| Spodoptera litura is an important agricultural pest,which is harmful to crops.At present,chemical control of its low instar larvae is effective,but it is easy to produce resistance and environmental risks.Plant essential oil has the characteristics of natural green,environmentally friendly and less impact on human health.In recent years,it has played an important role in pest control and has the potential to develop behavioral regulators.Based on the above background,this study took the female adults of S.litura as the object,and explored the olfactory behavioral response and peripheral olfactory perception mechanism of female adults of S.litura to some plant essential oils by electroantennography(EAG),behavioral test,essential oil composition analysis,odor induction test,prokaryotic expression and fluorescence competition binding test.The results are as follows :1.The antennal electrophysiological and behavioral responses of female adults of S.litura to different plant essential oilsThe results of antennal electrophysiological test showed that among the 14 plant essential oils,the female adults of S.litura produced olfactory electrophysiological responses to 13 of them.According to the absolute value of EAG response from high to low,they were honeysuckle essential oil,rosemary essential oil,menthol essential oil,bergamot essential oil,eucalyptus essential oil,green pepper aromatic oil,lemon essential oil,lavender essential oil,litsea cubeba essential oil,berberine essential oil,danpi essential oil,coral ginger essential oil and wood essential oil.The essential oil that did not produce olfactory electrophysiology was sweet orange essential oil.The plant essential oils with the top five absolute values of EAG response were selected to explore the behavioral choice response of female adults of S.litura.The results showed that honeysuckle essential oil had significant repellent effect on female adults of S.litura,while rosemary essential oil,mentha arvensis oil,bergamot essential oil,eucalyptus essential oil,had no significant behavioral effect on female adults of S.litura.2.Component analysis of honeysuckle essential oil and identification of essential oil active substancesThe components of honeysuckle essential oil were analyzed by gas chromatograph-mass spectrometer(GC-MS).Eight substances with the highest content were identified,which were Linalool,Phenylethyl alcohol,Benzyl acetate,Ethyl phenylacetate,4-tert-Butylcyclohexyl acetate,Butylated hydroxytoluene,Diethyl phthalate and α-hexylcinnamaldehyde.The results showed that the female adults of S.litura had obvious EAG response to Linalool,Benzyl acetate,α-hexylcinnamaldehyde,Ethyl phenylacetate and Phenylethyl alcohol,and the EAG response value increased with the increase of substance concentration,while there was no obvious EAG response to Butylated hydroxytoluene,Diethyl phthalate and 4-tert-Butylcyclohexyl acetate.3.Screening of target genes and bioinformatics analysisFor the 17 odorant binding protein(OBP)genes specifically expressed in the antennae of S.litura,it was found that only the relative expression of OBP21 gene increased significantly among the 17 OBPs by odor induction test.Then,the bioinformatics analysis of SlitOBP21 gene was carried out.The results showed that the coding region of SlitOBP21 gene contained 450 bases,encoding 149 amino acids.The relative molecular weight of the protein was 17.19 kDa,the isoelectric point was 4.69,the instability coefficient was 30.97,containing a signal peptide of 26 amino acid residues,with 4 major hydrophobic regions and 6 major hydrophilic regions.The results of amino acid multiple sequence comparison showed that SlitOBP21 was highly similar to the selected 10 related species OBPs,with homology between 72.41 %-98.66 %,and there were seven conserved cysteine sites,of which six cysteine sites met the structural characteristics of Classical-OBPs and belonged to Classic-OBPs.4.Cloning and prokaryotic expression of SlitOBP21 geneThe full-length gene of SlitOBP21 was successfully obtained by gene cloning.The soluble target protein was successfully obtained by pET-30 a vector and prokaryotic expression system.The molecular weight was between 15 kDa-25 kDa,which was consistent with the predicted protein molecular weight(about 21.5 kDa).It was verified by Western Blotting that the protein was the required protein,not a hybrid protein.5.The fluorescence competitive binding experiment of SlitOBP21 recombinant protein and honeysuckle essential oil active substance and behavioral verificationFirst,it was confirmed that the SlitOBP21 recombinant protein could bind well to1-NPN,and then the protein-probe complex was used for fluorescence competitive binding experiments.The results showed that SlitOBP21 protein did not bind to Linalool,Phenylethyl alcohol,Benzyl acetate and Ethyl phenylacetate,but only had strong binding ability with α-hexylcinnamaldehyde.The binding constant values was4.43 ± 2.07 μM.The results of behavioral verification showed thatα-hexylcinnamaldehyde had a significant attraction to female adults of S.litura.In summary,this study found that the female adults of S.litura had an EAG response to most plant essential oils,among which honeysuckle essential oil had a significant repellent effect on the female adults of S.litura.Honeysuckle essential oil is composed of eight main components,of which α-hexylcinnamaldehyde can strongly bind to the recombinant protein of SlitOBP21.The final behavioral validation showed that 1000 mg/L of α-hexylcinnamaldehyde can significantly attract female adults of S.litura,and this substance plays a behavioral regulatory role on female adults of S.litura. |
PDF Full Text Request