| Anoplophora glabripennis(Motschulsky)and Anoplophora chinensis(Forster)are Coleoptera,Cerambycidae,Anoplophora,which are important worldwide quarantine pests.The two occur in the same area,can harm the common host,and share the aggregate pheromone released by the male.To date,invasions have been demarcated and monitored using traps baited with an attractive mixture of volatile organic compounds(VOCs)produced by the host and two components of the maleproduced aggregation-sex pheromone,however,shows low efficacy of attraction.In insects,olfaction has several vital roles for survival and reproduction,including food selection,mate recognition,location of oviposition sites and predator avoidance.Elucidating the chemical communication mechanism of insects helps us to understand the relationship between insects and host plants,which providing new prospects for integrated pest management.Here,we studied the expression patterns and functions of key olfactory genes and proteins of A.glabripennis and A.chinensis.The main research results are as follows:1.Identification of olfactory genes of A.glabripennis and A.chinensis.A total of 124 olfactory-related protein genes were identified in the transcriptome of A.glahripennis antenna,including 42 odorant-binding proteins(OBPs),12 chemosensory proteins(CSPs),37 odorant receptors(ORs),4 ionotropic receptor(IR).11 gustatory receptors(GRs),one odorant-degrading enzymes(ODE),14 pheromone degrading enzymes(PDIEs)and 2 sensory neuron membrane proteins(SNMPs).We integrated the genome and transcriptome data of A.glabripennis and revised the descriptions of OBPs.a total of 61 AglaOBP genes were identified.A phylogenetic tree led to the identification of 15 ABPIIs,10 C lassic OBPs,29 Minus-C OBPs,and one Plus-C OBP,which including AglaOBP51 with additional cysteines.Notably,AglaOBP45 and AglaOBP46 showed higher sequence identity with PBP2 and PBP1 from Batocera horsfieldi.We also determined the transcriptomes of male and female adult antennae of A.chinensis,and identified 46 OBPs,16 CSPs,44 ORs,19 GRs,23IRs,and 3 SNMPs.Among 46 OBPs,23 had complete open reading frames(ORFs)?400 bp that included sequences encoding signal peptides.Phylogenetic tree results indicated that AchiOBP3 belongs to "Plus-C OBP";c27344.graph_c0 and c25366.graph_c0 clustered together with other coleopteran PBPs,suggesting that it may be a candidate PBPs genes of A.chinensis.12 CSPs are homologous genes of A.glabripennis and A.chinensis,while four CSPs are specifically expressed in the antennae of A.chinensis.2.Tissue expression analysis in the mRNA levels of olfactory genes from A.glabripennis and A.chinensisWe investigated the expression patterns of the AglaCSPs and and]2 AchiCSPs in three chemosensory tissues(antennae,legs,and maxillary palps)in both females and males using RT-PCR and qRT=PCR.We observed the highest expression of AglaCSP7,AglaCSP8,AglaCSP9,and AglaCSP11 in antennae.The AglaCSP7 and AglaCSP9 expression level was significantly higher in antennae than in all other tissues.The highest expression of AglaCSP3,AglaCSP6,AglaCSP 10 and AglaCSP12 was observed in the maxillary palp.Most CSPs are highly expressed in a variety of olfactory tissues,indicating that they participate in the olfactory recognition process of almost all tissues.AchiCSP10 was almost exclusively expressed in male antennae,suggesting that AchiCSP10 plays a role in recognizing female pheromones.Many highly expressed genes encoding CSPs belong to the homologous genes of A.glabripennis and A.chinensis,and four CSPs are specifically expressed in the antennae of A.chinensis,which may be related to the specific recognition of host plants.The results of expression profiling of 61 AglaOBPs showed that 12 AglaOBPs displayed higher expression in adult antennae.Meanwhile,seven AglaOBPs(AglaOBP3,AglaOBP33,AglaOBP41,AglaOBP45,AglaOBP47,AglaOBP48,and AglaOBP53)exhibited almost antennae-specific expression,indicating putative roles in odorant recognition in A.glabripennis,presumably detection of sex pheromones and/or host plant volatiles.Remarkably,AglaOBP45 and AglaOBP46 