ECPUB5 Polyubiquitin Gene in Euphorbia characias: Molecular Characterization and Seasonal Expression Analysis

The spurge Euphorbia characias is known for its latex, which is rich in antioxidant enzymes and anti-phytopathogen molecules. In this study, we identified a novel polyubiquitin protein in the latex and leaves, leading to the first molecular characterization of its coding gene and expressed protein in E. characias. Using consensus-degenerate hybrid oligonucleotide primers (CODEHOP) and rapid amplification of cDNA ends (5'/3'-RACE), we reconstructed the entire open reading frame (ORF) and noncoding regions. Our analysis revealed that the polyubiquitin gene encodes five tandemly repeated sequences, each coding for a ubiquitin monomer with amino acid variations in four of the five repeats. In silico studies have suggested functional differences among monomers. Gene expression peaked during the summer, correlating with high temperatures and suggesting a role in heat stress response. Western blotting confirmed the presence of polyubiquitin in the latex and leaf tissues, indicating active ubiquitination processes. These findings enhance our understanding of polyubiquitin's regulatory mechanisms and functions in E. characias, highlighting its unique structural and functional features.