Cloning And Function Analysis Of Five Odorant Binding Protein Genes In Sitodiplosis Mosellana (Gehin) (Diptera: Cecidomyiidae)

The orange wheat blossom midge,Sitodiplosis mosellana(Gehin),is a devastating wheat pest in China.Non-preference induced by volatile compounds is the main resistance mechanism.Previous studied showed that volatiles from wheat spikes play important role in host selection of this species.This study aims to clarify olfactory recognition mechanism of S.mosellana.In this study,five OBP genes in S.mosellana were cloned by reverse-transcription PCR(RT-PCR)and rapid amplification of cDNA ends(RACE)techniques,and relative proteins were obtained by prokaryotic expression system and enriched via Ni ion affinity chromatography.Further,fluorescence competitive binding experiment was used to determine binding properties of SmosOBPs with wheat volatiles,and Y-type olfactometer and EAG technique were used to determine behavioral and EAG responses of adult to volatile compounds.Results will help us to clarify the olfactory recognition mechanism of S.mosellana in host selection and provide a basis for green control technological.The main results are as follows:1.Five ORF sequences of high-expressed SmosOBPs(SmosOBP11、SmosOBP12、SmosOBP16、SmosOBP17 and SmosOBP21)from transcriptome of wheat midge had been cloned successfully.Their full-length of cDNA were 438 bp,438bp,498 bp,432bp and 405 bp,and the five genes encoded 168,168,165,143 and 134 amino acids,respectively.The predicted molecular weight/isoelectric point of those amino acids were 14.68 kDa/5.49,15.89 kDa/5.00,17.61 kDa/4.10,14.08 kDa/5.14 and 13.61 kDa/4.84.The analysis of amino acids revealed that five SmosOBPs had six conserved cysteine residues,owning the signal peptide of 16-24 amino acids in N-terminal regions.2.The analysis of SDS-PAGE showed that five proteins were all detected in inclusion body.When the concentration of IPTG was 0.5 m M,each proteins had the highest expression.Using Bicin-choninic Acid(BCA)protein assay kit,we measured the mass concentration of the enriched proteins,which was 4.14,5.63,5.48,4.28 and 17.99 μM in the above mentioned order.3.Fluorescent binding test results of SmosOBPs and 28 volatiles revealed that five SmosOBPs had no binding activities with alkane,parts of SmosOBPs had binding activities with aldehydes and terpenes compounds,and all SmosOBPs had binding activities with alcohols and esters compounds.SmosOBP12 showed strongest binding activities with 19 volatiles(Ki < 10.42 μM),meanwhile SmosOBP11 showed stronger binding activities with 18 volatiles(Ki < 23.74 μM).SmosOBP12 displayed the highest binding activities with 3-Hexanol(Ki = 3.33 μM)and Heptyl acetate(Ki = 3.34μM).SmosOBP11 had the highest binding affinities with Hexyl propionate(Ki = 3.47 μM).The best binding-activated volatiles of other three proteins(SmosOBP16,SmosOBP17 and SmosOBP21)were 3-hexanol(Ki = 13.28 μM),methyl salicylate(Ki = 20.13 μM)and cis-3-hexenyl acetate(Ki = 14.32 μM),respectively.4.The analysis of electroantennogram response of female adults to 25 wheat volatiles displayed that adults showed cis-3-hexenylacetate,methyl salicylate,heptyl acetate,1-octen-3-ol and 3-hexanol elicited the strongest eletrophysiological reaponses(relative EAG values over 1.0)of female antennae,while hexyl acetate,ethyl heptanoate,isooctyl acetate,pentyl pentanoate,D-panthenol,2-ethyl-hexanol,ocimene,α-cedrene,α-farnesene,α-pinene,α-humulene and β-caryophyllene evoked stronger eletrophysiological reaponses(relative EAG values over 0.3)of female antennae.Hexyl propionate,1-undecanol,3-carene 2-tridecanone,5-nonanone,3-hexanone and 2-hexanone elicited weak eletrophysiological reaponses(relative EAG values under 0.3)of female antennae,and dodecy aldehyde,pentadecane,dodecane and tridecane did not evoked eletrophysiological reaponses.5.The results of two-choice bioassays of 24 volatiles indicated that 9 volatiles had a significant attraction to females(P < 0.05),in contrast to control experiment.Those volatiles were 1-octen-3-ol,methyl salicylate,ethyl heptanoate,heptyl acetate,cis-3-hexenyl acetate,D-panthenol,hexyl acetate,3-carene and 3-hexanol.But α-farnesene and ocimene showed significant repellency to females(P < 0.05),the other 13 volatiles were no effect.