Analysis Of Binding Characters And Localization In Tissues Of Odorant Binding Proteins In Sitobion Avenae

Acute olfaction allows insects to orientate food and mates, and hide from predators quite accuratelyby discriminating semiochemical cues in natural environment. Odorant binding proteins (OBPs) are thefirst functional proteins which involved in the olfactory perception. According to the known pea aphidsequence, seven OBPs genes with complete coding sequence were obtained from Sitobion avenae byhomologous gene cloning, and they were deposited in GenBank (SaveOBP2, JN165749; SaveOBP3,JN165750; SaveOBP4, JN165751; SaveOBP5, JN165752; SaveOBP7, GQ847859; SaveOBP8,GQ888708; SaveOBP9, GQ847860). All the SaveOBPs were predicted to contain a highly conserveddomain of the PBP and GOBP superfamily separately except SaveOBP5. Two consistent evolutionarytrends of orthologous genes were observed from the phylogenetic trees which constructed by aphidOBPs available, one was S. avenae and Metopolophium dirhodum, the other was S. avenae andAcyrthosiphon pisum, which indicated that some particular relationships might exist between OBPgenes and the selective pressure from the host surroundings.Six expression plasmids contained separate SaveOBPs were reconstructed and they were expressedin Escherichia coli host cells by inducing. Fusion proteins with histidine tags were easily purified by theimmobilized metal nickel chelate affinity chromatography, and western blot confirmed that fusionproteins were totally expressed. The histidine tags were digested by the recombinant enterokinase (rEK),and the rest part of fusion proteins were quite close to the natural state of SaveOBPs. Fluorescencecompetition binding assays were applied to explore the binding characters of SaveOBPs for wheatvolatiles and aphid alarm pheromone. The six SaveOBPs showed a broad binding character for the smallodorants with C6–C8when they competed1-NPN. SaveOBP2was able to bind several components ofhost plant volatiles which were released from the 'Beijing837' wheat crops at micromolar level (thedissociation constants of inhibitors were at about10μM), among which benzaldehyde and someodorants belonging to green leaf volatiles like trans-2-hexenal, trans-2-hexen-1-ol and cis-3-hexen-1-olexhibited higher affinities. Meanwhile, SaveOBP7could bind trans-β-farnesene very strongly (thedissociation constant of inhibitor was at1.3μM), which indicated that SaveOBP7was an importantfunctional protein in recognizing aphid alarm pheromone. After comparing the binding abilities amongSaveOBP2, SaveOBP3, SaveOBP4, SaveOBP5and SaveOBP8, I found that they had obviousdiscrepancy in discriminating green leaf volatiles of C6–C8. SaveOBP2was pretty excellent in bindingalcohols, aldehydes and esters with small molecular weight, which suggested that SaveOBP2might playan important role in sensing host plant volatiles.The expression and localization in tissues of three genes (SaveOBP3, SaveOBP7and SaveOBP8)were investigated by real-time fluorescent quantitative PCR. These genes were thought to be involved inolfactory recognition, because they all expressed in the antennae. SaveOBP7was detected in neither legnor wing, but was predominantly expressed in the antennae. Both SaveOBP3and SaveOBP8wereexpressed in the whole body, and were much higher in the leg tissues, which implied special olfactoryfunctions they might have. Additionally, in the study of expression among different developmental stages, SaveOBP3, SaveOBP7and SaveOBP8were expressed in the first three nymphal periods withrather small differences, but the expression increased and reached a peak in the4th nymphal period, andthen it dropped back to the former amount in the adult period; SaveOBP3and SaveOBP8wereexpressed higher in the alate adults than the apterous ones, but SaveOBP7was equally expressed, theirdifferential expressions suggested that important roles might exist in the host-seeking of the alate adults.