Molecular Mechanism Of GNA Domain Of MNB1 Protein Regulating Cadmium Tolerance In Arabidopsis Thaliana

Previous studies have shown that by enhancing the expression of MAN3 gene in Arabidopsis thaliana,the activity of mannan hydrolase can be enhanced,and the mannose content in cell wall can be increased,thus enhancing the accumulation and tolerance of plants to heavy metal cadmium.It has been reported that the GNA domain of Ca MBL1,a homologous protein of MNB1,can specifically bind to mannose in pepper.GNA domain is also present in MNB1 protein in Arabidopsis thaliana.However,it is unclear whether the GNA domain of MN B1 protein is related to the molecular mechanism of MAN3-mediated mannose regulating cadmium tolerance in Arabidopsis thaliana.Therefore,on the basis of previous studies that have proved that MNB1 participates in the regulation of cadmium stress response,this topic further studies the molecular mechanism of GNA domain of MN B1 protein regulating cadmium tolerance in Arabidopsis thaliana.The results are as follows:1.To study whether MNB1 gene is induced by cadmium stress,MNB1 promoter was cloned and Pro MNB1:GUS transgenic plants were constructed.GUS staining analysis showed that under cadmium stress,the blue color of roots and leaves of transgenic seedlings was significantly stronger than that of the control,indicating that MNB1 gene was induced by cadmium stress.2.A series of MNB1 protein deletion e xpression vectors were constructed and prokaryotic expression of the proteins was carried out,and mannose-binding analysis was carried out.It was found that MNB1 protein containing only GNA domain can bind to mannose.From the biochemical point of view,it is proved that the GNA domain of MNB1 protein is the key domain for MNB1 protein to bind to mannose.3.Transgenic plants MNB1?/mnb1-1 and 35S:MAN3/MNB1?/mnb1-1 with GNA domain deletion were constructed and their phenotypes were analyzed under cadmium stress.Compared with wild type,MNB1?/MNB1-1 plants showed the same cadmium sensitive phenotype as mnb1-1 with or without mannose.35S: MAN3/MNB1?/mnb1-1 did not show cadmium tolerance as 35S:MAN3,35S:MAN3/35S:MNB1/mnb1-1,but showed cadmium sensitivity phenotype as mnb1-1.Therefore,it is further proved from the genetic point of view that the GNA domain of MN B1 protein participates in MAN3-mediated mannose regulation.4.The contents of GSH and PC and the expression level of genes involved in GSH-dependent PC synthesis pathway in wild type,MNB1?/mnb1-1 and mnb1-1 plants were further analyzed under cadmium treatment.It was found that these indexes were significantly lower in these mutant plants than those in wild type,indicating that the GNA domain of MNB1 protein-mediated cadmium tolerance is related to GSH-dependent PC synthesis pathway in Arabidopsis thaliana.In conclusion,our results prove that the GNA domain of MNB1 protein participates in the regulation of MAN3-mediated cadmium tolerance in Arabidopsis thaliana by binding to mannose.