Cloning Of Orco And CSPs Genes And Functional Study Of CSP3 Protein In Bactrocera Minax

Bactrocera minax is one of the important pests in citrus fruit trees in China.The adults lay eggs in the citrus fruit,and the larvae eat inside the fruit,which seriously affects the yield and quality of citrus.Due to the damage caused by the larvae of B.minax,the chemical control effect is limited,and it is easy to produce"3R"problems,so people focus on the prevention and control of adults.Using the sense of smell to trap pests is an efficient and environmentally friendly control strategy.Therefore,an in-depth study of the olfactory recognition mechanism of B.minax can not only deepen the understanding of the olfactory recognition pathway of B.minax,but also provide an important theoretical basis for the development of efficient attractants or repellents.Odorant receptor co-receptor(Orco)is a key protein in insect olfactory pathway.In insects,Orco and the specific odorant receptor ORx are often used as important functional proteins in the process of insect olfactory signal transduction in the form of heterodimer,which promotes chemical signals to be transduced into electrical signals and transmitted to the central nervous system of insects.Chemosensory protein(CSPs)has the physiological function of participating in the olfactory recognition process of insects.It can bind and transport odorant molecules through the receptor lymph to the olfactory receptor,and then trigger the corresponding olfactory behavior of insects.Insect odorant receptor co-receptors and chemosensory proteins are potential targets for pest control.Green control strategies can be developed for this target.In this study,one Orco gene(BminOrco)and four CSP genes(BminCSP1-BminCSP4)were obtained by head transcriptome analysis of B.minax,and the bioinformatics and tissue expression patterns of these genes were analyzed.According to the results of tissue expression pattern,the BminCSP3 highly expressed in antennae was prokaryotic expressed and purified,and the heterologous expression protein of the gene was obtained.The binding characteristics of BminCSP3 with 14 odorant compounds were analyzed by fluorescence competitive binding experiment.The main results are as follows:1.BminOrco and BminCSPs sequence analysisThe open reading frame of B.minax Orco gene(BminOrco)is 1431 bp in length and encodes 476 amino acids with 7 transmembrane domains,the amino terminal is located in the membrane and the carboxyl terminal is outside the membrane.It is predicted that the isoelectric point of the protein is 8.69,the molecular weight is 53.41ku,there is no signal peptide,and the average hydrophilicity is 0.296,which is a hydrophobic protein.The results of homology comparison and phylogenetic tree analysis showed that the amino acid sequence of BminOrco have high homology with Coleoptera,Diptera,Lepidoptera,Hemiptera,Hymenoptera and Orthoptera,and the Dmel Orco homology with D.melanogaster was the highest,reaching 86.42%.All Four CSP genes have the complete ORF,varying from 336 to 474 bp encoding111-157amino acids.All four deduced BminCSPs have the signal peptide,ranging from18 to 28 amino acids.The predicted mature protein sequences of the four BminCSPs ranges in length from 83 to 135 amino acids,with molecular weight of 9,616–14,901 Da and isoelectric points of 5.19–9.46(Table S2).In addition,all these BminCSPs bear the typical four-cysteine signature(C1-X6-C2-X6–18-C3-X2-C4)of the CSP family.The phylogenetic tree shows that the CSP gene of Diptera insects may come from three ancestral genes and then differentiate due to different selection pressures.Four BminCSP genes were obviously differentiated,and they were clustered on different evolutionary branches,indicating that they may have different functions.2.Analysis of tissue expression of BminOrco and BminCSPsThe distribution of BminOrco and BminCSP1-BminCSP4 in different tissues of adult B.minax was analyzed by real-time quantitative PCR.The results showed that the relative expression of BminOrco in adult antennae was significantly higher than that in other tissues,and slightly expressed in head(excluding antennae),thorax,abdomen,leg and wing,suggesting that BminOrco plays an important role in olfactory recognition of B.minax.The situation of BminCSPs was different:BminCSP3 was significantly up-regulated in antennae,BminCSP4 was significantly upregulated in head tissues(without antennae),while BminCSP1 and BminCSP2 were widely expressed in antennae,not only in antennae,but also in leg,wings,abdomen and thorax.The tissue expression pattern of BminCSPs further indicates that different BminCSPs may have different physiological functions.3.Cloning,prokaryotic expression and protein purification of BminCSP3 geneIn order to verify the olfactory function of BminCSP3 which is highly expressed in antennae,the cloning vector(p GEM-T/BminCSP3)and prokaryotic expression vector(p ET-32a/BminCSP3)of BminCSP3 gene were successfully constructed.After induced expression in E.coli BL21(ED3),purification of recombinant protein and removal of His-tag of recombinant protein by enterokinase,the target protein BminCSP3 with high purity was obtain.4.Analysis of binding characteristics of BminCSP3Using fluorescent probe 1-NPN,the affinity of BminCSP3 with 14 odorant ligands was analyzed by fluorescence competitive binding method.Most of the odor ligands selected in this experiment can cause the behavioral response of adult B.minax.The results of fluorescence competition experiment showed that the K_ivalues of BminCSP3and 14 odorant ligands were all more than 20μM,and it was generally believed that the K_ivalue was more than 20μM,and the affinity between protein and ligand was weak.It is suggested that although BminCSP3 is significantly up-regulated in the antennae of adult B.minax,it may not be involved in the olfactory recognition process of adult B.minax.It is speculated that odorant binding protein(OBPs)plays a major role in the first biochemical process between adult B.minax and external odorant,rather than chemosensory protein(CSPs).