| Monochamus alternatus Hope is the main vector insect of Bursaphelenchus xylophilus disease.The key approach to control this disease is preventing and controling M.alternatus.Olfaction is the basis of many physiological behaviors of insects.The research of insects olfactory mechanism is essential,which is beneficial to understanding the molecular recognition mechanism of insect chemical signals.However,the olfactory recognition mechanism of M.alternatus is still unclear.Odorant receptor co-receptor(Orco),which is highly conserved across different insect species,plays an important role in the olfactory system of insects and possesses certain research prospects.Studying insect olfaction and applying it to pest control has become an important method of pest management.In our study,we designed and developed some more efficient regulatory techniques to further study the olfactory recognition mechanism of M.alternatus,and clarify the olfactory-related receptors and their functions.Based on the research progress of olfactory receptors,we studied the genomic structure and expression characteristics of Orco in M.alternatus.The receptor gene Orco was studied by RNAi.The main conclusions are as follows:In order to systematically analyze the olfactory receptors,which was associated with olfactory recognition in M.alternatus,we used transcriptome sequencing to construct transcriptome of the antennae of M.alternatus adults.Then we obtained 53,015 Unigenes with an average length of 745 bp by transcriptome sequencing.8 CSPs,53 IRs,12 OBPs,37 ORs and 1 Orco were also obtained from the transcriptome of antennae of M.alternatus adults.The phylogenetic tree of Orco was constructed with Orco of other insects.The results indicated that comparing with Orco of other insects,Orco of M.alternatus had the closest relationship with the Orco of Anoplophora glabripennis,an insect belonging to Coleoptera.The fact was relatively consistent with its biological evolutionary status,which indicated that Orco was in the evolutionary process.The identification of Orco full-length sequence of M.alternatus provided a preliminary basis for further exploring the role and function of olfactory receptors in odor recognition.Orco gene of M.alternatus was successfully cloned by PCR technology.The total length of orco gene was 1,434 bp,encoding 477 amino acids.After cloning,identification and bioinformatics analysis of the orco gene of M.alternatus,we found that the Orco of M.alternatus is a hydrophobic protein with 7 transmembrane structures.And we predicted the Orco of M.alternatus had no signal peptide sites.At the same time,there were 21 N-glycosylation modification sites and 47 phosphorylation sites in the amino acid sequence of Orco,which illustrated the possible biology function of Orco of M.alternatus.The expression of Orco in the antennae of adult M.alternatus was analyzed by RT-qPCR.Orco was highly expressed in the antennae of adult M.alternatus,and was also detected in other parts such as head,chest,abdomen,foot and wings.The results showed that the highest expression of Orco was in the head,followed by the abdomen,wings,feet and chest.The expression of Orco at the end of adult stage of M.alternatus was significantly higher than that at other developmental stages.And the expression of antennae in females was significantly higher than that in males.The study on the expression pattern of Orco of M.alternatus can provide a theoretical basis for further identifying the function of Orco.The Orco gene of M.alternatus was silenced by RNAi technology.The successful application of RNAi technology in M.alternatus was proved by RT-qPCR.The expression of Orco was significantly reduced by injecting dsRNA.EAG and behavioral analysis showed that the gene silencing of Orco of M.alternatus reduced the potential changes induced by attractants,turpentine oil and sex pheromones,and also made the behavior of M.alternatus towards odor molecules random.The gene silencing of orco may cause some interferences to the olfactory recognition of M.alternatus.This study laid a foundation for further study the biological function of Orco in the process of olfactory recognition of M.alternatus,and revealed the olfactory conduction mechanism of M.alternatus by orco gene.Our research provided a potential application for the control of M.alternatus and helped to develop new technology for pest control.The research indicated that Orco may be a potential target to destroy the olfactory signals of insects because of the high conservativeness of Orco in different insects and its unique position in the olfactory recognition mechanism.Thus,both Bursaphelenchus xylophilus disease and the number M.alternatus can be efficiently controlled by olfactory disturbance.This will be a new method for integrated control of Bursaphelenchus xylophilus disease in the future.The expected expected results not only laid lay a solidfirm foundation for the development and utilization of new and highly effective insecticides,and attractants and re-pellent with Orco of M.alternatus as potential molecular target,but also provided scientific basis for studying the mechanism of host selection behavior mechanism and behavior regulation of M.alternatus,Besides,andthis result played an important role in exploration of the behavior regulation of M.alternatus and the prevention and control of Bursaphelenchus xylophilus disease in China. |
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