Molecular Characterization And Functional Analysis Of Chemosensory Proteins From The Nilaparvata Lugens (ST?L)

Complicate and sensitive olfactory system plays important functions in the behavior of herbivorous insects,such as recognition of semiochemicals and selection of host plants.The new effective pest monitoring and control strategies might be developed based on the substantial understanding of the insect olfactory mechanisms.It have been showed that several proteins involving the chemosensitive of insect,and one of these proteins,chemosensory proteins(CSPs),have been showed playing vital roles in the olfactory system in some insect species,while some other CSPs have been showed playing other physiological functions in some insects,such as development etc.And the exact function of CSPs is still not clear.The brown plant hopper(BPH)Nilaparvata lugens(St?l)is an important pest on rice,caused major threat to rice productivity.Many CSPs have been identified in the antennal transcriptome of BPH in our previous study,though their functions are not known.In order to illustrate the function of CSPs in BPH,the three chemosensory proteins(CSPs)of BPH were cloned.The expression patterns of the CSPs genes and the ligand binding properties with different odorants in addition with molecular docking were studied in this study.And also,the effects on the response of BPH to volatiles were studied by RNAi.The main results are listed below:1.Functional analysis of NlugCSP3In this study,we cloned NlugCSP3 gene to better understanding the physiological functions of CSPs in Nilaparvata lugens.Quantitative real-time PCR demonstrated that transcriptional levels of NlugCSP3 were high in mated 3-day old short wing female antennae as well as in abdomen and antennae of unmated 3-day long wing males.By taking in vitro binding assay,behavioral screening and fluorescence quenching assays together,we provided viable and an efficient method for screening active semiochemicals.Of the 25 ligands tested,results of both methods in vitro binding assay and behavioral screening indicated that nonadecane and 2-tridecanone exhibited high binding affinity with the NlugCSP3 and behaviorally attracted by the N.lugens.The high binding affinity to rice plant volatiles support a possible functional role of NlugCSP3 in the perception of general odorants.Binding mode analyses revealed much information on the interactions between ligands and NlugCSP3.Key residues involved in interacting with these high binding affinity ligands were pointed out.Considering individual energy terms and the interaction combination,it could be estimated hydrophobic interaction were key factors in determining the binding affinity of ligands to NlugCSP3.This study also investigated that nonadecane and 2-tridecanone clearly quenched the fluorescence of NlugCSP3 as a stable static quenching mode.The binding interaction is spontaneous and mainly promoted by hydrophobic interactions.This study is of guiding importance to screening potentially attractive semiochemicals by taking insights into fluorescence quenching assays.2.Functional analysis of NlugCSP8In this study,we cloned the full-length CSP8 gene from N.lugens.Protein sequence analysis indicated that NlugCSP8 shared high sequence resemblance with the CSPs of other insect family members and had the typical four-cysteine signature.Analysis of gene expression indicated that NlugCSP8 mRNA was specifically expressed in the wings of mated 3-day brachypterous females with a 175-fold difference compare to unmated 3-day brachypterous females.The NlugCSP8 mRNA was also highly expressed in the abdomen of unmated 5-day brachypterous males and correlated to the age,gender,adult wing form,and mating status.A competitive ligand-binding assay demonstrated that ligands with long chain carbon atoms,nerolidol,hexanal and trans-2-hexenal were able to bind to NlugCSP8 in declining order of affinity.By using bioinformatics techniques,threedimensional protein structure modeling and molecular docking,the binding sites of NlugCSP8 to the volatiles which had high binding affinity were predicted.In addition,behavioral experiments using the compounds displaying the high binding affinity for the NlugCSP8,revealed four compounds able to elicit significant behavioral responses from N.lugens.The in vivo functions of NlugCSP8 were further confirmed through the testing of RNAi and post-RNAi behavioral experiments.The results revealed that reduction in NlugCSP8 transcript abundance caused a decrease in behavioral response to representative attractants.An enhanced understanding of the NlugCSP8 is expected to contribute in the improvement of more effective and eco-friendly control strategies of BPH.3.Functional analysis of NlugCSP10In this study,we conducted the developmental expression patterns,competitive binding assay,and the binding sites,as well as knock-down assay by RNA interference both in vitro and in vivo to reveal the functions of NlugCSP10.The results showed that the expression of NlugCSP10 mRNA was significantly higher in males than in females and probably involved in locating suitable mates.The fluorescence competitive binding assay results demonstrated that the NlugCSP10 displayed the highest binding affinity with cis-3-hexenyl acetate,eicosane,and(+)-β-pinene.In order to clarify the binding mechanism,the key binding sites of NlugCSP10 were well predicted by threedimensional structure modeling and molecular docking and we found that hydrophobic and van der Waals interactions are both important in binding between NlugCSP10 and those three volatiles.Amongst the three compounds with the highest binding affinity at neutral pH 7.4,eicosane was shown attractant activity for N.lugens,while cis-3-hexenyl acetate and(+)-β-pinene exhibited repellent activity.The research has also shown that NlugCSP10 silencing achieved through RNAi that disrupted cis-3-hexenyl acetate communication.Overall,our findings showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow-release mediators that attract/repel N.lugens and subsequently improve the exploration of plans to control this insect pest.