Identification, isolation and characterization of defensin gene family in lentil (Lens culinaris L.)

Plant defensins are a family of cysteine-rich cationic antimicrobial peptides. They are small molecules with molecular mass between 5 and 7kD and eight conserved cysteines that are involved in disulfide bond formation. In the present study, using bioinformatics tools, the defensin gene family were isolated and characterized from lentil. All nucleotide sequences of the defensin genes of dicotyledon plants (583 sequences) were retrieved from GenBank. The retrieved sequences were aligned against lentil EST library (9539 ESTs) and the resulted sequences were pooled and assembled. By using BLASTn tool, the assembled (contigs) and non-assembled (singletons) fragments were aligned against GenBank nr database. The resulted contigs and singletons were further analyzed for presence of complete ORFs, functional domains, signal peptides, subcellular localization, physicochemical properties, amino acid frequency, antimicrobial activity and expression pattern. Finally, by using PCR, complete coding sequence of defensins were amplified and experimentally validated. The results determined the existence of 6 contigs that contained ORFs of 222 to 228 bp. The protein sequences of identified defensins contained Knot1 (Gamma-thionin) functional domain. These defensins had signal peptides and extracellular localization. Due to high frequency of hydrophobic amino acids, the identified defensins were hydrophobic and produced complex secondary structures. The results of antimicrobial analysis showed high antimicrobial activity in the identified defensins. The gene expression analysis, showed a different pattern of expression for defensins. The identification and characterization of antimicrobial genes is the first step in genetic engineering. For first time in this study, full length coding sequence of defensin gene family was isolated from lentil and structures, features and expression pattern of them were determined.