| Insect sensitive olfactory system is essential for location host plant and mating partners,avoidance of predators,and oviposition site selection.A variety of olfactory proteins are involved in the process of insect olfactory recognition.The olfactory mechanism can be illustrated by studying the olfactory system of insects.Studies on the olfactory perception mechanism of insects could provide new strategies and ideas for pest control.Bradysia odoriphaga is an agricultural pest insect affecting the production of Chinese chive and other liliaceous vegetables in China,and it is significantly attracted by sex pheromones and the volatiles derived from host plants.Moreover,B.odoriphaga exhibited a strong electroantennogram(EAG)response to biogenic volatiles.Despite these reports on chemosensory behavior,however,the mechanism by which B.odoriphaga recognizes these volatile compounds at the molecular level is still unknown.Elucidation of the molecular mechanisms of these behaviors and electrophysiological responses will provide a theoretical basis for the behavioral regulation of the insect.In the present study,we performed transcriptome analysis of the antennae and body of female and male adults of B.odoriphaga,respectively,and identified chemosensory genes(OBPs,CSPs,NPC2s,ORs,IRs,GRs and SNMPs).The transcript expression levels of putative chemosensory genes among different tissues(female antennae,male antennae,legs,wings,abdomens and thoraxes,and heads)were investigated by quantitative real-time PCR(qRT-PCR).We also cloned 3 male antennae enriched OBPs cDNAs in B.odoriphaga(BodoOBP2,BodoOBP4 and BodoOBP8)and expressed them in competent Escherichia coli cells.Ligand-binding assays were used to analyze the binding affinities of recombinant proteins to putative sex pheromones and host volatiles.Homology modeling and molecular docking analyses confirmed the interactions between BodoOBPs and odorant molecule.The main results are as follows:1.Four cDNA libraries were constructed based on the RNA from the female antennae(FA),male antennae(MA),female body(FB)and male body(MB),and sequenced on an Illumina HiSeq 4000.A total of 42.6 GB of clean data was obtained from the antennae and body transcriptomes of B.odoriphaga.After assembling all samples together,we identified 55,867unigenes with an N50 length of 2,806 bp,the average length was 1552 bp and GC content was38.08%.35,013(62.67%)of the total unigenes were annotated to Nr,Nt,SwissProt,InterPro,KEGG,COG,and GO databases,respectively.A total of 161 putative chemosensory genes were identified in the adult antennae,body and larval transcriptome of B.odoriphaga.Including candidate 49 OBPs,5 CSPs,6 NPC2s,71 ORs,18 IRs,5 GRs and 7 SNMPs were identified.Fifty-nine of the 60 carrier protein genes(except for BodoOBP32)have complete open reading frames(ORFs)with lengths ranging from 327 to 759 bp,codes for 108 to 252 amino acids.Nearly all full-length OBPs had a predicted signal peptide(a signature of secretory proteins)at the N-terminal region except for BodoOBP22/25.Based on the number and location of the conserved cysteines,all BodoOBPs could be divided into three groups:Minus-C OBPs group(BodoOBP14/23/26/31/33/41/42/43/44),Plus-C OBPs group(BodoOBP19/34),and the remaining OBPs belong to Classic OBPs group.Ninety of 94 receptor protein genes(except for BodoOR5,BodoIR1,BodoGluRIIa and BodoGR5)have intact ORFs with lengths ranging from1008 to 3144 bp,codes for 335 to 1047 amino acids.All the 7 SNMPs genes had intact ORFs with lengths ranging from 1296 to 1638 bp,codes for 431 to 545 amino acids.2.qRT-PCR and transcriptome abundance(FPKM)were used to analyze the expression characteristics of OBPs,CSPs and ORs among different tissues.Results indicated that 9 of the22 antennae-biased OBP genes(BodoOBP2/4/6/8/12/13/20/28/33)were predominantly expressed in the male antennae and might have potential functions in sex pheromone detection.In addition,13 other OBPs that were highly expressed in the