2.4. In silico analysis
The plasmid from the PCR positive clones was purified using the plasmid purification kit (Qiagen, USA) and sequenced (Eurofins, Bangalore, India). The nucleotide and the deduced amino acid sequences were analyzed using the BLAST program of NCBI (www.ncbi.nlm.nih.gov). The molecular weight, isoelectric point (pI) and the hydrophobic nature of the peptide were determined using Sequence Quickie Calc version 5.0 software. The nucleotide sequence obtained in the study was aligned with crustin sequences from other closely related species available in the database using the ClustalW2 online bioinformatics tool (http://www.ebi.ac.uk/Tools/clustalw2/index.html) to evaluate the percentage similarity. A phylogenetic tree was generated using the UPGMA programof MEGA5 software (http://www.megasoftware.net/) based on crustin sequences available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/).