Identification And Binding Characteristics Of Odorant-binding Proteins In Batocera Horsfieldi

Batocera horsfieldi(Hope)is an important stem borer.Due to its hidden larvae and long activity cycle,traditional chemical control methods cannot achieve ideal control effects.The sensitive olfactory system of insects plays an important role in various physiological activities.The main function of odorant-binding protein is to transport hydrophobic information chemicals through hydrophilic lymph.It is an important functional protein in insect olfactory system.If the molecular mechanism of olfactory perception of Batocera horsfieldi can be explored,it will provide an important theoretical basis for pest control strategies based on olfactory regulation technology.In order to more systematically explore the olfactory perception mechanism of Batocera horsfieldi and explore prevention and control technologies based on olfactory regulation,this study identified the olfactory-related genes of Batocera horsfieldi using the Illumina Hi Seq sequencing platform.And determined the binding characteristics of 4 Bhor OBPs to host plant volatiles.It lays a theoretical foundation for exploring the function of Bhor OBPs in the olfactory perception of Batocera horsfieldi,and provides a theoretical basis for screening its behavioral active substances.The main results are as follows:1.Transcriptome sequencing and identification of olfactory related genes in adult antennae of Batocera horsfieldi.A total of 17426 unigenes sequences were obtained by sequencing the antennae of the adult antennae of Batocera horsfieldi.By comparing the bioinformatics analysis with the NR database,116 olfactory-related genes were mined in the transcriptome of the adult antennae of Batocera horsfieldi,including 43 OBPs genes,12 chemosensory proteins genes,43 odorant receptors genes,12 ionotropic receptors genes and 3 sensory neuron membrane proteins genes.2.Analysis of tissue expression characteristics of Bhor OBPs in Batocera horsfieldi15 Bhor OBPs were randomly selected,and the Bhor OBPs in different tissues(Antennae,Labial palpus,Maxillary palpus,Mandible,Head,Thorax,Abdomen,Reproductive organ,Leg and Wing)were determined by fluorescence quantitative PCR technology expression.The results showed that most Bhor OBPs had the same tissue expression characteristics in male and female adults,and the expression abundance of Bhor OBPs was different in different tissues.Among them,Bhor OBP8,Bhor OBP28 and Bhor OBP29 are highly expressed in the adult antennae,Bhor OBP16,Bhor OBP27 and Bhor OBP33 are highly expressed in the labial palpus and maxillary palpus,and Bhor OBP19,Bhor OBP24 and Bhor OBP30 are expressed in various adult tissues.The highly expressed Bhor OBPs in the olfactory organs may play an important role in the olfactory recognition of Batocera horsfieldi.3.Isolation and identification of volatile compounds from branches and leaves of Fraxinus chinensis.Using dynamic headspace adsorption and GC-MS techniques,the volatiles from the branches and leaves of Fraxinus chinensis,the host plant of Batocera horsfieldi,were isolated and identified.The results showed that 34 kinds of volatile compounds were isolated and identified under the experimental conditions,including 14 kinds of alkenes,9 kinds of alkanes,8 kinds of esters,and 1 kind of ketones,aldehydes and alcohols.Through comparative analysis of the volatiles of the spawning host plant Populus and the supplementary nutrient host plant Rosa multiflora,it was found that 11 volatiles were the common host volatiles of the above 3 species.4.Prokaryotic expression,protein purification and determination of binding properties of Bhor OBPsThrough prokaryotic expression and protein purification technology,the in vitro recombinant proteins of Bhor OBP16,Bhor OBP28,Bhor OBP30 and Bhor PBP2 were successfully obtained.The binding properties of the above 4 recombinant proteins with 11 host plants shared volatiles and 3 sex pheromones were determined by fluorescence competition binding experiments.The results showed that under p H 7.4,recombinant Bhor OBP16 was combined with 3-Carene,alpha-Pinene,D-Limonene,beta-Pinene,and recombinant Bhor OBP28 was combined with(E)-beta-ocimene,alpha-Pinene,DLimonene,beta-Pinene,recombinant Bhor PBP2 with 3-Carene,alpha-Pinene,D-Limonene, beta-Pinene,Hexanal has binding ability(Ki < 50 μmol/L);recombinant Bhor OBP30 has no binding ability to all 14 tested compounds(Ki > 50 μmol/L).The compounds with binding ability to the above-mentioned Bhor OBPs were screened(Ki < 50 μmol/L),and fluorescence quenching experiments were performed respectively.The results showed that recombinant Bhor OBP16 with 3-Carene,alpha-Pinene,DLimonene,recombinant Bhor OBP28 with alpha-Pinene,D-Limonene,beta-Pinene,recombinant Bhor PBP2 with 3-Carene,alpha-Pinene,D-Limonene,beta-Both Pinene and Hexanal were statically quenched,indicating that the recombinant protein and the compound formed a stable “protein-ligand” complex.The binding mechanism of the abovementioned Bhor OBPs and ligands was analyzed by homology modeling and molecular docking,and the results showed that the hydrophobic interaction force and hydrogen bonding force played an important role in the binding process of the above-mentioned Bhor OBPs and ligands.The compounds that were statically quenched with the above Bhor OBPs were screened,and circular dichroism experiments were performed respectively.The results showed that the main secondary structure of recombinant Bhor OBP16,Bhor OBP28 and Bhor PBP2 was α-helix.Under neutral conditions,the α-helix of recombinant Bhor OBP16 increased gradually with the increase of the concentration of ligands 3-Carene;with the increase of the ligand concentration of alpha-Pinene,the α-helix first increased and then decreased.The α-helix of recombinant Bhor OBP28 decreased gradually with the increase of the concentration of ligands alpha-Pinene,D-Limonene and beta-Pinene.The α-helix of recombinant Bhor PBP2 decreased gradually with the increasing concentrations of ligands 3-Carene,alpha-Pinene,D-Limonene,beta-Pinene and Hexanal.All of the above ligands can cause conformational changes in the secondary structure of recombinant Bhor OBPs.In conclusion,this study identified 116 olfactory-related genes of Batocera horsfieldi,clarified the tissue expression characteristics of 15 Bhor OBPs,and the binding characteristics of 4 recombinant Bhor OBPs with 11 host volatiles and 3 sex pheromones of Batocera horsfieldi.were studied,five compounds with potential behavioral activities were screened.The research results lay an important theoretical basis for exploring the functions of olfactory-related proteins of Batocera horsfieldi,and provide an important theoretical basis for screening potential behavioral regulators.