| Insects rely primarily on sophisticated olfactory system to distinguish key signals emitted from environment,which regulate a series of their behaviors,such as locating habitats,searching for food source,finding mates and oviposition sites.Odorant receptors(ORs)play a vital role in the olfactory perception of chemical cues.Therefore,the identification and functional characterization of ORs will help to understand olfactory mechanisms in insects.The reverse chemical ecology concept indicates that active chemicals were high-throughput screened by olfactory genes in order to regulate insect olfactory behavior.This method grently improves the efficiency for accurately screening insect behavioral regulators.In this study,we analyzed the phylogenetic tree of ORs in Diptera insects,and identified their conserved clades of ORs.Based on reverse chemical ecology concept,PCR amplification was used to clone the full-length c DNA of conserved OR genes.Real-time quantitative PCR(RT-q PCR)was used to evaluate the expression levels of associated OR genes in different tissues of female and male of Eupeodes corollae.Functional characterization of associated ORs was analyzed by using the Xenopus occyte expression system combined with twoelectrode voltage clamp technique.Obtained odorant ligands were selected to preform electroantennogram(EAG)assay and Y-tube experiments.This study clarified the molecular mechanism of olfactory recognition of E.corollae,and provide a theoretical basis for screening and developing efficient attractants of E.corollae.Main results were shown below.1.The OR of the conserved clade of the phylogenetic tree of ORs in E.corollae was identified and cloned.The phylogenetic analysis showed that Ecor OR4 clustered with homologous genes in Diptera,such as Dmel OR13 a,representing a conserved clade.The fulllength sequence of Ecor OR4 gene was cloned and obtained.Nucleotide sequence of Ecor OR4 is 1275 bp and encodes 424 amino acids.Sequence analysis showed that Ecor OR4 and other homologous genes in Syrphidae and Trypetidae had 7 transmembrane domains with 53.88%-80.13% identities of amino acid sequence.This result further indicated that Ecor OR4 clade was quite conserved sequence.2.The tissue expression pattern of Ecor OR4 gene was analyzed.The results of RT-q PCR showed that Ecor OR4 gene was mianly expressed in the antennae,with no significant between the female and male.Very low expression was observed in other tissues,such as heads(without antennae),thoraxes and abdomens,and legs.3.In vitro function of Ecor OR4 was characterized.The results recorded by two-electrode voltage clamp showed that Ecor OR4 specifically responded to 1-octen-3-ol with an average current of 259.05±27.96 n A,and weakly to cis-3-hexenyl acetate,trans-3-hexen-1-ol and trans-2-hexen-1-ol.No responses were tested by other chemicals.This result suggested that Ecor OR4 is the major receptor to detected 1-octen-3-ol.4.The antennal response of E.corollae to 1-octen-3-ol was measured.The EAG results showed that 5 serial concenteations of 1-octen-3-ol can evoke EAG responses of both virgin and maded E.corollae.The antennal responses gradually increased as the concentrations of1-octen-3-ol from 0.01μg/μL to 100μg/μL.There were no significant differences of EAG responses between both sexes of virgin hovorflies to 1-octen-3-ol.However,the antennal responses of mated female responds to 1-octen-3-ol significantly higher than mated male.The antennae of mated female were more sensitive than the antennae of virgin female to the1-octen-3-ol at concentrations of 100μg/μL,10μg/μL and 1μg/μL.No significant difference of EAG response was found between virgin female and mated female with 0.1μg/μL and0.01μg/μL.5.The behavioral response of E.corollae to 1-octen-3-ol was determined.The results of behavioral experiment showed that 0.1 mol/L 1-octen-3-ol could attract mated female hovorflies with a singnificant preference,but no attraction was observed in virgin female,virgin male and mated male. |
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