Genome-wide Identification And Tissue Expression Analysis Of Odorant Receptor Genes In Anopheles Sinensis

Olfaction plays a crucial role in mediating mosquito to find a food source,a mate,an oviposition site or avoid predators.The process of olfactory signal transduction mainly relies on diverous olfactory genes including at least odorant-binding proteins(OBPs),odorant receptors(ORs),ionotropic receptors(IRs),and odorant degrading enzymes(ODEs).Among these factors,ORs play a central role in chemosensory signal transduction processes.Therefore,characterization and functional exploration of mosquito ORs will provide a valuable theoretical basis for development of new control strategies of mosquitoes.Currently,the odorant receptors have been identified in genome-wide level in Anopheles gambiae,Aedes aegypti,Culex quinquefasciatus,Aedes albopictus and other mosquitoes.However,the Anopheles sinensis OR genes are still unknown.In this study,we systematically conducted a genome-wide characteristion of ORs family of genes in An.sinensis,and analyzied their expression patterns in different tissues of female and male An.sinensis to find the sex and tissue specific expression genes.The main contents and results are as follows:(1)Identification of OR genes in the genome of An.sinensisThe OR genes from An.gambiae,Ae.aegypti,Cx.quinquefasciatus and Drosophila melanogaster genome were used as query sequences to search the An.sinensis genome by homological BLAST and HMM methods.As a results,58 candidate OR genes were identified.All of the genes had full-length amino acid sequences,and no pseudogenes were found.The An.sinensis OR genes were named As OR1-As OR77 according to the homologies of An.gambiae OR genes.(2)Identification of orthologous genes of An.sinensis OR genesUsed all candidate AsORs as query sequences to search the genome of the An.gambiae,Ae.aegypti and Cx.quinquefasciatus by BLASTP,TBLASTn and HMM tool,with these comprehensive approaches,we identified 42,18 and 13 orthologous genes from An.gambiae,Ae.aegypti and Cx.quinquefasciatus,respectively.There were 6 genes had 1:1:1 orthologous genes in An.sinensis,An.gambiae and Cx.quinquefasciatus,9 genes had 1:1:1 orthologous genes in An.sinensis An.gambiae and Ae.Aegypti,8 genes had 1:1:1:1orthologous genes in this four mosquitoes.(3)Characterization,gene structure and genomic distribution of An.sinensis OR genesAnalysis of characterization of AsORs we found that these ORs have 40-54 KDa of molecular weight,5.36-9.50 of isoelectric point,no signal peptide.All ORs are hydrophobic proteins,and located in plasma membrane.Most AsORs have seven transmembrane helixes,motif SYS and WY near the extreme C-terminus.The 58 As OR genes had 1-7 exons each,the majority of these 58 AsORs possessed 2-6 exons(accounting for 70%),the maximum value of intron is 3790 bp and the minimum is23 bp,most of introns were 1-100 bp long.AsORs were mapped onto 18 different scaffolds,the scaffold 14 contained the largest number(13 genes)and the scaffolds 9had only 1 As OR gene,most of the AsORs were tandemed arrangement in the genome.(4)Phylogenetic analysis and select porce analysis of AsORsAll amino acid sequences of ORs in An.sinensis,An.gambiae,Ae.aegypti and Cx.quinquefasciatus were used to perform phylogenetic analysis.The result showed that one clade included 40 genes belongs to Ae.aegypti and Cx.quinquefasciatus,another one obtained 23 genes belongs to An.sinensis and An.gambia,the rest genes were scattered throughout the phylogenetic tree.The evolutionary tree showed the bootstrap values >70%,every of the clade had different number of ORs gene,among them,the largest containing 45 genes,and the least only gathered 7 genes.We calculated Ka/Ks for these 1:1 ortholog pairs in reference of counterparts of An.gambiae to investigate the selective pressure of As OR genes,all 58 As OR genes had a Ka/Ks < 1,which suggests the purifying selection for all of these genes.(5)Expression of the AsORs in different tissuesThe expression of AsORs was detected by RT-PCR in the female and male mosquitoes' antenna,maxillary palp,proboscis,leg and in the 4thinstar larvae.The results showed that 47 and 37 As OR genes were expressed in female and male mosquito respectively,and 16 genes were expressed in larvae.Compared with male mosquitoes,10 AsORs were specifically expressed in female mosquitoes,and only As OR9 specifically expressed in male mosquitoes.Among the female mosquitoes,there are 44 genes expressed in the antennae,11 in the maxillary palp,13 genes in the proboscis and 3 genes in the legs.In male mosquitoes,there are 30,17,7 and 0 As OR genes expressed in antenna,maxillary palp and proboscis,respectively.No As OR genes were detected in male legs.In this paper,we identified the ORs gene at the level of whole genome,bioinformatics analysis and tissue expression analysis were helpful to understand the evolution the ORs gene of Anopheles sinensis and offer reference genes for study of the olfactory mechanism.