Identification Of Olfactory Related Genes And Ligand Binding Properties Of Pheromone Binding Proteins From Greater Waxmoth Galleria Mellonella L.

The olfactory system is essential for insects’ survival and reproduction and the olfactory related proteins,such as pheromone binding proteins(PBPs),play an important role in the olfactory perception.The wax moth Galleria mellonella is a main pest affecting Apis cerana.This study assessed it and firstly investigated the types and ultrastructures of olfactory sensilla in G.mellonella antennae by scanning electron microscopy.Then,we used transcriptome sequencing on antennae of G.mellonella to identify several olfactory gene families and the differentially expressed genes(DEGs).The c DNA sequences of three PBPs and one atypical odorant receptor(Orco)from G.mellonella were amplified by RT-PCR,respectively.Expression levels of these four genes at different development stages(egg,larva,pupa and adult)and in different tissues(antennae,head,thorax,abdomen,legs and wings)of adults were detected by real-time quantitative PCR.The recombinants Gmel PBPs were successfully expressioned and purified,and the binding characteristics of PBPs to odor ligands were investigated by fluorescence competitive binding test.The main results were as follows:1.The scanning electron microscopy examinations revealed that the antennae of male and female G.mellonella were both filiform,and consisted of scape,pedicel and flagella.The flagellum was divided into50~60 sub-segments,and the length was 5.5~7.5 mm,which varies greatly between male and female moths.There are seven kinds of sensillum on G.mellonella antennae: sensilla trichoid(type I and type II),sensilla chaetica,sensilla basiconis(type I and type II),sensilla coeloconica,sensilla styloconica,sensilla auricillica and sensilla furcatea.The distributions of each kind of sensillum were similar.2.According to the antennal transcriptome of male and female G.mellonella,we have identified 52 OBPs,36 CSPs,80 ORs,56 IRs and 2 SNMPs.Comparative analysis of the expression level between male and female antennae,66 up-regulated and 48 down-regulated genes were found in female antennae.3.The cDNA sequence of Gmel PBP1,Gmel PBP2 and Gmel PBP3 were cloned from the antennae of G.mellonella by RT-PCR.Their ORFs were 492 bp,510 bp and 492 bp,amino acids(AA)were 163,169 and 163,estimated molecular weight were 18.79 k D,19.12 k D and 18.35 k D,respectively.Sequence analysis showed that Gmel PBP1,Gmel PBP2 and Gmel PBP3 belong to the Classical OBP.Bioinformatics analysis showed that Gmel PBP1,Gmel PBP2 and Gmel PBP3 were all secreted proteins with a signal peptide at the N-terminus,and have a conserved domain of the PBP-GOBP superfamily.The pairwise sequence alignment results of Gmel PBP1,Gmel PBP2 and Gmel PBP3 showed that the homology was low,which was about 49.70%.4.Real-time PCR data revealed that the expression level of Gmel PBP1,Gmel PBP2 and Gmel PBP3 expressed predominantly in antennae,in smaller amounts in legs,and in negligible amounts in thorax,abdomen and wings.The expression profiles of Gmel PBP1,Gmel PBP2 and Gmel PBP3 appeared an upward trend with the days of age increasing,among which the 1-day-old moths had the highest expression level.The expression level of Gmel PBP2 and Gmel PBP3 in the female moth were significantly higher than in male moth,suggesting that they might be involve in the identification of sex pheromone from male moths.5.The pET/Gmel PBP1,p ET/Gmel PBP2 and p ET/Gmel PBP3 had been constructed successfully and expressed in E.coil chemically competent cells induced by IPTG.The results of SDS-PAGE showed that the size of recombined proteins were consistent with the predicted molecular weight.After purified by affinity chromatography,the concentration of the target proteins were 1.1 mg/m L,1 mg/m L and 1.4mg/m L,and the purity were all more than 85%.The results of fluorescence competitive binding assay showed that Gmel PBP3 had binding properties with rolene,n-octanol and other plant volatiles(Ki < 50),while Gmel PBP1 and Gmel PBP2 had almost no binding properties with 18 odors(Ki > 50),suggesting that there may be multiple modes of action of Gmel PBPs.6.The cDNA sequence of olfactory receptor Orco was obtained from G.mellonella,and named as Gmel Orco(Gen Bank accession number is KT020861).Sequence analysis revealed that the open reading frame of Gmel Orco is 1425 bp in length,encoding 474 amino acid with 7 putative transmembrane domains,the protein had molecular mass of 53.36 k D and isoelectric point of 8.44.The sequences obtained are c DNA sequences of Orco orthologue chemoreceptor gene.The expression levels of Gmel Orco in different tissues(antennae,head,thorax,abdomen,legs and wings)and different stage were detected by Real-time PCR.Results showed that Gmel Orco were also dominantly expressed in antennae,the expression profiles of Gmel Orco appeared an upward trend with the age increased before emergence.However,the expression level decreased after emergence.The expression profiles of Gmel Orco in the adult female moths was higher than that in male moths;Gmel Orco was also expressed in eggs with a similar expression level at the3-day-old of adult.In this study,the identification and expression characteristics of olfactory related genes from G.mellonella were studied based on the transcriptional sequence,not only to reveal the olfactory behavior reaction of the wax moths in nature,to provide a theoretical basis for the development of the attractants and repellents with good effects on both sexes,but to expand the comprehensive prevention and control of G.mellonella.