Interaction Between Calmodulin And ERECTA

Our previous study suggested that the calmodulin (CaM) existed in apoplast of plant cells and had various biological functions in plants. Based on these studies, we put forward a view that the biological functions of extracellular CaM may act as a polypeptide signal. So Identification of calmodulin-binding proteins helps to understand the biological process regulated by extracellular CaM.During the research of extrecellular CaM and its binding protein, ERECTA was chosen as a candidate, because it has putative calmodulin-binding domains, transmembrane domains and a secretory signal peptide analyzed by bio-informatic tools, indicating its possible membrane localization and interaction with CaM .First, ERECTA fused with YFP was transformed into onion epidermis cell. Under laser confocal microscopy, it specifically localized in plasma membrane. Using CaM2 antibody and immunohistochemical method, we studied the organic specially distribution of CaM2 in shoot meristem of Arabidopsis , the result indicated that CaM2 distributed more in cells that are predict to divided . The cellular and organic overlap distribution of CaM2 and ERECTA provides preliminary information for their interaction.Then, the interaction and binding domain was approved in split ubiquitin system and SPR system. Furthermore, we have screened ERECTA and CaM2 double mutant, extrecellular CaM2 overexpression transgenic lines for their genetic interaction study.In addition, our lab got an Arabidopsis mutant lfr-1 with a distinct phenotype in leaf and flower development. In order to study its function and molecular mechanism, recombination expression plasmid was constructed and transformed into the host bacteria strain Rosetta. Then IPTG was used to induce the recombinant protein expression in engineering strain. The GST:LFR fusion protein was existed in soluble form with a relative molecular mass 77 kDa, which is fit with the molecular weight supposed from gene coding frame. After purification by GST-tag affinity chromatography and electroelution, the fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen, the antiserum was obtained and further purified by decreased nonspecific bacteria and GST-tag antibody with method of immuno-precipitation. Western blot analysis showed that the purified antiserum, raised against the recombination LFR protein in rabbits, could react to the recombinant protein expressed in Rosetta specifically. And then the nuclear proteins of Arabidopsis wild type and mutant were extracted and separated by SDS-PAGE. Western blot assays revealed that there was a protein band, with a relative molecular mass 50 kDa, indicating that antiserum could react to the native protein expressed in Arabidopsis specifically.