Rapid Screening of Antimicrobial Peptide Genesfrom Oilseed Rape and its Antimicrobial Activities Analysis

Antimicrobial peptides (AMPs) are widely distributed in nature and play a critical role in the innate immunity of host defense systems. The number of described AMPs has increased over recent decades; however, the recent generation of huge amounts of genomic, transcriptomic, proteomic and ESTs data enables novel strategies for the discovery of new candidate AMPs. In this study, 1308 AMP genes were predicted from ESTs and genome data of oilseed rape with the bioinformatics analyses, in which, 40 AMP genes were cloned and analyzed their antimicrobial activities. The average length of candidate AMP genes is 135bp, and they encode about 45 amino acids. Using the overlap PCR, the 40 AMP genes were synthesized artificially and cloned into pET30a - EDDIE - GFP expression vector. His-EDDIE-AMP fusion proteins were expressed in Escherichia coli. Purified His-EDDIE-AMP inclusion bodies were diluted in optimized refolding buffer and incubated to enable self-cleavage to occur. Antimicrobial activity of 40 recombinant AMPs was detected, and the result showed that 32 recombinant AMPs had specific bactericidal activities against Gram positive bacteria or Gram negative bacteria. The antifungal activities of AMPs from oilseed rape are being analyzed now. In conclusion, the method based on bioinformatics analyses is useful for high- throughput screening plant AMPs genes from genome and EST sequence data.