Functional Study Of General Odorant Receptor Genes Of The Oriental Fruit Moth,Grapholita Molesta Busck

The oriental fruit moth,Grapholita molesta Busck(Lepidoptera: Tortricidae),is a pest that causes serious damage to pome and stone fruits worldwide.The first and second generations larvae damage extremely young shoots of peach,nectarine,and plum trees,while the third and fourth generations larvae mainly feed on the pulp,including that of pear and apple,which has a typical harmful habit of host-switch.The previous works showed that changes in the components of host-plant volatiles were one of the primary reason that G.molesta switched among host plants.Olfactory sensitivity enabled G.molesta to detect and discriminate a variety of chemical compounds.Several olfactory-related genes(for instance pheromone binding proteins,PBPs;odorant binding proteins,OBPs;chemosensory proteins,CSPs and odorant degrading enzymes,ODEs)have been previously studied in G.molesta;however,the functions of odorant receptors(ORs)are not well understood.Therefore,we used the antennal transcriptome of female adults of G.molesta,and five general OR genes of G.molesta were cloned and analyzed.The expression levels of Gmol ORs at different developmental stages(egg,larva,pupa,and adult),in different tissues of adult(antenna,head with antenna removed,thorax,abdomen,leg and wing),and in antennae of females of different ages(1,3,5 and 7 day-old)were detected by quantitative real-time PCR(q RT-PCR).The function of Gmol ORs was studied by Xenopus oocyte expression system,which was further verified by RNAi.The main results are as follows:1.On the basis of the antennal transcriptome of G.molesta adult females,the open reading frame of Gmol ORs were cloned(including Gmol OR7,MH844555;Gmol OR9,MK548580;Gmol OR12,MK910370;Gmol OR20,MH898864;Gmol OR21,MH898865).Five Gmol ORs had 8,7,7,7 and 5 transmembrane domains,respectively,which had typical characteristics of ORs.Multiple sequence alignment and phylogenetic tree analyses indicated that five Gmol ORs were low homology,and only with an identity of 31.26%.The Gmol ORs clustered into five different groups,explicating that they highly differentiated in evolution.2.q RT-PCR results showed that Gmol ORs expressed in different developmental stages of G.molesta,Gmol ORs expressed higher in adults than in other life stages(P < 0.05).Gmol ORs highly expressed in the antennae of adults,with extremely higher expression level in the antennae of adult females than in those of males(P < 0.01).Gmol ORs very weakly expressed or even not expressed in other tissues.This result implied that Gmol ORs might be involved in the identification of host-plant volatiles.Gmol ORs expressed in the antennae of adult females of different ages,with a peak in the antennae of 3-day-old adult females.It is speculated that Gmol ORs might play an important role in host-plant location and spawning site selection.3.Xenopus oocytes expression system results revealed that Gmol OR9 broadly tuned to eight of 47 host-plant components: esters(Z-3-hexenyl acetate,butyl propionate,ethyl hexanoate and ethyl heptanoate)and alcohols(1-hexanol,Z-3-hexenol,2-ethyl-1-hexanol,and linalool).The response of Gmol OR9 to 2-ethyl-1-hexanol was significantly higher than that to the seven other odorants.Gmol OR12 responded to five host-plant volatiles,including alcohols(decanol)and aldehydes(heptanal,octanal,nonanal and decanal),among them,nonanal was significantly higher than to the four other volatiles.Gmol OR20 and Gmol OR21 had no responses to any of the odorant compounds tested in the Xenopus oocyte expression system,suggested that they might respond to other odorants that were not tested in the present study.4.The function of Gmol OR7 and Gmol OR9 were studied by RNA interference and electroantennogram(EAG),and the results revealed that the expression levels of Gmol OR7 were notably reduced(P < 0.05)in ds OR7-injected females and males compared to control.EAG responses of ds OR7-injected females treated with butyl acetate were significantly reduced compared to control(P < 0.05).Compared with the control,the expression levels of Gmol OR9 were significantly reduced(P < 0.05),and the EAG response to Z-3-hexenyl acetate was markedly reduced in ds OR9-injected females(P < 0.05).Gmol OR7 and Gmol OR9 were verified to involve in the identification of butyl acetate and cis-3-hexene acetate,respectively.