Molecular cloning and characterization of CtCuZnSOD gene from Cyamopsis tetragonoloba under drought stress

CuZnSOD plays a crucial role in mitigating drought-induced oxidative stress by serving as the primary defense against reactive oxygen species (ROS). This study identifies and characterizes CuZnSOD in Cyamopsis tetragonoloba, revealing tissue-specific expression of the CtCuZnSOD gene. Its significant upregulation under drought stress, particularly in leaf tissues, underscores its essential role in the plant's adaptive defense mechanism. A key achievement of this research was the successful cloning of the full-length CtCuZnSOD gene, which consists of a 453 bp open reading frame (ORF) encoding a 150 amino acid protein. The expression of the recombinant protein in E. coli led to the purification of a protein with a molecular weight of 15.22 kDa. Remarkably, the enzyme displayed thermostability, retaining over 20 % of its activity at 80 °C, and functioned effectively across a wide pH range, with optimal activity at pH 5.0. Its inhibition by potassium cyanide and hydrogen peroxide confirmed its classification as CuZnSOD. The enzyme demonstrated remarkable stability, retaining activity even in the presence of strong denaturants such as urea, SDS, DTT, and β-mercaptoethanol. This robustness, confirmed by in silico analysis, underscores its significance for diverse applications. This study underscores the pivotal role of CuZnSOD in bolstering plant resilience against environmental stressors, particularly drought conditions. Additionally, the CtCuZnSOD enzyme's stability and resilience under harsh conditions render it a highly valuable candidate for applications in both agricultural biotechnology and enzyme technology, where the presence of stable enzymes is critical for effectiveness.