Identification and Characterization of Oryza cystatin II Protein from Oryza Sativa L Indica Rice and its Role as a Potential Bioinsecticide Against Rice Pests

Abstract

Rice (Oryza sativa L.), a vital staple food crop for billions of people worldwide, faces consistent challenges from pests and diseases that significantly impact crop productivity and quality. While conventional pesticides provide temporary protection from pests and diseases, their detrimental environmental effects demand the development of sustainable pest management strategies. Oryzacystatins (OCs), a unique class of protease inhibitors from Oryza sativa L., specifically target cysteine protease enzymes, affecting insect growth and development. This thesis investigates the expression, purification and functional characterization of oryzacystatin II (OC-II) gene, from Oryza sativa L. Indica rice, intending to evaluate its potential as a bioinsecticide against rice pests. The first objective focused on sequential and structural analysis, unravelling the intricate relationships between eleven reported oryzacystatins and their potential interactions with cysteine protease enzymes from major rice pests. Through extensive analysis of sequence heterogeneity, structural variability, and functional significance, key regions (N-terminal glycine residue, the central conserved QVVXG motif, and the C-terminal AVVXXXPW region) responsible for mediating efficient protease inhibition were elucidated. Molecular docking and Molecular dynamics (MD) simulations, provided compelling evidence and validation of the stability and potential of oryzacystatins to interact and inhibit cysteine protease enzymes. The OC-II gene, isolated from Indica rice varieties (IR22 and IR64), cloned, sequenced and deposited into the NCBI database. The coding sequence (CDS) region of the OC-II gene was identified, chemically synthesized and cloned into the linearized pET28a (+) plasmid vector and transformed into E. coli newlinevii newlineBL21(DE3) bacterial system. 1mM IPTG was identified as optimum concentration for the higher expression followed by Ni-NTA column purification.