Screening For Proteins Interacting With AtBAG5 By Yeast Two-hybrid System

Programmed cell death (PCD) plays an indispensable role in development and physiology, but it is unclear whether the mechanisms governing PCD in both plants and animals are similar.Although it is important to note that none of the core machinery members (e.g. caspases, Bcl-2 family proteins) have been identified in plant. Other similarities of animal and plant PCD include DNA fragmentation, cell shrinkage, chromatin condensation, cysteine protease activation and generation of reactive oxygen species, indicating the existence of common elements in both systems.We hypothesize that plant genomes may have functional equivalents of Bcl-2 family proteins that share little or no sequence similarity with animal counterparts.BAG (BCL2-associated athanogene) family proteins were originally identified in mammals due to their ability to associate with the antiapoptotic protein, BCL2, and promote cell survival. Further studies revealed that BAG family genes exist in a number of eukaryotes whose members share at least one copy of a 50 amino-acid conserved domain (BAG domain) that interacts with heat shock protein 70 (HSP70/HSC70). BAG proteins may serve as bridging molecules that recruit HSP70/HSC70 to a specific target protein. As a consequence, this family of cochaperones functionally regulates diverse cellular pathways, such as programmed death (PCD) and stress responses.The Arabidopsis BAG gene family shows remarkable structural and functional similarity to its mammalian counterparts. we hypothesize that the Arabidopsis BAG proteins may play a similar function like their animal counterparts in PCD.Then we choice AtBAG5 as bait to inditified proteins interacting with it by yeast two-hybrid.We also have subcloned CDS of AtBAG5 into pET-32a vector.These plasmids were used to express the recombinant protein in host BL21(DE3) E.coli cells. Insoluble recombinant protein were purified .Seven true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Most of the positive colonies coding genes related with stress responses.