antennae(BodoOBP1/5/7/10/11/15/18/22/24/26/41/43/46)might be associated with functions in general host odorant perception.Four of 9 leg-specific BodoOBPs(BodoOBP9/35/38/39)were significantly higher expressed in the male legs implying that these four OBPs might also function in the recognition of sex pheromone compounds.In the present study,five OBP genes(BodoOBP17/30/32/37/44)were abundantly expressed in the wings,and three OBP genes(BodoOBP14/23/31)were enriched in the heads,suggesting that these genes might also participate in taste functions.In addition,BodoOBP29/36 were significantly more highly expressed in the female abdomens indicating that these two genes might be involved in the synthesis and release of sex pheromones,or in the detection of egg-laying substrates.Results showd that BodoCSP3/5 were antennae enriched and might be involved in the chemosensory process.BodoCSP1 and BodoCSP2 were significantly more highly expressed in the legs and heads,respectively,and BodoCSP4 was more highly expressed in antennae and heads.In addition,the expression levels of BodoOR17 showed no significant differences among antennae,heads,and legs.The other BodoORs were significant highly expressed in the antennae.3.BodoOBP2/4/8 were predominantly expressed in the male antennae and might have potential functions in sex pheromone detection.Using antennae cDNA as template,the BodoOBP2/4/8 gene was successfully cloned,and the recombinant plasmids pET32b-BodoOBP2,pET32b-BodoOBP4 and pET32b-BodoOBP8 were successfully constructed.Then,the recombinant plasmids were transformed into chemically competent Escherichia coli BL21(DE3)cells,and fusion protein expression was successfully induced under appropriate conditions.pET32b-BodoOBP2 and pET32b-BodoOBP4 were mainly expressed in the inclusion body.Through the refolding of the inclusion body,and purification involved two rounds of Ni~+ion affinity chromatography,and the His-tag was removed using recombinant enterokinase.In addition,pET32b-BodoOBP8 was highly expressed in supernatant,and purified by Ni~+ion affinity chromatography and recombinant enterokinase.Ligand binding assays using 1-NPN as the fluorescent probe.The binding properties of BodoOBP2/4/8 protein to 13 volatile compounds(putative sex pheromones,host plant volatiles and biogenic volatiles)were determined by fluorescence competitive binding assays at different pH values(pH=5.0and 7.4).The results showed that the recombinant BodoOBP2/4/8 displayed strong binding ability with the putative sex pheromone 1-octanal,nonanal and linolenic acid.They also have certain binding capacity with the host plant volatile compounds,allyl methyl disulfide,dimethyl trisulfide,diallyl disulfide and dipropyl disulfide.Moreover,BodoOBP8 has the strongest binding ability with linolenic acid(Ki=0.51?M).4.Because of high sequence similarity with the BodoOBP2/8 protein,the template structure of CquiOBP1 was selected for homology modelling using the automated-mode build homology model protocol of SWISS-MODEL.The results showed that BodoOBP2/8 consists of 6?-helices and three disulfide bonds.All six antiparallel?-helices contributed to the formation of a compact hydrophobic binding pocket,and an opening is formed on the surface of the bonding pocket.The surface of the binding pocket is hydrophobic,and the lipid soluble ligand molecules are fixed in the binding pocket by hydrophobic force.Amino acid residues Tyr72,His120,Tyr121 and Phe122 formed hydrophilic regions on the binding pocket surface.Molecular docking of putative sex pheromones linolenic acid and octanal with BodoOBP2 was performed.The docking results showed that linolenic acid and octanal exhibit stretched conformations in the BodoOBP2 binding pocket.In addition,the amino acid residues Met54,Gln81 and Phe125 formed hydrophilic regions on the binding pocket surface of BodoOBP8.Linolenic acid exhibit curved conformations in the BodoOBP8 binding pocket,and hydrogen bond interaction with the amino acid residues His113. |
PDF Full Text Request