were expressed in adult antennae,and AglaOBP45 was expressed at a 200-fold higher level in antennae than in other tissues.3.Purified and protein expression pattern of AglaOBP45 and AglaOBP46Cloning and sequence analysis of two candidate PBPs genes AglaOBP45 and AglaOBP46 were performed.The results showed that the full-length open reading frame(ORF)of AglaOBP45 and AglaOBP46 consisted of 408 and 411 nucleotides,respectively.Which also encoding 135 and 136 amino acid residues,respectively.AglaOBP45 and AglaOBP46 were cloned into the pET-30a(+)plasmid vector,for overexpression of recombinant proteins.Induction with IPTG resulted in a protein band on SDS-PAGE at about 12 kDa,consistent with the expected size.Western blot analysis showed that AglaOBP45 was significantly expressed in the A.glabripenni antennae of both male and female,and the color of the protein band indicated that the antennae of males were higher than that of females;AglaOBP46 was expressed in the antennae,leg and maxillary palps of male and female,which almost the same as expression results of mRNA levels.4.The three-dimensional structure modeling and molecular docking of AglaOBP45 and AglaOBP46Homology modeling of AglaOBP45 and AglaOBP46 revealed that both of them have six ?-helices,and these helices and the folds connecting them constitute their 3D structure.The evaluation scores for Procheck.VERIFY 3D and ERRAT indicated that the quality of both two AglaOBPs structures were high.34 volatiles from host tree and seven pheromone substances of A.glubripennis were selected and docked with AglaOBP46 and AglaOBP45 respectively.The results showed that the distribution of the binding free energy of AglaOBP45 and AglaOBP46 with the ligand molecule was relatively narrow.AglaOBP45 has the lowest free binding energy to two components of female-produced contact sex pheromone,which included(Z)-9-tricosene and(Z)-9-pentacosene.The binding energy to a-pinene is also lower.The free binding energy of AglaOBP46 with long-leafene,limonene and dodecanol is the lowest,indicating that they have the strongest binding ability.Binding pattern analysis with proteins and compounds showed that the binding cavity is mainly composed of hydrophobic residues,which facilitates the binding of hydrophobic odor molecules.In addition,hydrogen bonding,van der Waals forces,and hydrophobic interactions affect the affinity of AglaOBP45 and AglaOBP46 to odor molecules.5.Binding ability analysis of AglaOBP45 and AglaOBP46 with ligand compoundsLigand molecules with higher affinity to AglaOBP45 and AglaOBP46 in molecular docking were selected,and their binding ability was further analyzed by fluorescence competition binding assay.The ligand molecule mainly includes two kinds of aggregate pheromone components,five kinds of contact pheromone components,ten kinds of alcohols,seven kinds of olefins,four kinds of aldehydes and four kinds of esters.The experimental results show that neither AglaOBP45 nor AglaOBP46 can effectively bind to two components of male-produced aggregate pheromone,and AglaOBP46 does not bind to all contact pheromone components.However,AglaOBP45 only binds effectively to the(Z)-9-tricosene and(Z)-9-pentacosene,may participate in the recognition of these components.AglaOBP45 is mainly expressed in male antennae,and the binding constants with ocimene and camphene are 1.02?mol/L and 1.96?mol/L,respectively.Which is showed that AglaOBP45 is also an important protein for recognizing two ligands.AglaOBP46 has the strongest binding ability to 1-dodecanol,the binding constant is 0.74?mol/L,and followed by benzylalcohol and 2-pentanol,and the binding constants are 0.82?mol/L and 2.12?mol/L,respectively.Based on the comprehensive analysis,we believe that both AglaOBP45 and AglaOBP46 are involved in the olfactory response of A.glabripennis,but AglaOBP46 does not bind to its pheromone components.AglaOBP45 is mainly expressed in the antennae and can be combined with the female-produced contact pheromone,which involved in the recognition which pheromones. |
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