| Sex pheromone components of Conogethes punctiferalis includ(E)-10-hexadecenal(E10-16: Ald),(Z)-10-hexadecenal(Z10-16: Ald),(Z3,Z6,Z9)-tricosatriene(Z3,Z6,Z9-23: HC),(Z9)-heptacosene(Z9-27: HC)identified in female adults and a(E)-2-methyl-2-butenoic acid(a tiglic acid)identified in male adults.Among these components,E/Z-10-16: Ald are highly volatile and work in a long distance.And odorant binding proteins(OBPs)of these two aldehydes have been identified recently.While binding proteins for remaining three components,i.e.two hydrocarbons and a tiglic acid with low volatility and active in a short range are yet to be identified.In this study,RNA-seq and quantitative PCR were used to analyze the differentially expressed odorant-binding protein genes in the antennae of male and female adults of C.punctiferalis.Then recombint proteins were obtained and subjected to fluorescence competitive binding experiments.Finally,binding details were analyzed with homologous modeling and molecular docking.Main results are as follows:1.Differentially expressed odorant-binding protein genes in antennae of male and female adultsA total of 139 differentially expressed genes were found by male and female antennal transcriptome analysis.Among those 77 genes were detected highly expressed in male antennae and 62 genes were highly expressed in female antennae.Then 8 OBP genes with full-length sequences were futher obtained by sequence alignment with our previous full-length transcriptome data of C.punctiferalis.Among 8 OBP genes,Cpun PBP1,Cpun OBP2,Cpun PBP5 and Cpun GOBP1 genes were highly expressed in female antennae,while Cpun PBP2,Cpun OBP5,Cpun OBP8 and Cpun OBP13 showed male-biased expression.Expression levels of those genes were subsequently verified with a real time quantitative PCR and the sex expression patterns obtained by above transtriptom analysis were finally confirmed except for Cpun OBP8,expression of which showed no significant difference between two genders.2.Phylogenetic analysis of sex-biased OBP genesA phylogenetic analysis for above OBP genes was performed with candidate genes of Bombyxi mori and Cnaphalocrocis medinalis.Among 8 OBP genes,Cpun PBP1,Cpun PBP2,Cpun PBP5,Cm PBP1,Cm PBP5,Bmor OBP3 and Bmor OBP4 clustered in a large cluster,within which Cpun PBP1 and Cm PBP1 clustered in a small branch with a highly self-spreading value of 86%.Meanwhile,Cpun PBP5 and Cm PBP5 also clustered into a cluster with a highly self-spreading value of 97%.Cm GOBP1,Cm GOBP2,Bmor OBP1 and Bmor OBP2 clustered into a large cluster,among which Cpun GOBP1 and Cm GOBP1 closely clustered with a self-spread value near 98%.Thse 8 OBP genes of the C.punctiferalis showed high homology with those of Bombyx mori and Cnaphalocrocis medinalis and belonged to the typical OBPs gene family.3.Gene cloning and prokaryotic expressionFour genes including Cpun PBP1,Cpun PBP2,Cpun OBP2 and Cpun OBP5 were successfully cloned and then subjected to prokaryotic expression.Soluble recombinant proteins were successfully obtained with molecular weights of 15.89 k Da,16.20 k Da,14.17 k Da and 14.02 k Da,respectively,which match well with those predicted with softwares.4.Fluorescence competitive binding experiment of OBPs and short range pheromonesGood binding affinities of four recombinant proteins,Cpun PBP1,Cpun PBP2,Cpun OBP2 and Cpun OBP5,to 1-NPN were confirmed firstly.Then proteins were then subjected to fluorescence competitive binding experiment.Results showed that Cpun PBP1 strongly binded to Z3,Z6,Z9-23: HC with a binding constant of 6.50±0.32μM.The binding constants of Cpun PBP2 with Z3,Z6,Z9-23: HC and Z9-27: HC were1.30±0.13 μM and 11.64±0.82 μM,respectively,and the binding constants of Cpun PBP2 with tiglic acid were 26.50±3.16 μM.The binding constants of Cpun OBP2 with Z3,Z6,Z9-23: HC and tiglic acid were 3.675±2.33 μM and 7.34±2.53 μM,respectively.Cpun OBP5 did not bind to all tested pheromone components.5.Homologous modeling and molecular docking of OBPs and short range pheromone componentsHomologous modeling and molecular docking of proteins were performed by softwares of Swiss-Moedel and Auto Dock Tools.Modeling results showed that each of Cpun PBP1,Cpun PBP2 and Cpun OBP2 had 7 α helices,which formed hydrophobic binding cavities.As to binding sites,a residue of ASN46 of Cpun OBP2 was thought to bind to tiglic acid with a hydrogen bond,which was speculated to form a main binding site.Meanwhile,although no hydrogen bonds were detected in each of Cpun PBP1,Cpun PBP2 and Cpun OBP2 with hydrocarbon pheromones component(Z3,6,9-23:HC and Z9-27: HC),most of the amino acid residues formed hydrophobic forces and van der Waals’ forces with two hydrocarbons,which probably benifited stable binding as well.Generally,in this study,transcriptome sequencing was used to obtain the OBP genes with sex-biased expression patterns.Then through prokaryotic expression and fluorescence competition experiment,Cpun PBP1,Cpun PBP2 and Cpun OBP2 were identified as binding proteins of short range pheromone components of C.punctiferalis.And the hydrogen bond,the hydrophobic force and van der Waals’ forces were thought to be involved in binding mechanisms. |
