Gene Cloning And Functional Characterization Of Olfatory Receptors In Apolygus Lucorum And Adelphocoris Linedatus

Insects are most successful animals whose lives intertwine with human beings.They negatively affect human society when insects become agricultural pests that damage crops or vectors of diseases.By contrast,many other insects are beneficial and essential for human lifes,like the honey bee and Silkworm.Insect prominence among other animals is mostly due to a key physiological element for their survival and reproduction:a sophisticated olfactory system.With this system,insects can precisely perceive the multiple environmental odorants and subsequently accomplish various physiological and behavioral responses,such as food,oviposition sites,locating mating partners and avoidance of predation during the long time of biological evolution.Several proteins are involved in chemo-reception,such as odorant receptors(ORs),ionotropic receptors(IRs),odorant binding proteins(OBPs),chemosensory proteins(CSPs),sensory neuron membrane proteins(SNMPs)and odorant degrading enzymes(ODEs).In recent years,with the rapid development of DNA sequencing technology and molecular biology,it becomes possible to investigate the insect olfactory system more deeply,which is also in the popular field of entomology research today.Based on the insect olfactory mechanism,we can set olfactory genes as target and screen effective odors,which lay an important theoretical basis for designing a more efficient regulation of insect behavior.Here,by combined the use of traditional chemical ecology,molecular biology,bioinformatics and electrophysiological techniques,we identified and functionally analyzed a number of olfactory receptor genes which involved in sex pheromone and plant volatile recognition of the important agricultural pest Apolygus lucorum and Adelphocoris lineolatus.The main results are as follows:1.The pheromones in A.lucorum and the response of EAG and behaviorIdentification the sex pheromones is a prerequisitie to investigate the insect control by sex pheromones.By GC-MS,we identified the main content from the extract in male and female A.lucorum in this chapter,which was(E)-4-Oxo-2 hexenal?Hexyl butyrate and(E)-2-Hexenyl butyrate.In the EAG experiment,both male and female A.lucorum could response each sex pheromone or combinations with no difference.In the behavior experiment,the combination of(E)-4-Oxo-2 hexenal and(E)-2-Hexenyl butyrate could attract male adults at low concentration,while being repellent at high concentration.Hexyl butyrate was repellent to male adults.In this study,we verified the composition of sex pheromones from A.lucorum,which could lay the foundation to the research of olfactory molecular recognition mechanisms in the future.2.Antennae transcriptome analysis and identification of putative odorant receptors in A.lucorum and A.lineolatusWe carried out a next generation sequencing project on two cDNA library constructed from the antennae of A.lucorum and A.lineolatus using Illumina HiSeqTM2000 platform.The analysis of the sequencing data resulted in about 4.6?4.8G reads,56,210 Unigenes in A.lucorum and 95,262 Unigenes in A.lineolatus.Among these Unigenes,most sequences blasted to Tribolium castaneum and Bombyx mori.In the GO classification,high percentages of Unigenes were associated with chemosensation related sub-category such as binding and catalytic activity.Based on the transcriptomic analysis,a total of 79 and 68 ORs were for the first time obtained from A.lucorum and A.lineolatus by Blastx.In the phylogenetic tree,traditional ORs shared high homology between A.lucorum and A.lineolatus,while Orcos in different specises were high conservative and clustered in one subgroup.RT-PCR was conducted in A.lucorum to examine the accuracy of the assembly and annotation of the transcriptome.The result showed that all the AlucORs were expressed in antennae,and few ORs were highly expressed in male antennae.Obtaining of numerous olfactory genes in this two related mirid bugs will lay the foundation for further research in the olfactory molecular mechanisms.3.Identification,expression pattern and functional analysis of pheromone receptors from A.lucorumBased on the prediction from transcriptome analysis of A.lucorum,we cloned 3 PRsORF from the antennae of A.lucorum.Tissue expression patterns showed that the 3 PRs was male antennae-biased and almost non expression in other tissues.Temporal expression patterns showed that AlucOR3 and AlucOR42 were abundantly expressed from the adult stage,but AlucOR25 were expressed in every stage from nymths to adults.Functional analysis in heterologous expression system Xenopus oocytes demonstrated that 3 PRs were all tuned to Hexyl butyrate and(E)-2-Hexenyl butyrate;AlucOR25 was also tuned to pheromone analogs.Dose response showed that the sensitivity to pheromones of AlucOR3 was higher than other two.This chapter provided the basis for studying the recognition mechanism of sex pheromones in A.lucorum.4.Identification,expression pattern and functional analysis of odorant receptors from A.lucorumThe odorant receptor repertors(ORs)housed within the dendritic membrane of sensory neurons is one of the primary determinants of the odor recognition.ORs could be classified into pheromone receptors(PRs)and non-pheromone receptors(non-PR ORs).Based on the data from transcriptome of A.lucorum,we cloned 4 ORs full lengths.Tissue expression patterns showed that 4 ORs were highly expressed in antennae,low expressed in proboscises and legs,and no expression in other tissues.Temporal expression patterns showed that 4 ORs were low expressed in nymth stages and abundantly expressed from adult stage.Functional analysis of the four AlucORs was conducted in heterologous expression system Xenopus oocytes.AlucOR28 was sensitively tuned to cis-3-Hexenyl acetate,with other isomers and 3 lipids.AlucOR30 was slightly responded to(1S)-(-)-Verbenone.However,AlucOR12 and AlucOR18 didn't respond to any chemicals tested in this study.These results provided the basis for the molecular recognition mechanisms of traditional olfactory receptors from A.lucorum.5.Identification and functional analysis of odorant receptors from A.lineolatusBased on the transcriptome from A.lineolatus,we predicted and cloned the full length of 1 Orco gene(AlinORl)?3 PR genes(AlinOR4?AlinOR6?AlinOR7)and 2 OR genes(AlinOR8?AlinOR9).We examined that these 6 ORs were all expressed in antennae by RT-PCR.Functional analysis in heterologous expression system Xenopus oocytes demonstrated that 3 PRs were all tuned to Hexyl butyrate and(E)-2-Hexenyl butyrate;AlinOR4 was also tuned to pheromone analogs and alcohols.Dose response showed that the sensitivity to pheromones from biggest to smallest was:AlinOR6>AlinOR7>AlinOR4.Meanwile,AlinOR8 responsed to 7 acetates?alcohols and benzene compounds.AlinOR9 was only tuned to benzaldehyde and acetophenone.In conclusion,we identified 79 ORs from A.lucorum and 68 ORs from A.lineolatus based on the transcriptome analysis.We cloned the full cDNA lengths of 3 PRs and 4 ORs from A.lucorum and examined the expression profile in different tissues and development stages.From A.lineolatus,the full cDNA lengths of 3 PRs and 4 ORs were obtained.All these ORs were functionally studied by heterologous expression system in Xenopus oocytes.The results will provide considerable basis for further elucidation of the molecular mechanisms of sex pheromone and plant volatile recognition in A.lucorum and A.lineolatus,and provide important reference to develop efficient behavioral attractants and disruptants for pest control.