Construction And Primary Expression Of The Gene Encoding Calmodulin Binding Protein In Arabidopsis Thaliana

Camodulin(CaM),as an important second messenger. The cytosolic free calcium level leads to binding of Ca²⁺ sensor protein and its target proteins initiating a variety of cellular processes. CaM is a major calcium sensor protein, exist in all eukaryotic cells , including in plants.In plant cells, there were three intracellular signal transduction pathways:G-protein and phospholipid signaling systems, cyclic nucleoside cAMP and cGMP signaling systems and Ca²⁺ signaling system. CaM plays important roles in Ca²⁺ signaling pathway,different CaM isoforms interacted with its target proteins. In Arabidopsis, CaMl-CaM7 were typical CaM isoform proteins.Use pET prokaryotic expression system , induction and expression the vector pETCaM₂ by IPTG in E. coli BL21 (DE3). Separation and biotinylated CaM₂ by chromatography. for the preserve in order to analysis of the interaction of our target protein and CaM.According to the bioinformatics analysis in Arabidopsis, We found a gene(At5g62390) encoding for calmodulin-binding protein. Further analysis showed that it contained two motifs for Ca²⁺dependent binding: 1-5-10 and 1-8-14, one motif IQ for Ca²⁺ independent binding. At5g62390, as a template, start at C-terminal, amplified different cDNAs contain different domains by PCR. Construction different vectors by sub-clone. The results showed that the cDNAs were respectively right amplified by Agarose gel electrophoresis. Identification of recombinant plasmids showed correct by digested with enzyme and by sequencing.During the process of studying the conditions of protein expression, we found no expression at different IPTG-induced concentration and different temperature on the different fragments. In the same experimental conditions, we use the gene small 480bp fragment of recombinant expression vector, successfully on expression. This work laid the foundation for our further study.