Functional Analysis Of Odorant-binding Proteins From Cnaphalocrocis Medinalis (Guenée)

In recent years,the area of rice pests and diseases has continued to expand,and pests have become resistant to major pesticide species.Finding efficient and safe ways of biological control has become a research hotspot.Insect olfactory plays an important role in the whole life process of insects,the odor-binding proteins(OBPs)are involved in the first step in the identification of foreign substances.The study of OBPs is of great significance for pest control.In this paper,Cnaphalocrocis medinalis was studied,six OBPs genes of Cnaphalocrocis medinalis were cloned,and two OBPs were expressed and purified.Based on the analysis of OBPs gene expression and its behavioral correlation,the function of OBPs was explored.Through behavioral experiment verification,we find 5 kinds of host volatiles with behavioral activity.In order to provide a basis for the identification of the molecular mechanism of host location,and the synthesis of high-performance behavioral regulators.The main results are as follows:1.Scanning Electron Microscopic Observation of wings and Legs of Cnaphalocrocis medinalisThrough scanning electron microscopy,we found two kinds of sensilla,stinging sensillum and sensilla auricillica on the male and female wings of Cnaphalocrocis medinalis.The sensilla basiconc,sensilla trichodea,sensilla squamiformia,sensilla chaetica,and Bohm mane were found on the Legs of Cnaphalocrocis medinalis.The foreleg sensation was more than hindleg and the hind leg was more than the midleg.There is no difference between male and female.2.Cloning and sequence analysis of CmedOBPs gene in Cnaphalocrocis medinalisIn this experiment,six genes including CmedOBP5,CmedOBP8,CmedOBP10,CmedOBP13,CmedOBP14,and CmedOBP20 were successfully cloned.Sequence analysis revealed that there are 6 conserved cysteine sites in CmedOBP5,CmedOBP8,and CmedOBP14,which are consistent with the characteristics of Classic OBPs.CmedOBP13 has 7 Cys sites and CmedOBPIO has 15 conserved Cys sites,which are Plus-C OBPs.CmedOBP20 has 15 conserved Cys sites belongs to Minus-C OBPs.The predicted signal peptides were:CmedOBP5,CmedOBP8,CmedOBPIO,CmedOBP13,CmedOBP14,and CmedOBP20 contained 18.17,20,22,25,and 16 signal peptides respectively.3.Temporal and spatial expression of CmedOBPs gene in Cnaphalocrocis medinalisFor the above six genes,after mating of male and female adults,the mated and unmated,each day-old segment and each tissue site were analyzed by fluorescent quantitative methods.The results showed that CmedOBP13,CmedOBP14,and CmedOBP20 were highly expressed in antennae(At)of 3 days old adults,and there were differences between males and females,which may play a major role in chemical sensing.The expression levels of the other three genes at the main tissue sites differed by adult age,sex,and mating status.CmedOBP5 is mainly expressed in the wings(W)of 1 day old male and female adults;CmedOBP8 is mainly expressed in male and female fat bodies(Fa);CmedOBP10 is mainly expressed in the unmatched 5-day male and female head(H),possibly exercising other functions.4.Analysis of the binding characteristics of CmedOBP5 and CmedOBP14Recombinant plasmids pET-22b-(+)-CmedOBP14,pET-22b-(+)-CmedOBP5,pET-30 a-(+)-CmedOBP16,pET-30a-(+)-CmedOBP20,pET-28a-(+)-CmedOBP10 and pET-28a-(+)-CmedOBP13 were constructed.After expressed in expression strains,they were pu rified by ion chromatography column chromatography to obtain pure proteins CmedOB P5 and CmedOBP14.Fluorescence competition binding experiment results showed that the binding force of CmedOBP14 and 37 kinds of rice volatiles was stronger than that of alkanes and hexanal under neutral conditions than under acidic conditions.The binding of CmedOBP5 to 14 volatiles was higher at pH=7.4 than that of pH=5.0 except for trans-2-Hexanal,(-)-Limonene,3-decene,Nerolidol.This may be related to the different pH will cause OBPs different conformational changes.Five kinds of substances with strong binding ability to CmedOBP5((+)-Cedrol,Sabinene hydrate,Hexadecane,Cis-3-hexenol,Caryophyllene)were selected for fluorescence quenching experiments at 16? and 27?.The results showed that(+)-Cedrol and cis-3-hexenol with CmedOBP5 were 1:1 stable static binding,The binding constants and binding sites were calculated,and it was found that the forces were all hydrophobic forces.5.Behavioral active substance screening of Cnaphalocrocis medinalisThrough the results of fluorescence competition experiments,the volatiles with strong binding ability to CmedOBP14 were screened,among them,13 volatiles have been reported to have strong electrophysiological activity on Cnaphalocrocis,medinalis.In order to further understand the relationship between volatiles and CmedOBP14,RNAi technology was used to down-regulate the expression of CmedOBP14,and the electrophysiological differences of volatile for Cnaphalocrocis medinalis were detected by the Electroantennography(EAG).The results showed that the reaction of Nerolidol,(-)-Limonene;?-ionone,Hexadecan and Henicosane were decreased after RNAi.Electrophysiologically active volatiles may not necessarily cause behavioral responses in insects.In order to further verify the function of CmedOBP14,H-tube behavioral experiments of Cnaphalocrocis,medinalis were performed using the above five volatiles.The results showed that it exhibited tropism for Nerolidol,Limonene,Hexadecane and Henicosane,and exhibited evasion against ?-ionone.After RNAi,Cnaphalocrocis medinalis had no effect on the selection of Nerolidol.Decreased attraction of(-)-Limonene,Hexadecane and Henicosane to Cnaphalocrocis medinalis.