Cloning,Prokaryotic Expression And Purification Of OBPs In Agasicles Hygrophila(Coleoptera:Chrysomelidae)

[Objective]Insects by perceiving volatile chemical information substances in the surrounding environment,the identification and location of host plants,the selection of spouses and oviposition sites are realized.Agasicles hygrophila is specific natural enemies of the global malignant weed Alternanthera philoxeroides.Olfaction plays an important role in Agasicles hygrophila specific recognition the host plants of Alternanthera philoxeroides.Odorant binding proteins are important olfactory related proteins that participate in the identification of odorants.By heterologous expression of Agasicles hygrophila OBPs is helpful to the study of the identification and location of Alternanthera philoxeroides and then realize the host specificity.[Methods]Three sequences of OBPs were obtained by gene cloning,the inclusion body proteins of Agasicles hygrophila OBPs was obtained by prokaryotic expression.The purification was achieved by Ni-IDA affinity chromatography.The physicochemical properties of proteins were analyzed by ExPASy-ProtParam.Multiple sequence alignment of protein were analyzed by DNAMAN.MEGA 5.02 was used to construct the evolutionary tree of OBPs.RT-qPCR was used to determine the expression of OBPs in the male and female adult worms with the same sex in different tissues and different tissues of the male and female adults.The data were analyzed by SPSS 22.0 and the difference was analyzed by One-way ANOVA and T test respectively.[Results]The construction of gene cloning and prokaryotic expression vector for AhygOBP1,AhygOBP2 and AhygOBP3 was realized,the protein expression of AhygOBPI was induced by IPTG.The protein purification of AhygOBP1 was realized by Ni-IDA affinity chromatography OBPs are acidic small molecular weight hydrophilic proteins.AhygOBPI belongs to plus-C OBPs,AhygOBP2 and AhygOBP3 belong to minus-C.The trend of OBPs clustering is obvious.The highest expression levels of AhygOBPI and AhygOBP3 in the antennae of female and male were significantly different from those of other tissues.The highest expression of AhygOBP1 in the male's foot was significantly different from that of other tissues,and the highest expression of AhygOBP2 in the female's antennae was significantly different from that of other tissues.There was significant difference in the expression of AhygOBPI between male and female heads,but there was no significant difference in the expression of AhygOBP2 and AhygOBP3 in the same tissues between females and males.[Conclusion]Bioinformatics analysis of OBPs in lotus leaf is important for understanding its physical and chemical properties and evolutionary relationship.The expression pattern of OBPs is important for the study of its distribution and function.The protein purification of Agasicles hygrophila OBPs laid the foundation for the study of fluorescence competitive binding experiment,It has important significance to the interpretation of Agasicles hygrophila on the sense of smell to achieve recognition and localization of host plants to achieve specialization,it's also important to study the ecological security risk of imported species.