In vitro and in silico characterization of Solanum lycopersicum wound-inducible proteinase inhibitor-II gene

Plant proteinase inhibitors (PIs) are antimetabolic defensive proteins conferring resistance in plants against a variety of competing organisms such as bacteria, viruses, fungi, attacking nematodes, and insects. In the fields of plant biochemistry and molecular genetics research, tremendous success has been achieved in generating transgenic crops that have defensive approaches against biotic challenges. In this study, in vitro and in silico analysis was carried out for a wound-inducible PI-II gene isolated from 4 selected varieties (Roma, Nagina, Moneymaker, and Rio Grande) of Solanum lycopersicum L. Around 684 bp of PI-II gene was amplified, sequenced, translated, modeled to protein structure, and phylogenetically analyzed. The sequence analysis by BLAST showed high similarity scores (99%, 97%, 96%, and 94%) for Moneymaker, Roma, Rio Grande, and Nagina, respectively, with the original PI-II gene sequence from Solanum lycopersicum var. cerasiforme (GenBank accession no. AY007240) selected for primer designing. Sequenced data were translated to protein sequences, and translated sequences were modeled to 3-dimensional structures with iterative threading assembly refinement (I-Tasser) software. Phylogenetic analysis was carried out using Molecular Evolutionary Genetic Analysis software. Comparative phylogenetic analysis with 26 other complete coding sequences of PI from dicotyledonous plants was also done with in vitro analyzed PI-II genes from selected tomato varieties. In silico insight into the phylogenetic evaluation revealed that 30 PIs from different plants share a common root of evolutionary origin. Furthermore, 3-dimensional protein modeling by Ramachandran plot analysis revealed that PI from S. lycopersicum 'Roma' has the best quality structure with 85% of residues in most allowed regions.