Identification And Function Analysis Of Olfactory Proteins In Galeruca Daurica (Coleoptera:Chrysomelidae)

Galeruca daurica(Coleoptera:Chrysomelidae)has become a new insect pest on the Inner Mongolia grasslands in recent years,feeding on Allium plants,Liliaceae,such as Allium mongolicum,A.ramosum and A.polyrhizum,and causing serious damage on the grassland ecosystem.Olfactory sensory system plays an important role in the normal life activities of insects.Insects contact and analyze the volatile substances by olfaction to communicate information.However,there has been no reports on the chemosensory systems of this pest up to now.In this study,we identified lots of olfactory protein genes from the G.daurica transcriptome,and analyzed the expression profiles of odorant binding protein genes and chemosensory protein genes,and the binding properties of the recombinant proteins.Our results lay a necessary foundation for the futher study on the moleculer mechanism of chemosensation in G.daurica,and have important roles in revealing its disaster mechanism and controlling it by exploiting the attractant or repellent.The main results are shown below.1.Observation on the microstructure of antennal sensilla in G.dauricaThe morphology of antennal sensilla in G.daurica was investigatied by scanning electron microscopy.There are five classes of sensilla on the antennae of G.daurica,including sensilla trichodea,sensilla chaetica,sensilla basiconica,sensilla campaniformia and Bohm bristle.2.Identification and analysis of olfactory protein genes in G.dauricaWe identified 66 olfactory protein genes from the transcriptome database of G.daurica assembled in our lab by using bioinformatics analysis,including 29 odorant binding proteins(OBPs),10 chemosensory proteins(CSPs),21 olfactory receptors(Ors)including an olfactory receptor co-receptor(ORco),and 6 sensory neuron membrane proteins(SNMPs).All 28 OBP genes have the full-length open reading frames(ORFs)except GdauOBP29,encoding proteins in length from 119 to 202 amino acids.GdauOBP1-12 belong to Classic OBPs and the others belong to Minus-C OBPs.All 10 CSP genes have the full-length ORFs,encoding proteins in length from 98 to 273,which contain four conserved cysteines.The ORFs of OR genes are incomplete,encoding 55-259 amino acids.Among the 6 SNMP genes,GdauSNMP1a and GdauSNMP1b have the full-length ORFs,which encoding proteins contain two transmembrane domains and six conserved cysteine residues.The other four GdauSNMPs have the incomplete ORFs,which encoding proteins contain one transmembrane domain.The best results of BlastX and phylogenetic analysis showed that the relationship of the olfactory proteins are closer between G.daurica and other Coleoptera insects,such as Colaphellus bowringi,Pyrriumta maculicollis,and Pyrrhalta aenescens.GdauORco forms independently a branch with the ORcos of Lepidoptera,Diptera,Hemiptera,Orthoptera,and other Coleoptera insects in the phylogenetic tree,exhibiting extremely conservative evolutionary mechanism.3.Expression profiling of olfactory protein genes in G.dauricaRT-PCR and qRT-PCR showed that plenty of the GdauOBPs and GdauCSPs were expressed in G.daurica adult antennae,and there were significant differences in expression levels between males and females.Most of OBP and CSP genes were broadly expressed in heads,thoraxes,abdomens,legs and wings of adults.The expression levels of OBP and CSP genes varied in different development stages of G.daurica.Over half of the genes were expressed at the highest levels in adult antennae with significant differences between female and male.Meanwhile,the expression levels of most OBP and CSP genes were relatively low in eggs and pupae.4.Molecular cloning,prokaryotic expression and protein purification of OBPs and CSPs in Galeruca dauricaRACE technique was used to clone the full-length cDNA of GdauOBP1,GdauOBP6,GdauOBP10,,GdauOBP209 GdauCSP4 and GdauCSP5,as well as the ORF and 3' non-coding region of GdauOBP15,which verified the integrity of ORFs.The expressions of recombinant proteins,pET-28a(+)/OBP1,pET-2 8a(+)/OB P6,pET-28a(+)/OBP10,pET-28a(+)/OBP15,pET-28a(+)/OBP20,pET-28a(+)/CSP4 and pET-28a(+)/CSP5,were induced by constructing prokaryotic expression system,and purified by using the Ni-NTA Agarose affinity column.5.Identification of volatile components from the main host plant of G.daurica and EAG testThirty-two volatile components were identified from the main host plant,A.mongolium,and 13 major compounds were measured for EAG determination.The results showed that 7 compounds elicited relatively strong eletrophysiologocal responses,such as diallyl sulfide,diallyl disulphide,diallyl trisulfide,(Z)-2-hexen-1-ol,2-hexenal,methyl benzoate and hexanal.Moreover,the EAG values in female were higher than those in male to most compounds.When the concentrations of the compounds were 0.1 mol/L and 1 mol/L,the EAG values were relatively higher.6.Fluorescence-based ligand binding assay of GdauOBPs and GdauCSPsThe binding abilities of 7 OBPs and CSPs were determind using 1-NPN as the fluorescence probe and 13 ligands as competitors.GdauOBP1 and GdauOBP10 showed weak binding affinity with all tested components(Ki>30 ?M).GdauOBP6 showed strong binding affinity to dimethyl disulfide,hexanal,2-hexenal and(Z)-2-hexen-1-ol,with the Ki values between 25.65 ?M and 29.29 ?M.GdauOBP15 bound to dimethyl disulfide specifically with the Ki value of 23.09 ?M.GdauOBP20 showed strong binding affinity to p-xylene and 1,3,5-cycloheptatriene with the Ki values of 22.91 ?M and 26.55?M,respectively.The binding affinities of the two CSPs to the host plant volatiles varied greatly.GdauCSP4 showed a broad binding profile with 9 compounds with the Ki values between 12.06 ?M and 21.32 ?M,among which the ligand with the strongest binding ability was methyl benzoate,followed by hexanal.However,GdauCSP5 could only bind dimethyl disulfide and 2-hexenal with the Ki values of 26.47?M and 25.28 ?M,respectively.All of 7 recombinant proteins showed weak bingding affinities to diallyl sulfide,myrcene and diallyl trisulfide(Ki>30 ?M).