Identification Of Odorant Receptors Responding To Myrcene And Ethyl Butyrate In Diaphorina Citri

Citrus huanglongbing(HLB)is a devastating disease of citrus industry,and citrus psyllid,Diaphorina citri Kuwayama,is a principal vector of Candidatus Liberibacter asiaticus.Because chemicals have not been found to prevent the HLB effectively yet,so the control of citrus psyllid has become the most effective way to suppress the regional expansion of HLB.At present,the chemical prevention is still the main method to control citrus psyllid.Besides,the research of green prevention and control technology based on efficient attractants is the focus of international scholars.In this study,we analyzed the expression profiles of different odors stimulation and different developmental stages of citrus psyllid by RT-qPCR based on the genomic and transcriptomic data of Diaphorina citri,which laid a foundation to explore potential molecular targets.The full length of DcitOR18 and DcitOrco were cloned by RT-PCR,and were coexpressed in mammalian expression system(HEK293 cells).Calcium imaging was employed to test the binding activity of DcitOR18 to myrcene.Our results provided theoretical basis for developing new attractants or repellents to control citrus psyllid.The main results were as follows: 1 Expression profiles of DcitORs in Diaphorina citriExpression profiles of 40 odorant receptor genes in citrus psyllid were analyzed by RT-qPCR upon the stimulation of 5% myrcene and ethyl butyrate for 5 h,respectively.This led to the initial screening of 8 DcitORs reacting with the two tested odors.The expression of 7 odorant receptor genes(DcitOR4,DcitOR7,DcitOR9,DcitOR17,DcitOR18,DcitOR21 and DcitOR28,respectively)was down-regulated upon the stimulation of myrcene.However,only the expression level of DcitOR18 and DcitOR46 were down-regulated after the exposure to ethyl butyrate.It is worth noting that the number of odorant receptors initially screened by myrcene was significantly higher than ethyl butyrate,suggesting that citrus psyllid is more sensitive to myrcene.In addition,DcitOR18 reacted to both myrcene and ethyl butyrate,indicating that DcitOR18 plays an important role in olfactory system of citrus psyllid.Furthermore,the expression patterns of atypical odorant receptor DcitOrco and 8 candidate odorant receptor genes in different developmental stages of citrus psyllid were analyzed by quantitative PCR.The results showed that the expression of DcitOrco was very low and remained relatively stable in nymph and adult.The higher expression levels of 8 DcitORs in 3-4th instar nymph than adults suggested that the citrus psyllid nymph may have more sensitive olfaction to myrcene and ethyl butyrate.2 The cDNA cloning of DcitOR18 and DcitOrco in Diaphorina citriBasing on the genome and transcriptome data of Diaphorina citri,the full length sequences of DcitOR18 and DcitOrco were obtained by RT-PCR.Sequence analysis revealed that the open reading frame of DcitOR18 is 1401 bp in length,encoding 467 amino acid residues.Meanwhile,DcitOrco is 1386 bp in length,encoding 461 amino acid residues.Using TMHMM software to predict structural characteristics of two odorant receptors,it was found that DcitOR18 has only 4 transmembrane regions,while DcitOrco has 7 transmembrane domains that according with the structural features of odorant receptors.Multiple sequence aligment and phylogenetic tree analysis found that the amino acid sequences of Orco is highly conserved in different insects,and the homology is as high as 70%.DcitOrco is quite conserved in evolution and its affinity is closer to the Orco of the aphids.3 Heterologous expression of DcitOR18 and DccitOrco and functional analysis in Diaphorina citriOn the basis of mammalian expression vector pcDNA3.1(+),the red fluorescent protein gene named mcherry was fused in it,and the recombinant plasmid pcDNA3.1(+)-mcherry was constructed.Subsequently,DcitOR18 and DcitOrco were cloned into pcDNA3.1(+)-mcherry,and the recombinant plasmid pcDNA3.1m(+)-DcitOR18 and pcDNA3.1m(+)-DcitOrco were successfully constructed.Using heterologous expression system,the recombinant plasmid pcDNA3.1(+)-mcherry was transfected into HEK293 cells,and red fluorescence signal was detected clearly,indicating that mcherry protein was successfully express in HEK293 cells.Furthermore,these two above recombinant plasmids were cotransfected into HEK293 cells,and the fluorescence in HEK293 cells obviously increased responding to myrcene by using calcium imaging.Although the fluorescence of only transfected recombinant plasmid of pcDNA3.1(+)-mcherry was found increased,it weaker than that cotransfected pcDNA3.1m(+)-DcitOrco and pcDNA3.1m(+)-DcitOR18.In order to obtain better evidence,we attempted to generate a stable cell line expressing DcitOrco in HEK293.The overexpression of DcitOrco upon to 1400 folds in mRNA level,but not detected in protein level,which laid a foundation for general functional analysis of odorant receptor in citrus psyllid.