Expression And Binding Specificity Analysis Of Odorant Binding Proteins In Sitophilus Zeamais Motschulsky(Coleoptera:Curculionoidea)

Sitophilus zeamais is a beetle species belonging to the order Coleoptera and family Curculionidae.S.zeamais is known as an important stored grain pest in the world and the most harmful to China,which attack not only crops,but stored cereal products,including wheat,rice,sorghum,oats,barley,rye,buckwheat,peas and cottonseed.For the process of feeling external environment and development of growth,olfactory system of insect plays an important role in host location,spouse identification,food selection and oviposition site selection.Odorant binding proteins(OBPs)are particularly important for the process of insect chemical information recognition,but the research on function of OBPs in S.zeamais(SzeaOBPs)is still blank.Studing on the function of OBPs for S.zeamais is to provide reference as well as target for the prevention and control of maize weevil.The odorant binding proteins of S.zeamais had preliminarily been studied in this study,the results were as followed:1.The expression characteristics of SzeaOBPs at transcriptional and protein levels were detected by using qRT-PCR and Western blot(WB).SzeaOBPl and SzeaOBP28 were expressed both in low-instar larva(LL)and eclosion adult(EA)at the transcriptional level.At LL stage,the expression of SzeaOBP10 and SzeaOBP40 were more prominent than in other developmental stages,while at mature larva(ML),only SzeaOBP28,SzeaOBP38 and SzeaOBP40 were slightly expressed.Similarly,only SzeaOBPl,SzeaOBP4,SzeaOBP28 and SzeaOBP38 were slightly expressed at pupae stage.WB analysis showed that SzeaOBPl was highly expressed in LL,ML,pupae and EA,but less expressed in male and female adults.The expression level of SzeaOBP28 was highest in EA and male adult stage,followed by pupae and female adult stage,and lowest in LL and ML stage.2.Sequence analysis and prokaryotic expression of odorant binding protein were performed,SzeaOBPl sequence is consists of 417 bp and SzeaOBP28 sequence consists of 432 bp,encoding 138 amino acids and 143 amino acids,respectively.SzeaOBPl belongs to Minus-C OBPs and SzeaOBP28 belongs to Classic OBPs.Recombinant plasmids pET-SzeaOBPl and pET-SzeaOBP28 were successfully constructed,and the recombinant proteins SzeaOBPl and SzeaOBP28 were expressed and purified.3.The ability of recombinant proteins bind to host plant volatiles was measured by using fluorescence competitive binding assays,then verified the results via behavioural experiments by using Y-tube olfactometer.The results showed that 22 host volatiles could bind to SzeaOBPl(recombinant protein)effectively,the best binding ability was cetanol with Ki value of 2.748±0.139 ?M,and myrcene had worst binding ability with Ki value of 30.27±0.798 ?M.Only seven host volatiles bind to SzeaOBP28(recombinant protein)effectively,among them 4-hydroxy-3-methoxybenzaldehyde exhibited a good binding capacity with Ki value of 20.23±1.918 ?M,and 1-pentanecarbaldehyde had a poor binding capacity with Ki value of 48.70±0.852 ?M.The results of behavioral experiments showed that male and female maize weevils responded strongly to 4-hydroxy-3-methoxybenzaldehyde,and the attraction of 2-methylbutyraldehyde and cetanol to male and female was also significantly different,but no significant difference between male and female selection.