Isolation, cloning and molecular characterization of defensin gene(s) in black pepper (Piper nigrum L.)

Abstract

Plant defensins are cationic antimicrobial peptides that function in the innate immune response in plants. Many plant defensins having antimicrobial activity are molecularly characterized in different plants. However, defensin genes present in black pepper are not completely characterized. The study entitled “Isolation, cloning and molecular characterization of defensin gene(s) in black pepper (Piper nigrum L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture Vellayani, Thiruvananthapuram during 2021-2022. The objective of this study was isolation, cloning and molecular characterization of antimicrobial peptide defensin gene(s) from black pepper variety Panniyur 1. In this study, the full-length gene sequence of a novel defensin gene termed PnDef was isolated for the first time from black pepper var. Panniyur 1 using gene specific primers. The full length of PnDef gene is found to be 356bp. The coding sequences of PnDef was deduced using Genscan software, which was found to contain 231bp open reading frame encoding a protein of 76 amino acid residues. The blast analysis of PnDef amino acid sequence showed 72.60% similarity with the defensin protein of Pyrus ussuriensis × Pyrus communis. The phylogenetic analysis showed a close relationship of PnDef to defensin protein from Rosaceae family. The clustal analysis of PnDef revealed eight conserved cysteine residues in similar positions. The putative PnDef mature protein showed striking similarity with other plant defensins representing a small molecular weight of 5.213 kDa, theoretical isoelectric point (pI) of 8.95 with an aliphatic index and GRAVY of 60.26 and 0.154 respectively. It has a potential 29 residue signal peptide and the remaining 47 residues belong to the mature peptide region. The secondary structure analysis of PnDef revealed that it has 46% alpha helix and 25% of β-sheet structures. The three-dimensional structure modeling of PnDef showed a striking resemblance (81%) with the antifungal defensin of Medicago trancatula implying it may have similar antimicrobial activity. PnDef was found to have three conserved disulfide bonds formed between six cysteine residues. In silico analysis of the PnDef indicated antibacterial, antiviral and antifungal properties of the peptide.Using the sequence data identified in this study one can further go for expression profiling of black pepper defensin in different plant parts under biotic and abiotic stress conditions, recombinant protein can be isolated by inserting into the expression vector and using the recombinant protein one can identify the inhibition activity of the protein against plant pathogens under in vitro studies. Molecular docking can also be done prior to the in vitro studies using the 3D structure of black pepper defensin to identify potential pathogen targets.