Molecular Cloning,Expression And Function Analysis Of Pheromone Binding Protein 1 (PBP1) Of Cyrtotrachelus Buqueti Guerin-Meneville

Insect olfactory system is a chemical monitor with strong specificity and sensitivity,and it can identify information specifically on chemical substances which in environment,are regarded as an important information on locating food sources,mate-finding,copulation and spawning,and so on.Cyrtotrachelus buqueti is an important forestry drilling pest of southern China,and this can seriously affect/inhibit the development of bamboo industry by harming a variety of sympodial bamboos.In the process of generations of insect reproduction,how the male and female individuals can accurately search and locate by olfactory system is the key to effective communication between the male and female individuals.In this study,according to the results of the previous Illumina HiSeq sequencing screening,we cloned one pheromone binding protein gene of C.buqueti successfully by RT-PCR method,named ChuqPBP1.We used SYBR Green I dye method to analyse CbuqPBP1 expression profiles of different organs of the male and female adults by qPCR and the protein was expressed and purified.The expression patterns were analysed by the binding characteristics of the CbuqPBP1 with the main body of volatiles and host volatiles using fluorescence binding assay.The main research results were as follows:1 Two pairs of primers were designed based on the results of the previous Illumina HiSeq sequencing screening of C.buqueti(GenBank number:SAMN06176790).A new cDNAs of C.buqueti called CbuqPBP1(GenBank Number:KU845733.1)was cloned by using RT-PCR method and encoded 143 amino acids,including 17 aa of signal peptide.The molecular weight of the predicted protein of CbuqPBP1 was 15.8411 kDa and the isoelectric point that contained six conserved Cys was 4.60,and this gene had typical characteristics of primary structure of insect PBP.The results of multiple sequence alignment and phylogenetic tree analysis were showed that the similarity of the deduced CbuqPBP1 amino acid sequence to Coleoptera and Lepidoptera was 38.47%and 52.39%,respectively.The similarity with PBP2 and PBP3 of Anomala rufocuprea was higher than that of other insects and they were on the evolution of the same branch.2 The distribution of CbuqPBPl in different tissues(antenna,head,thorax,abdomen and leg)of male and female adults of Cbuqueti was studied by qPCR technique.The results were showed that the CbuqPBPl was expressed in antenna,head and thorax in both male and female,and was not expressed in abdomen and leg.Addtionally,the CbuqPBPl in antenna was highly expressed while it was rarely expressed in head and thorax.In the expression of organs,CbuqPBPl in male was expressed higher than the female.3 The CbuqPBP1 was constructed into prokaryotic expression vector pET-28a(?),transferred into E.coli BL21(DE3),and then successfully expressed by IPTG,and a 16kD protein band was detected by SDS-PAGE.The gene was expressed in the supernatant after induction(37 ?,3 h,IPTG 1 mmol/L).After affinity chromatography on nickel ions,the samples were filtered and concentrated to obtain a high purity target protein and its concentration was 345 ng/?L,which laid a foundation for further fluorescence competitive binding assays.4 The binding affinity of CbuqPBP1 and 14 kinds of sex pheromone of C.buqueti,3 kinds of plant volatiles of Neosinocalamus affinis and 3 kinds of mixture volatiles was studied by fluorescence competitive binding assays,chosing 1-NPN as fluorescent probes.The results were showed that:CbuqPBP1 could effectively combine with 11 kinds of sex pheromone and 3 kinds of plant volatiles.The binding ability of CbuqPBP1 with bis(2-ethylhexyl)phthalate and dibutyl phthalate performed the best among these chemical substances,and the binding constants were 6.49 ?M and 11.10 ?M,respectively;The binding ability to benzaldehyde,linalool and indole performed worse,and the binding constants were 28.43 ?M,29.77?M and 32.90 ?M,respectively;The binding affinity of benzaldehyde,linalool and indole was increased by 62.3%,65.1%and 51.7%,respectively when the three chemical substances were mixed based on 1:1 ratio.Overall,the CbuqPBP1 was a pheromone binding protein gene of C.buqueti.and played an important role in feeling pheromone.In addtion to this,the addition of plant volatiles may have synergistic effect on male mate-finding.