Functional Characterization Of Odorant Receptors In Holotrichia Oblita Faldermann(Coleoptera:Scarabaeida)

Holotrichia oblita(Coleoptera: Scarabaeidae)is one of the dominant species of underground pests in China and is mainly harmful to crops,forest trees and fruit trees.While the larvae are concealed in the soil therefor difficult to control,it is relatively easier to control the adults when they emerge from the soil to feed on the above-ground parts of their host plants.The adults’ ability to locate host plants and spouses relies on a highly sensitive and specific olfactory system to recognize complex volatile odorant molecules in its surroundings.A variety of olfactory proteins are involved in the recognition of odorant molecules in insects,and odorant receptors are particularly attractive because their specificities on recognizing odorant substances.In this study,we cloned five odorant receptor coding genes that are highly expressed in the antennae of Holotrichia oblita.The functions of these five odorant receptors were first studied by two-electrode voltage clamp in vitro using a heterologously expression system in Xenopus oocytes and further verified in vivo by RNA interference and insect behavioral tests.The main results are as follows.1.Cloning of five genes endocing odorant receptors and their expression profiles in different tissuesThe full-length coding gene sequences of HoblOR1,HoblOR2,HoblOR7,HoblOR8 and HoblOR23 were cloned by PCR,and the predicted molecular weights of the proteins were 46.2 k Da,42.0 k Da,43.7 k Da,44.3 k Da,48.0 k Da and each had 6,6,7,5 and 8 transmembrane domains.They have the typical structural characteristics of ORs.The expression of these five ORs in different tissues(antennae,head(resection antennae),thorax,abdomen,foot and wing)of male and female adults of Holotrichia oblita were determined by RT-PCR.The results showed that HoblOR1,HoblOR2,HoblOR7,HoblOR8 and HoblOR23 genes were mainly expressed in male and female antennae.While HoblOR1 and HoblOR8 were highly expressed in both male and female t antennae with no significant difference in expression,HoblOR2,HoblOR7 and HoblOR23 were significantly more expressed in female antennae than in male antennae,presumably related to host plant recognition.Furthermore,HoblOR1,HoblOR2,HoblOR7 and HoblOR23 genes were also highly expressed in the abdomen of female adults,suggesting that they may also be related to the selection of oviposition sites.2.Screening for active compounds by EAGThe EAG assay was used to measure the EAG responses of male and female adults of Holotrichia oblita to 36 host plant volatiles,among which 34 showed active responses.The female ORs responded to all 34 compounds,among which the reactions to cis3-hexenyl acetate,phenylethyl alcohol,1-heptanol and phenylacetaldehyde were relatively stronger.Male ORs responded only to 16 compounds and reacted strongly to 1-hexanol,nonanal and cis-3-hexene-1-ol.3.Functional characterization of odorant receptors in in Xenopus oocytesThe full-length sequences of the five HoblORs were successfully cloned into the expression vector p GEMHE.c RNA was synthesized and expressed in Xenopus oocytes.Then the functions of five HoblORs were studied by a two-electrode voltage clamp system.The results showed that HoblOR1 has electrophysiological reactions to dibutyl phthalate,1-heptanol,and cis-3-hexenol;HoblOR2 has electrophysiological reactions to β-caryophyllene,trans-2-hexen-1-ol,and cis-3-Hexenol,cis-3-hexenyl acetate,and octanal;HoblOR7 has electrophysiological reactions to 1-hexanol,linalool,decane and geraniol;HoblOR8 has electrophysiological reactions to phenylacetaldehyde and nonanal;HoblOR23 has electrophysiological reaction to methyl anthranilate,α-ionone,phenethyl alcohol and octane.4.Confirmation of odorant receptors function in Holotrichia oblitaUsing RNAi,combined with EAG and Y olfactometer,we verified the function of odorant receptor in vivo in Holotrichia oblita.The results showed that the relative EAG responses and tropism to 1-heptanol were significantly reduced in both male and female adults of Holotrichia oblita after HoblOR1 was knocked-down.HoblOR2 was involved in the recognition of β-caryophyllene by both male and female adults.However,after HoblOR2 RNAi,the EAG responses to trans-2-hexen-1-ol increased in both male and female adults.After HoblOR7 RNAi,the EAG responses and chemotaxis of male and female adults to linalool were decreased.After HoblOR8 RNAi,the EAG response and tendency of female to phenylacetaldehyde and the EAG response and tendency of male to nonanal were reduced.After HoblOR23 RNAi,the EAG response and chemotaxis of females to methyl 2-aminobenzoate decreased.The results of behavioral,electrophysiological and functional validation of odorant receptors for host plant volatiles recognition confirmed the functions of five odorant receptors of Holotrichia oblita.