Studies On Identification And Structure And Function Of Insect Membrane Proteins Of Olfactory Receptor

Insects got extraordinarily sensitive olfaction during the long history of evolution,which contributed to their feeding,mating,escaping from enemies and host-seeking.As for mosquitoes,olfaction was utilized to seek blood-meal host from human and mammalian to facilitate the oviposition after mating.Plenty of diseases including malaria,dengue fever,yellow fever,West Nile virus,filariasis and Zika virus can be spread when mosquitoes bit their hosts.We analyzed olfactory organs,ionotropic receptors(IR),olfactory receptors(OR)and olfactory signaling mechanism of Anopheles sinensis and Drosophila melanogaster.However,the olfactory molecular mechanism of Anopheles sinensis in China is still unclear although they are the main member to transmit malaria.Scanning electronic microscope was used to measure type,morphology,distribution and amount of sensilla in Anopheles sinensis antennae.Four type of sensilla of Anopheles sinensis in China were reported here,sensilla trichoid,sensilla chaetica,sensilla basiconic and sensilla coeloconic included.There were much difference among four sensilla about amount and distribution.Insects recognized amine and lactic acid mainly rely on ionotropic receptors instead of traditional olfactory receptors,while research on Anopheles sinensisIR still insufficient.35 IR genes were identified in Anopheles sinensis from the alignment of Anopheles gambiae and Drosophila melanogaster,which showed diverse and conserved phyletic evolution.Among 35 IR genes,7 of them were expressed in adult antennae of female Anopheles sinensis and shared some conserved domain.Those 7 IR genes performed differently in response to blood-meal,which has never been reported before.The blood-meal induced different expression level indicated that 7 IR may regulate the host-seeking behavior of mosquitoes.IR and OR were significant receptor proteins in insect olfactory system to manipulate variant signaling pathway but also work cooperatively each other,with difficulty to functional express.Four systems has been used to screen appropriate way to synthesize membrane protein OR42a and ORCO,including E.coli system,Sf9-baculovirus system,wheat germ and E.coli cell-free protein synthesis system.A comprehensive platform has been established to express OR42a and ORCO through combination of expression system,detergents,gravity affinity chromatography,size exclusion chromatography and ion-exchange column chromatography,which not only promoted to decipher olfactory mechanism of insects but also provided novel sights for membrane proteins research.ORCO and OR42a were mainly composed of ?-helix conformations.?-helices accounted for 71% of OR42a and 66% of ORCO,which demonstrated that ?-helices may play key role in ligand-binding.In addition,about 20% were proved coils in OR42a and ORCO,which may contribute to maintaining functional configuration.Ligand-binding ability of OR42a and ORCO was characterized in vitro for the first time on basis of right configuration.Only the combination of ORCO and OR42a could respond to propyl acetate,which conformed to the insect olfactory receptor heteromeric theory.Neither ORCO nor OR42a could recognize the ligand when alone,thus concluding that OR dimer is ligand-gated ion channel.Esters are member of human volatile odorant.The knowledge of recognition of esters by mosquito and fruit fly can facilitate the prevention and control of mosquito and further understanding of insect behavor.