| Honeybee exhibits complex and sensitive olfactory system, which plays aphysiological role in colony life, such as honeybee can perceive chemical odorsinvolved in the colony and distinguish different volatiles in the outer environment.As an indigenous bee species only live in China, Apis cerana cerana Fabricius isgood at discovering sporadic nectar for the honey products, meanwhile it also playsan important ecological role as pollinators of mountain plants and low-temperatureflowering plants species in China. The behavior characteristics of A. cerana ceranamight have close relationship with its olfactory related proteins in its olfactorysystem. In this paper, the odorant binding proteins AcerOBP11and the olfactoryreceptor Orco gene, AcerOrco from honeybee A. cerana cerana were cloned byRT-PCR. Meanwhile their expression profiling at different developmental stages andin different tissues were studied by real-time PCR, respectively. The recombinantAcerOBP11was separated and purifed by Escherichia coli gene expression systemand the artificial antigen was designed and synthesized, thus, high specific antibodiesof this two proteins were prepared, and the sub-cellular localization of AcerOBP11and AcerOrco on the forager’s antennae were detected by immunofluorescence，theprimary results as follows:1. The full-length cDNA sequences of odorant binding protein11(OBP11) geneand olfactory receptor Orco gene were obtained, their open reading frame isrespectively427bp and1434bp in length, and their GenBank ID is KC818631andJF968610.1, respectively. The nuclear acid sequences analysis showed AcerOBP11encoding142amino acid residues, its protein has six conservative cysteines andcontains a conservative domain that comprised by six α-helices, phylogentic treeshowed that AcerOBP11has similar relation with pheromone bind proteins fromother insects in Hymentoptera, which implies AcerOBP11maybe a novel putativePBP of A. cerana cerana；Meanwhile, AcerOrco encoding477amino acid residues, which was predicted as having seven-transmembrane domains and four hydrophilicregions outside the cell membrane, and its amino acid sequences have high similaritywith other Orco in other insects, so it suggests that AcerOrco should belong to onekind of highly conservative olfactory receptors.2.The expression profiling of AcerOBP11and AcerOrco at differentdevelopmental stages and in different tissues was measured by Real-time PCR, theresults showed that AcerOBP11and AcerOrco were expressed in the egg, larval andpupal at very low levels. The expression profiling in different tissues revealed thatAcerOBP11was only expressed in the antennae, legs and wings in three differentages. However, AcerOBP11was highly expressed in the antennae and wings of the1day-old worker, while in forager it was only highly expressed in antennae; ToAcerOrco, the expression profile in different tissues revealed it was highly expressedin antennae and legs in nurse bees, with the highest level in the antennae of the1day-old worker, while in forager it was highly expressed in antennae, head (withoutantennae), thorax, abdomen and wings.3. The recombinant AcerOBP11protein was obtained using constructedprokaryotic expression vector pET32-AcerOBP11, and the recombinant protein waspurified by nickel agarose gel chromatography. The binding properties of therecombinant purified AcerOBP11with some odorants was investigated by thefluorescence competition binding assays using1-NPN as fluorescent reporter. Theresults showed that AcerOBP11had strong binding capability withPhenylacetaldehyde,3,4-Dimethylbenzaldehyde,4-Allylveratrole and β-Ionone,which are belong to plant volaties. In addition, AcerOBP11can effectively bind withthe worker’s pheromone Farnesol. It suggest that the AcerOBP11is a pheromonebinding protein (PBP) and can selectively bind with plant volatile and somepheromone.4. The subcellular localization of AcerOBP11and AcerOrco on the chemosensillain the foragers’ antennae were labelled by immunofluorescence using polyclonalantibodies against AcerOBP11and AcerOrco, respectively.. The results displayedthat AcerOBP11was specially expressed in the1stflagellar segment of antenna andthe joint of every segment; AcerOrco was largely expressed with pairs in theforagers’ antenna, especially in the1stflagellar segment of the antenna. Furthermore, AcerOrco was seemingly to mainly express in the outer dendrite(OD) neurons ofsensilla trihodea and the dendrites of sensilla placodea on antennae. Doubleimmunofluorescence revealed co-localization of AcerOBP11and AcerOrco insensilla basiconcum and sensilla placodeum. |
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