Isolation and In Silico Characterization of Defensin Genes from Some Indigenous Plant Species of Pakistan

Aims: Damage of crop plants due to pathogenic attacks, postharvest crops spoilage and lethal effects of chemical pesticides has enforced scientists to find some potential natural alternative. Plants produce different types of antimicrobial peptides including defensins in response to biotic and abiotic stresses. Defensins are small cysteine rich, cationic peptide with 40-45 amino acid residues with a variety of biological activities. Hence defensins have pharmaceutical and agricultural significance especially wide range of antifungal activities.

Methodology: Nine plant species viz Brassica napus, Brassica nigra, Conyza bonariensis, Alhagi marorum, Sonchus arvensis, Brassica compestris, Eruca sativa, Cirsium arvensis and Brassica juncea were selected for the study. Four set of primers were applied on extracted genomic DNAs and four amplified genes were isolated from different plants species by PCR. The amplified genes were cloned via pTZ57R/T in E. coli. The cloned fragments were sequenced and characterized by different bioinformatics tool such as coding length and peptide sequence, site of cell aggregation, peptide physico-chemical properties, antimicrobial properties and gene expression pattern.

Result: The amplified products from B. napus, B. nigra, B. compestris, E. sativa and B. juncea were about 330 bp in length and showed upto 85% nucleotide homology to the reported defensins. Deduced amino acid sequence from Bn-Def, Es-Def Bj-Def and Bc- Def showed the conserved defensin domain. These sequences were also characterized for different characteristic like charged amino acids, pI value, shelflife and stability. Sonchus arvensis didn’t give any specific product by prescribed set of primers. However the products from Alhagi marorum was about 770 bp and B. napus was of 270 bp but both give homology below 30%.

Conclusion: This study showed that in silico characterization of deduced antimicrobial peptides from different plant species has depicted these as an effective alternative to communal therapeutics. However, further work is needed to verify defensins specific activity at protein level.