Research and Development Centre of Biorational Pesticides, Northwest Agriculture and Forestry University, Yangling 712100, China
Research Center of Biopesticide Technology and Engineering, Yangling 712100, China
: Received: 24 August 2016 / Approved: 25 August 2016 / Online: 25 August 2016 (10:11:13 CEST)
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Hou, C.; Wang, J.; Wu, H.; Liu, J.; Ma, Z.; Feng, J.; Zhang, X. Molecular Cloning and Expression Analysis of COX II from Sitophilus zeamais, a Potential Action Site of AITC. Preprints2016, 2016080203 (doi: 10.20944/preprints201608.0203.v1).
Hou, C.; Wang, J.; Wu, H.; Liu, J.; Ma, Z.; Feng, J.; Zhang, X. Molecular Cloning and Expression Analysis of COX II from Sitophilus zeamais, a Potential Action Site of AITC. Preprints 2016, 2016080203 (doi: 10.20944/preprints201608.0203.v1).
COX II containing a dual core CuA active site is one of the three core subunits of mitochondrial Cco, which plays a significant role in the physiological process. In this report, the full-length cDNA of COXⅡ gene was cloned from Sitophilus zeamais, which had an ORF of 684 bp encoding 227 amino acids residues. The predicted COXⅡ protein had a molecular mass of 26.2 kDa with pI value of 6.37, and multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXⅡ had high sequence identity 78.51% with the COXⅡ of other insect species, especially similarity to sitophilus oryzae. This gene was subcloned into the prokaryotic expression vector pET-32a, and induced by IPTG in E.coli Transetta (DE3) expression system. Finally the COXⅡ with 6-His tag was purified using affinity chromatography with Ni2+-NTA agarose. WB showed the recombinant COXⅡ was about 44 kD, and the concentration of fusion protein was 50μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXⅡ could catalyze the oxidation of substrate Cytc, and influenced by AITC. It was found that AITC could form a hydrophobic region with COXⅡ protein via molecular docking, besides, a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results will provide valuable information for elucidating the role of COXⅡ in Sitophilus zeamais responses to AITC, meanwhile, it will helpful to carry out a point mutation in AITC binding sites for the future research.