In Vitro Odorant Binding Profiles Of The Olfactory Receptor Of Bactrocera Dorsalis:Comparison Of Two Detection Methods

Olfactory receptors（ORs）play a central role in odorant recognition,the functional study of ORs has attracted extensive attention.Due to the large number and high divergence of ORs in insect olfactory sensilla,the"deorphanization"of insect ORs has been a hot topic.However,as ORs are membrane receptor proteins with complex structures,it is still a big challenge to develop a simple,rapid and reliable in vitro binding assay method for insect ORs.Even in the post-genomic era,due to the constraint of OR"deorphanization"method,the ligand-receptor pair identification of insect ORs has been extremely difficult.Currently,there are three in vitro expression systems of insect ORs,including Xenopus oocytes,mammalian cells and insect cells.Among them,Xenopus oocyte expression system combined with the two-electrode voltage clamp recording technique is the gold standard for the detection of odorant binding ability of ORs in vitro.Nevertheless,this system depends much on the quality of Xenopus oocytes.Moreover,since its operation is complicated and time-consuming,it is hard to achieve high throughput measurement.In contrast,calcium imaging based on HEK293 cell expression system is regarded as a simple,rapid and high throughput method which is expected to be used for high-throughput screening of odorant binding patterns of insect ORs.Therefore,the present study focused on the comparison of the two in vitro binding activity assay systems targeting the ORs of Bactrocera dorsalis,providing an important reference for the development of a simple and efficient"deorphanization"method of insect ORs.The main results are as follows:1.Construction of heterologous expression plasmid for Bdor ORxBased on the high-quality genome and transcriptome data of B.dorsalis available in the laboratory,the group have annotated 73 OR-like genes.Using the primers we designed,full-length ORF sequences of 38 ORs（including ORco）were obtained by nested PCR.Subsequently,the 38 ORs were successfully subcloned into the expression vector p T7Ts for Xenopus oocytes by enzyme digestion,ligation and were further confirmed by Sanger sequencing.2.In vitro responses of Bdor ORx to odorants based on Xenopus oocyte expression systemUsing a two-electrode voltage clamp recording technique based on Xenopus laevis oocyte expression system,we measured the in vitro binding ability of 38 Bdor ORx to 22odorants,and found that 10 ORs could bind with 12 odorants.Among them,eight ORs（Bdor OR4,Bdor OR6,Bdor OR10,Bdor OR11,Bdor OR18,Bdor OR36,Bdor OR43a-1,Bdor OR47）showed dose-dependent binding ability to odorants such as 1-octen-3-ol,benzothiazole and citral.The other two ORs（Bdor OR5 and Bdor OR63a-1）showed weak binding ability with odorants such as n-hexanol,ethyl butyrate and diethyl maleate at the concentration of 100μM.3.In vitro responses of Bdor ORx to odorants based on transient expression system of HEK293 cellsThe above results were verified by calcium imaging technology based on the transient expression system of HEK293 cells,and the overall results were similar.In this process,two expression vectors of mammalian cells were used.One is pc DNA3.1（+）vector with a fluorescent protein tag（m Cherry or m Orange）to improve the observability.The other one is the p CMV-BI vector containing the bidirectional promoter cassette,which can drive the expression of the two genes at the same time.As the results indicated,only four（Bdor OR11,Bdor OR18,Bdor OR36,Bdor OR47）of the eight ORs showed dose-dependent responses to the corresponding odorants.In order to improve the heterologous expression efficiency of ORs,the two ORs（Bdor OR11 and Bdor OR36）genes were constructed with minimal N-terminal peptide tags,which were synthesized and subcloned into the p CMV-BI vector.However,the results showed that Ca²⁺response was not significantly increased.