Studies On The Function Of High Expression Of Odorant Binding Protein CpomOBP14 In The Antennae Of Codling Moth

The codling moth(Cydia pomonella L.)seriously threaten the production security of apple fruit.Odorant Binding Proteins(OBPs)can bind and deliver the insect pheromones and plant volatiles to the receptors,participate in the initial step of recognizing of environmental semiochemicals,and play an important role in insect host location or oviposition.However,the role and function of OBPs of codling moth in recognizing environmental semiochemicals are still unclear.We found that CpomOBP14 gene was highly expressed in the antennae of both male and female moths by previous transcriptome analysis.To better understand the role and function of CpomOBP14 in recognizing environmental semiochemicals,in present study,we systematically studied the evolution and function of CpomOBP14 gene through gene cloning,expression profile analysis,phyletic evolution,prokaryotic expression,protein purification,fluorescence competitive binding experiment,homology modeling and molecular docking,as well as other techniques and methods.The main conclusions are as follows:1.The full length of CpomOBP14 gene was cloned by PCR technology,which is comprised of a 432 bp coding sequence(CDS)encoding 143 amino acids residues.CpomOBP14 containing 6 conservative cysteine residues,which belonged to the Classical OBPs subfamily.Phylogenetic analysis showed that CpomOBP14 was closely related to the orthologous OBPs from other lepidoptera insects.The expression profiles of CpomOBP14 gene in different developmental stages and different tissues of male and female adults were studied by fluorescence quantitative PCR.Stage-specific expression profile indicated that CpomOBP14 transcript level in adults was significantly higher than that in other developmental stages,and the expression level in males was significantly higher in females(P<0.05).Tissue-specific expression profile showed that the expression level of CpomOBP14 gene in antennae was significantly higher than that in other tissues,and its transcript level in male antennae was significantly higher than that in female antennae(P<0.05).The result suggested that CpomOBP14 maybe play an important role for male codling moth in recognizing environmental semiochemicals.2.The purified protein of CpomOBP14 was obtained by prokaryotic expression and protein purification.Furthermore,1-NPN probe was used to investigate the binding properties of CpomOBP14 to 19 odorant compounds.The results showed that three compounds,including Ethyl 2-trans-4-cis-decadienoate,cis-3-Hexenyl benzoate and(E,E)-α-farnesene,had the binding ability to CpomOBP14,and the dissociation constants were: 9.89 μM,23.93 μM and 67.52 μM,respectively.The result indicated that CpomOBP14 maybe play an important role in the recognition of these three host plant volatiles in codling moth.3.We reconstructed the 3D modeling of CpomOBP14 by the method of homologous modeling,and evaluated the reliability of the model by Procheck,Verify＿3D and ERRAT indicators.Subsequently,we studied the binding ability and binding mode between CpomOBP14 and Ethyl 2-trans-4-cis-decadienoate,cis-3-Hexenyl benzoate and(E,E)-α-farnesene by molecular docking technique.The result showed that all three semiochemicals could bind with CpomOBP14.Their binding energies are-5.56 kc J/mol(Ethyl 2-trans-4-cis-decadienoate),-5.39 kc J/mol(cis-3-Hexenyl benzoate)and-5.85 kc J/mol((E,E)-α-farnesene),respectively.The results of this study provide a theoretical foundation for virtual screening and development of behavioral regulatory compounds by reverse chemical ecology methods.