Analysis Of Odorant Receptor Genes Differentially Expressed In Antennae Of Male And Female Adults Of Conogethes Punctiferalis

The yellow peach moth(Conogethes punctiferalis)is an important lepidopteran pest in many fruit trees and crops.As a borer,larvae of the insect are difficult to control with traditional methods.Therefore,behavioral regulators such as sexual attractants are important alternatives for control of C.punctiferalis.Based on olfactory perception mechanism of insect pests,a theory of “reverse ecology” provides valuable guidance for finding potential chemicals as behavior regulators.The application of the theory requires a comprehensive understanding of the peripheral olfactory system of insect pests,especially the odorant receptors(ORs),which are key players in olfactory system.But inadequate understanding of odorant receptors of C.punctiferalis limites the development of efficient behavioral agents.In this study,based on the full-length transcriptome of C.punctiferalis,the differentially expressed odorant receptor genes were screened by antennae transcriptome sequencing of female and male adult,and phylogenetic relationship of OR genes was analyzed.Then,several odorant receptors were expressed by Xenopus laevis oocytes heterologous expression system.Finally,a two-electrode voltage clamp system was used to detect the binding ability of odorant receptors to pheromone components of C.punctiferalis.Main results are as follows:1.Analysis of antennae transcriptome and differentially expressed ORs of C.punctiferalisThrough transcriptome sequencing and comparative analysis,139 genes differentially expressed between the antennae of male and female moth were obtained.Among 139 genes,77 ones were male-biased expressed and 62 genes were female-biased.Among all differentially expressed genes,a total of 15 genes were annotated as odorant receptors,among which full length of 10 genes were obtained based on our previous full length transcriptome data.Three of the 10 OR genes,Cpun OR3、Cpun OR5 and Cpun OR34 were verified highly expressed in male antennae and seven OR genes including,Cpun OR14,Cpun OR15,Cpun OR16,Cpun OR26,Cpun OR30,Cpun OR53 and Cpun OR54,were highly expressed in female antennae,through quantitative PCR trial.2.Cloning and bioinformatics analysis of full-length c DNA of odorant receptor genes differentially expressed in male and female antennae of C.punctiferalisBy designing specific primers,Cpun Orco,Cpun OR3,Cpun OR5,Cpun OR14,Cpun OR15,Cpun OR16,Cpun OR26,Cpun OR30,Cpun OR34,Cpun OR53 and Cpun OR54 were cloned from the c DNA of the antennae of the male and female moth.The open reading frame of each OR gene ranged from 330 to 1377 bp.Each OR gene encoded amino acids ranging from 316 to 474.Online prediction showed that all ORs had no signal peptides,and only Cpun Orco and Cpun OR5 had complete seven transmembrane domains.Phylogenetic tree of OR was constructed and the results showed that,Cpun Orco was associated with Orco of Lepidopteran species including Ostrinia furnacalis,Cnaphalocrocis medinalis,Helicoverpa armigera,Plutella xylostella,Mythimna separata and Bombyx mori and clustered in one branch.Sequence alignment results of these Orco showed that the sequence similarity was more than 90%.Both Cpun OR3 and Cpun OR5 were clustered with the sex pheromone receptor genes of Cnaphalocrocis medinalis.While Cpun OR30 and Cpun OR34 were clustered with the odorant receptor of Ostrinia furnacalis.The self-spreading values of remaining ORs are relatively low.3.Heterologous expression and functional analysis of odorant receptor genesCpun Orco-p T7 TS and expression vectors Cpun OR3-p T7 TS,Cpun OR5-p T7 TS,Cpun OR26-p T7 TS and Cpun OR30-p T7 TS were successfully constructed by double enzyme digestion ligand.Both c RNA of Orco and OR genes were then synthesized and co-injected into Xenopus laevis oocytes.Cpun OR3,Cpun OR5 and Cpun OR30 were successfully expressed and subjected to the two-electrode voltage clamp trial.Results showed that Cpun OR5 binds to male pheromone,the tiglic acid.The binding reaction of Cpun OR3 and Cpun OR30 with pheromone ligand was not detected.Generally,results of this study showed that antennal odorant receptor genes of C.punctiferalis were similar to those of Lepidopteran species,some of which were expressed in a sex biased way.Among these ORs,Cpun OR5 binds to the male sex pheromone of tiglic acid.Combind with the fact of high expression abundance of Cpun OR5 was detected in both male and female adult.These results indicat that Cpun OR5 is expected to be a potential target of sexual attractant for both male and female adults.Further research will be expected to find more similar targets,so as to promote the research and development of behavior regulators of C.punctiferalis and related insect pest species.