Abstract: Objective: To investigate the antioxidant function of the prokaryotic expression product of the heme-binding protein (HBP) gene, rHBP2, cloned from red rice. Methods: We conducted molecular cloning of target genes and construction of expression vectors, transformation and expression in Escherichia coli, followed by isolation of the target protein rHBP2 and identification of its heme binding capacity and antioxidant activity in vitro. Additionally, we assessed the effect of feeding rHBP2 on oxidative and heat stress responses and oxidative stress-responsive gene expression in Caenorhabditis elegans N2. Results: Sequence and molecular structure analysis showed that the full-length coding sequence (CDS) of rHBP2 was 651 bp, encoding a protein containing 216 amino acid residues. The conservativeness of its amino acid sequence exceeded 90% among the compared rice plants. The molecular structure of rHBP2 exhibited both internal and external compatibility, and the pocket formed by several folded sheets might provide an important site for heme binding. The typical alpha helix was located on the periphery, potentially facilitating other potential interactions. rHBP2 was successfully expressed in E. coli, with a molecular weight of approximately 25 kDa. RHBP2 demonstrated a strong binding affinity for 1.0 mmol/L hemin and high hydroxyl radical scavenging activity, which scavenged nearly 30% of hydroxyl radical at a concentration of 1 mg/mL rHBP2. After 1.5 h exposure to H2O2 and 8 h exposure to 35 ℃, the survival rates of nematodes fed rHBP2 were 5.7 and 2.4 times higher than those of the control group, respectively, indicating that rHBP2 significantly enhanced nematode tolerance to oxidative and thermal stress. Moreover, nematodes fed rHBP2 exhibited significantly lower endogenous reactive oxygen species (ROS) levels and higher expression levels of oxidative stress-responsive genes such as SOD-3 and CAT-1. These results suggested that rHBP2 had potent antioxidant function. This study provides an experimental basis for discovering and utilizing antioxidant proteins from red rice.

Key words: red rice; antioxidant; heme-binding protein; Caenorhabditis elegans