PDC1, a corn defensin peptide expressed in Escherichia coli and Pichia pastoris inhibits growth of Fusarium graminearum.

摘要

Plant defensin corn 1 (Pdc1) gene was amplified from corn genomic DNA with the primers designed from a corn EST sequence homologous to a barley defensin gene. The cloned gene contains two exons and an intron. The deduced 9KDa PDC1 peptide has a sequence that is identical to corn gamma2-zeathionin and has the typical features of a plant defensin, including a signal sequence of 35 amino acids, followed by a characteristic defensin domain of 47 amino acids containing 8 cysteines. The defensin protein was expressed from the cloned cDNA introduced into two different expression systems; prokaryotic, Escherichia coli and eukaryotic, yeast (Pichia pastoris). The PDC1 protein was purified with a nickel resin column and was tested for its antifungal activities using the pathogen Fusarium graminearum. The protein expressed in both E. coli and P. pastoris had antifungal activity, however the protein expressed in P. pastoris was more efficient in inhibiting growth of F. graminearum. FTIR analysis of PDC1 protein expressed in the two expression systems showed that expression in P. pastoris gave a product with more beta-sheets and less random unordered structure than when it was expressed in E. coli. In addition, removal of the His-tag used for purification increased the fungicidal activity of the PDC1 protein. The data presented here suggest that the defensin PDC1 peptide of corn could be effectively used to restrict the disease caused by F. graminearum.