Expression and Functional Characterization of Oryzacystatin II Protein from Oryza sativa L. Indica Rice and Its Potential Role as a Bioinsecticide Against Major Insects of Rice.

Rice ( Oryza sativa L.) is a crucial staple food crop globally, facing significant challenges from various pests that affect crop productivity and quality. Conventional pesticide usage has limitations, necessitating the development of sustainable pest management strategies. This study focuses on the expression, purification, and functional characterization of Oryzacystatin II (OC-II), a protein derived from O. sativa L. Indica rice, with the intent to evaluate its potential as a bioinsecticide against rice pests. The OC-II gene was expressed and purified, and purification confirmed its molecular weight (∼12 kDa) and protein sequence through LC-MS/MS analysis and Western blotting. The IC 50 value of OC-II was calculated as 0.06 μM, and the inhibition was identified as a competitive inhibition. The protein exhibited efficient control of both pests at the nymph and adult stages, with lower probing marks observed on treated plants. The inhibition of cathepsin B enzyme activity in insects further confirmed the bioactivity of the OC-II protein. Molecular docking and molecular dynamics simulations provided insights into the interaction between the OC-II protein and cathepsin enzymes reported in BPH and WBPH. Further investigations can focus on optimizing production methods and exploring the specificity and efficacy of the OC-II protein against other crop pests to enhance its practical applications.