC)were used to further study the interaction between the two proteins.The salt-alkali resistance of interaction gene was preliminarily studied.The specific research results are as follows:AtPHB6 gene was cloned from A.thaliana.Bioinformatics analysis showed that AtPHB6 was closely related to AtPHBl and AtPHB2 of the same genus ?.The expression of AtPHB6 increased after stress treatment.The resistance of overexpressed AtPHB6 strains was higher than that of wild-type strains,while the resistance of mutants was lower than that of wild-type strains at seed germination stage and late germination stage.AtSOT12,an interacting protein of AtPHB6,was screened out.Cotransformation in yeast cells demonstrated that AtPHB6 and AtSOT12 interact.The interaction between the two proteins was further confirmed by in vitro Pull-down and BiFC test.Subcellular localization showed that both AtPHB6 and AtSOT12 were localized in the nucleus and cytoplasm.The expression of AtSOT12 increased after stress treatment.The resistance of overexpressed AtSOT12 strains was higher than that of wild-type strains at seed germination stage and late germination stage.These results suggest that the two proteins may work together to increase plant resistance.