Expression and functional characterization of oryzacystatin II protein from Oryza sativa L. Indica rice and its potential role as a bioinsecticide against rice pests

Abstract

Abstract 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 Oryza sativa L. Indica rice, intending to evaluate its potential as a bioinsecticide against rice pests. The OC II gene was cloned into the pET28a (+) vector and transformed into BL21 DE3 bacterial system for protein expression. The expressed protein was purified and confirmed its molecular weight (~ 12 kDa) and protein sequence through LC-MS/MS analysis, and western blotting. IC50 value of OC II and E-64 was calculated as 0.06 µM and 0.14 µM for inhibiting 0.02 ng/µL cathepsin B enzyme respectively. The inhibition was identified as a competitive inhibition with increasing Km values regarding increasing OC-II concentrations while the Vmax remains 254.5 ΔRFU/minute. The Ki value of OC-II was determined as 5x10^-8 M (0.05 µM). In vivo, studies were conducted to assess the efficacy of OC-II protein against major rice pests, BPH and WBPH. 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 further confirmed the bioactivity of OC-II protein. Molecular docking and molecular dynamics simulations provided insights into the interaction between OC-II protein and cathepsin enzymes reported in BPH and WBPH. The successful expression, purification, and characterization of OC-II, along with its promising bio-insecticidal activity, provide valuable insights into its potential application as an eco-friendly alternative to conventional pesticides. Further investigations can focus on optimizing production methods and exploring the specificity and efficacy of OC-II protein against other pests to enhance its practical application.