| The insect’s highly sensitive olfactory system plays an important role in behavioral activities such as searching host plants,foraging,selecting spawning sites,population survival and reproduction.The most important organ in the insect olfactory system is the antennae,it was equipped with olfactory sensilla and contain various olfactory-related proteins,which regulate the behavioral activities of insects by participating in the identification of external odorant compounds.Research finding that the multiple olfactory-related proteins is involved in insects identifying processes of external odorant information molecules,the main components include ORs（odorant receptors）,IRs（ionotropic receptors）,OBPs（odorant binding proteins）,CSPs（chemosensory proteins）and SNMPs（sensory neuron membrane proteins）.In this study,by sequencing the transcriptomes of the antennae of the male and female adults of Empoasca onukii Matsuda,we identified the olfactory-related genes of the E.onukii and used qRT-PCR for the corresponding expression level and expression profile analysis;it will lay a foundation for screening the olfactory receptor genes with significantly high expression in the adult antennae and cloning to verify the function of olfactory receptors genes in the olfactory recognition pheromone of the E.onukii in the later stage.The outcome research work are as shown below:1.The cDNA library was constructed by using the total RNA from the antennae of the male and female adults of the E.onukii.Using the DNBSEQ（DNA Nanoball Sequence）sequencing platform,a total of 37.98 GB of data was obtained,after assembling and removing redundant data,getting a total of 84,213 unigenes with a total length of 88,322,799 bp and an average length of 1,048 bp,with N50 length of 2,136 bp and GC content of 39.06 %.In a total of 84,213 functional unigenes identified,nearly 41,772（49.60 %）unigenes annotated in the seven databases: NR,NT,GO,KEGG,KOG,Pfam,Swiss Prot,the annotation results were39,631（47.06 %）unigenes in NR,there are 12,375（14.69 %）unigenes in NT,23,617（28.04 %）unigenes in GO,32,181（38.21 %）unigenes in KEGG,29,240（34.72 %）of the KOG with unigenes,27,400（32.54 %）unigenes in Pfam and 30,180（35.84 %）unigenes in Swiss Prot.A total of 25 olfactory-related genes were identified by sequencing the antennae transcriptome of the male and female adults of the E.onukii,including 12 ORs,8 IRs,3 GRs and 2 SNMPs.In the acquired genes,except Eonu Orco,Eonu OR5,Eonu OR6,Eonu OR8,Eonu IR1,Eonu IR2 and Eonu IR6,the remaining have a whole open reading frame（ORF）with ORF length ranging from 339 bp to 2,697 bp and encoding 113 to 899 amino acids.All two SNMPs genes had complete ORF with ORF length between 1,437 bp and 2,439 bp,encoding479 to 813 amino acids.2.The expression characteristics of ORs,IRs,GRs,and SNMPs in different tissues of the E.onukii were analyzed using quantitative PCR.The results showed that 12 ORs were significantly expressed in the female and male adults antennae.Among them,Eonu Orco was relatively highly expressed in the antennae and highly expressed in the female antennae,it is speculated that the gene might play a vital role in localized host plant.Six ORs genes（Eonu OR1/2/4/6/8/10）were significantly highly expressed in the male antennae,it is acknowledged that they may play an important role in the recognition of sexual pheromone molecules.Eonu OR3 was significantly highly expressed in the female leg,suggesting that it plays a potential role in identifying host volatile or non-volatile compounds,Eonu OR5 genes and Eonu OR9 genes were significantly highly expressed in the female head,Eonu OR11 gene was significantly highly expressed in the female body,along with higher expression levels in the female head and male antennae.In addition,a non-tissue-specific gene Eonu OR7 was identified,which was expressed in at least three tissues of the E.onukii,and expression levels did not form significant differences between these tissues.Eight IRs genes were significantly expressed in the female and male adult antennae,three of these IRs genes（Eonu IR1,Eonu IR25 a,Eonu25a-like）were significantly highly expressed in the male antennae.Two IR genes（Eonu IR6 and Eonu NMDAR3）were significantly highly expressed in the female antennae,and there was no significant difference in the other tissues.It is speculated that these genes may play an important role in the selection of spawning sites as well as in the identification of host general odor molecules.In addition,Eonu IR2 gene was significantly highly expressed in the female head;Eonu NMDAR2 gene was significantly highly expressed in the female leg,it is speculated that this gene may play an important role in identifying the host volatile or non-volatile compounds;Eonu NMDAR1 is a nontissue-specific gene.Three GRs genes are widely expressed in various tissues of the E.onukii.Among them,Eonu GR1 is highly expressed in the female body,Eonu GR2 is relatively highly expressed in the male body and Eonu GR3 was highly expressed in the male antennae.For SNMPs,Eonu SNMP1 is a non-tissue specific gene and Eonu SNMP2 was significantly highly expressed in the female head.These results will lay a biological foundation for the future study of olfactory receptor gene functions and developing behavioral regulators targeting olfactory receptors of the E.onukii. |
