Cloning, sequence analysis, tissue expression profiling and prokaryotic expression of odorant binding protein genes MaltOBP2 and MaltOBP6 from Monochamus alternatus (Coleoptera: Cerambycidae)
[Aim] Odorant binding proteins (OBPs) play an important role in olfactory recognition process.This study aims to explore the structure and function of odorant binding proteins of Monochamus alternatus.[Methods] The cDNA and deduced amino acid sequences of MaltOBP2 and MaltOBP6 genes were analyzed by bioinformatics methods.The expression levels of the two genes in different tissues and developmental stages of M.alternatus were detected by real-time RT-PCR.Prokaryotic expression vector was constructed to express the recombinant proteins.[Results] We successfully cloned two odorant binding protein genes from M.alternatus,which were named as MaltOBP2 (GenBank accession no.KP120891) and MaltOBP6 (GenBank accession no.KP120892),with the opening reading frame of 402 bp and 408 bp in length,respectively.Their translated amino acid sequences contain four conserved cysteines,suggesting that they belong to the Minus-C OBP subfamily.MaltOBP2 and MaltOBP6 have six deduced α-helix regions,and the sites of α-helix regions are very similar between each other.However,the type and polarity of their putative ligand binding sites are completely different.Tissue expression profiles showed that MaltOBP2 and MaltOBP6 were differentially expressed in the head,antennae,maxillary (labial) palpi,abdominal apex and legs of M.alternatus adults,with the highest expression level in the head.The expression levels of MaltOBP2 and MaltOBP6 in antennae were not higher than those in other tissues.Developmental expression profiles showed that MaltOBP2 and MaltOBP6 had the highest expression level in the antennae of pupa and the head of larva,respectively.Prokaryotic expression vectors pET32a-MaltOBP2 and pET32a-MaltOBP6 were successfully constructed and the recombinant proteins were successfully expressed in Escherichia coli.Low temperature (16℃ and 20℃) induced higher levels of the expression of recombinant proteins in the supernatant,and long induction time (12 h) increased the expression of the protein.[Conclusion] In this study,the full-length cDNA of two Minus-C OBP genes were cloned from M.alternatus.Their encoded proteins have different physiological functions due to their different ligand binding sites.Expression profiling results suggested that these two OBP genes are not only involved in the olfactory recognition of M.alternatus,but also in other physiological functions such as taste sensing and chemical sensing.Our results lay the foundations for studying the structure and function of MaltOBP2 and MaltOBP6,and are also helpful to our understanding of chemical sensing in M.alternatus.
Monochamus alternatusodorant binding protein (OBP)gene cloningexpression profilingprotein structureprokaryotic expression
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