[Cloning and expression of cDNA for maize nonspecific lipid transfer protein as well as calmodulin-binding activity analysis of the expression product].

Abstract

Maize nonspecific lipid transfer protein (Zm-nsLTP) was cloned and expressed to investigate its CaM-binding activity. The cDNA of Zm-nsLTP was amplified using RT-PCR (Fig.1), and then inserted into the vector pET32a(+). The recombinant vector pET-Zm-nsLTP was expressed in E. coli BL21(DE3)trxB(-). Results of CaM-gel overlay assays (Fig.2) and CaM-sepharose pull-down experiments (Fig.3) indicated that recombinant Zm-nsLTP was bound to CaM in a Ca(2+)-independent manner, which is in accordance with the way that CaMBP-10 and Arabidopsis non-specific lipid transfer protein-1 (At-nsLTP1) are bound to CaM. The CaM-binding domain in Zm-nsLTP was mapped to the region of 47-60 amino acids (Fig.3), and online sequence analysis using Predict Protein program predicted that it has a BAA structure (Fig.4,5).