The Immuno-gold Localization And Immunohistochemical Analysis Of ECBP21 In Angelica Dahurica

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 C aM m ay act a s a p olypeptide s ignal. During t he r esearch o f extrecellular CaM, we found that CaMBPs may exist in cell wall. Tang et al (1996) detected and purified an extracellular calmodulin-binding protein from extracellular extracts of suspension-cultured cells of Angelica dahurica, which is an Ca2+ dependent CaMBP with a molecular weight of 21kD(named ECBP21). Mao et al (2002) cloned and identified the full-length cDNA for ECBP21. Sequence analysis shows that the N-terrninal 1-25 amino acid sequence is a predicted signal peptide. These studies provided preliminary evidence for the existence of the extracellular CaMBPs. For further identification the existence of the extracellular ECBP21, we used immunogold localization technology to study the subcellular localization of ECBP21. First, ECBP21 expressional vector was constructed and transformed into E.coli. Recombinant ECBP21 protein was induced and obtained by cutting SDS-PAGE gel. The high specialties and liters antibody against ECBP21 was prepared. Then using ECBP21 antibody and immunogold transmission electron microscopy method, we studied the subcellular localization of ECBP21.The results indicated that the gold particles were mainly localized in the cell wall in callus cells and rachis cells of Angelica dahurica. These results indicated that ECBP21 mainly localized in cell wall, which provide a direct evidence of the extracellular existence of ECBP21. Furthermore, using ECBP21 antibody and immunohistochemical method, we studied the organic specially distribution of ECBP21, the results indicated that ECBP21 distributed in all organize, but it distributed more in leave N flower rachis than in leafstalk and root. ECBP21 distributed more in vascular bundle and split sect such as cambium and stigma, and mainly in cell wall area. The organic speciality distribution of ECBP21 provides preliminary information for further studying of its developmental role.