Abstract: 【Objective】The objective of this study is to clone full-length cDNA of cytochrome C oxidase subunit Ⅲ (COXⅢ), which is a possible action site of allyl isothiocyanate (AITC), then analyze the sequence and prokaryotic expression．【Method】Firstly, finding the known part of the gene of COXⅢ in the NCBI, the full sequence of Sitophilus zeamais COXⅢ was cloned by RT-PCR and RACE techniques. The sequences of COXⅢ, phylogenetic relationship and properties of S. zeamais COXⅢ, the three-dimensional structure were analyzed by a variety of bioinformatics softwares such as DNAMAN8.0, MEGA5.1, GENEDOC, ProtParam and TargetP 1.1 Server et al. Using pEASY-Blunt E1, pET28a, pET30a, PET32a and pET42a as a fused expression vector, a recombinant plasmid pEASY-Blunt E1-COXⅢ, pET-28a-COXⅢ, pET30a-COXⅢ, pET32a-COXⅢ, pET42a-COXⅢ were constructed. Then inducing its expression in Escherichia coli BL21 (DE3) with IPTG at different concentrations, times and temperatures. SDS-PAGE was used to detect the fusion protein expression and the protein expression was verified by Western blot.【Result】The full-length sequences of COXⅢ from S. zeamais was obtained, and its ORF is 792 bp encoding 263 amino acid residues, the predicted molecular weight is 30 938.1 Da, isoelectric point pI is 6.51, the prediction formula is C₁₄₉₂H₂₁₅₄N₃₃₈O₃₆₂S₁₀, the unstable factor is 34.49, and the total average hydrophilic coefficient is 0.492. It is a stable hydrophobic protein. Phylogenetic tree showed that S. zeamais had the closest evolutionary relationship with S. oryzae at the amino acid level. Prokaryotic expression results showed that efficient expression of COXⅢ protein could be realized after induced with 1 mmol·L^-1 IPTG in E. coil BL21 (DE3) for 24 h at 18℃. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. Western blot confirmed that the molecular weight of the recombinant COXⅢ is about 62 kD, consistent with the predicted result.【Conclusion】The full-length sequences of COXⅢ from S. zeamais was successfully obtained, and the prokaryotic expression of its encoding protein was achieved, which will lay a foundation for further function research of S. zeamais COXⅢ, and provide a theoretical basis for the research of the mechanism of the action on controlling S. zeamais by using isothiocyanates compound.

Key words: Sitophilus zeamais, cytochrome C oxidase subunit Ⅲ, RACE technique, bioinformatics analysis, prokaryotic expression