Interaction Between The BAG Containing Protein And Heat Shock Protein Hsc70 Showed By Co-immunopreciptation Method In Arabidopsis Thaliana

The BAG proteins are an evolutionarily conserved family,and are distinguished by a common conserved region located near the C terminus termed the BAG domain(BD). Animal proteins are distinguished by a conserved BAG domain that directly interact with Hsp70/Hsc70 ATPase domain to promote substrate relaease and they are chaperone regulators.They can participate in modulating a number of diverse physiological processes in animals,including apoptosis,proliferation,differentiation,stress responses and the cell cycle.Multifunctional BAG family members are also found in plant and remarkably similar to their animal counterparts,as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development,but we do not kown the mechanism how they act in plant.In this passage,we reviewed the studies of BAG proteins in recent years,then took AtBAG4 for an example and investigated the binding between the AtBAG4 and heat shock protein Hsc70/Hsp70 in Arabidopsis thaliana.The cDNA sequences encoding Arabidopsis thaliana AtHsc70,AtBAG4,AtBAG7 was specificlly inserted into prokaryotic expressing vector pET32a(+)and we named these reconstructed plasmids pET-AtHsc70,pET-AtBAG4,pET-AtBAG7 and so on. After identifying the correction of reconstructed plasmids,we transformed them into E.coli BL21(DE3).The proteins we want was expressed effectively after IPTG induction, and the recombinant proteins were purifield by Ni-NTA chelating column.Then polyclonal antibody was prepared by immunizing rabbit using purified protein.Western blot confirmed that the prepared antiserum was able to recognize the native proteins from Arabidopsis thaliana tissues,and they are useful tools for our next research.In order to study the possible molecular interaction between the AtBAG4 and Hsc70 proteins in Arabidopsis thalina.We extracted full proteins from young Arabidopsis thalina in native conditions,and added purified anti-AtBAG4 or anti-AtHsc70 IgGs to these extractions,then used ProteinA to harvest the complex that containing antibodies and antigens.In vivo AtBAG4 was examined when we use anti-AtHsc70 to participate, and AtHsc70 was also found in anti-AtBAG4 expriment.It is mean that the interaction between the AtBAG4 and Hsc70 proteins really exists.Our result is consistent with previous result that deprived from the yeast two-hybrid system,but the different is,we provided the expriment proofs that there is the in vivo interaction between the AtBAG4 and Hsc70 proteins in Arabidopsis thalina,and may be some funtion relations between them.At the same time,another finding that the AtBAG4 mutant are more sensitive to salt stress told us AtBAG4 play an important role in response to salt stress.Beause AtBAG4 interact with Hsc70,bag gene responsiveness to salt stress was of interest.Our results provide useful evidences for further studies of potential role of interaction between the AtBAG4 and Hsc70 proteins in the response to salt stress.