Olfactory Function Analysis Of OBPs In Aenasius Bambawalei(Hayat)

Aenasius bambawalei Hayat is an important parasitic natural enemy of phenacoccus solenopsis and it has better biological control potential for it,but the host localization mechanism of A.bambawalei is not clear.It was generally believed that odorant binding proteins(OBPs)played an important role in transporting odor molecules.If the binding characteristics between OBPs and related odor molecules can be clarified,it will provide an important theoretical basis for the analysis of the host localization mechanism of A.bambawalei.Based on the obtained transcriptome data,we cloned Abam OBPs genes and constructed gene expression measurement of AbamOBP17 and Abam OBP28 genes,further purified AbamOBP17 and Abam OBP28 in vitro and obtained recombinant pure proteins,on this basis,the fluorescence competitive binding assay of Abam OBPs with 27 cotton volatiles were carried out,then,the circular dichroism(CD)spectra of Abam OBPs with eight volatiles were carried out,finally the behavioral activity compounds were screened by Y tube olfactometer and RNA interference.The results provided a theoretical basis for exploring the function of Abam OBPs.The main results were as follows:1.Gene clone and gene expression measurement of A.bambawaleiAbamOBP17 and Abam OBP28 genes were successfully cloned by RT-PCR.The open reading frames of AbamOBP17 and Abam OBP28 genes were 357 bp and 402 bp,they encoded 118 and 133 amino acid residues respectively.AbamOBP17 belonged to Minus-C OBPs and contained signal peptides with 28 amino acid residues,while Abam OBP28 belonged to Classical OBPs and contained signal peptides with 18 amino acid residues.The expression levels of this two genes in different tissues of A.bambawalei were measured by real-time fluorescence quantitative PCR.The period was 1 day after the emergence without mating,and the tissues were head,thorax,abdomen,leg,wing and antenna respectively.The results showed that both AbamOBP17 and Abam OBP28 genes were expressed in all tissues.The expression level of AbamOBP17 gene was the highest in the head of male adults,followed by the head and wing of female adults and the wing of male adults,and lower in other parts.However,the expression of Abam OBP28 gene was the highest in the antennae and wings of female adults,followed by the antennae and wings of male adults and the head of female adults,and lower in other parts.2.Expression,purification and binding affinities of Abam OBPsThe recombinant plasmids p ET32b-AbamOBP17 and p ET32b-Abam OBP28 were constructed,the prokaryotic expression results showed that Abam OBPs were largely expressed in the supernatant.The recombinant pure proteins of AbamOBP17 and Abam OBP28 were successfully obtained by nickel column affinity chromatography.The binding affinities of AbamOBP17 and Abam OBP28 to 27 kinds of cotton volatilities was studied by fluorescence competitive binding experiment.The results showed that Abam OBP28 had binding ability to 26 kinds of cotton volatilities,moreover,the binding ability of Abam OBP28 to the ligands was mainly influenced by the type of ligand rather than the p H.However,AbamOBP17 just showed bind ability to diethyl sebacate.The results of the fluorescence competitive binding experiment were analyzed,and 8 ligands with strong binding ability under p H 7.4 condition were screened for latter circular dichroism(CD)spectrum.The results showed that when the external p H was changing,the number of ?-helices and the structural conformation of Abam OBPs were also changing.When the ligands were bind to Abam OBPs,it also had significant changes in the number of ?-helices,which will affect the structural conformation of the Abam OBPs.The CD spectrums obtained by the combination of Abam OBPs and ligand were different from ordinary OBPs,there was a "red shift" phenomenon.3.Screening of behavioral activity compounds and functional verification of AbamOBP171-octen-3-one,diethyl sebacate and 2,4,4-trimethyl-2-pentene with strong binding ability to Abam OBPs were screened to conduct behavioral experiments.The results showed that the female adults showed no behavioral response to 1% and 1‰ concentrations of diethyl sebacate,while the male adults showed obvious preference to both concentrations of diethyl sebacate.The male and female adults showed no behavioral response to 1% and 1‰ concentration of 1-octen-3-one.1% concentration of 2,4,4-trimethyl-2-pentene was repell to the female adults,while 1‰ was attract to them.The male adults showed obvious preference to both concentrations of 2,4,4-trimethyl-2-pentene.RNA interference results showed that after the injection of ds AbamOBP17 at the head of male adults,48 h and 72 h later,the head was sampled for the real-time fluorescence quantitative PCR,the result showed the expression level of AbamOBP17 gene in head was significantly decreased.Then,the behavior experiment was verified by Y tube olfactometer and it was found that the attraction of 1% concentration of diethyl sebacate to male adults disappeared,the results showed that diethyl sebacate had an important effect on the orientation of the male adults.Therefore,this study successfully screened 2,4,4-trimethyl-2-pentene and diethyl sebacate as two behavioral activity compounds,and verified the olfactory function of AbamOBP17.