Preliminary Study On The Function Of Highly Expressed Odorant Binding Proteins SlitOBP11 And SlitOBP23 In Spodoptera Litura Larvae

Odor-binding proteins are the first step to participate in the biochemical reaction of insect olfactory recognition of odorants,reverse chemical ecology uses OBPs as the target to screen pest behavioral active substances,according to the preference of insects for specific odors,attractants can be designed to attract pests.S.litura is an omnivorous pest,which is harmful to larvae.Therefore,controlling S.litura during its larval stage can not only reduce losses but also achieve better control effects.If the binding mechanism between S.litura odorant binding protein and ligand can be clarified,it will provide new targets for the development of new and high-efficiency attractants using OBPs to screen and pest control strategies based on olfactory regulation.To this end,this study based on the published data of the antennae transcriptome of the adult and larvae of S.litura,two OBPs genes,SlitOBP11 and SlitOBP23,were analyzed,screened and cloned.The expression of OBPs gene were detected and analyzed by real-time fluorescence quantitative PCR;SlitOBP11 and SlitOBP23 proteins were successfully expressed and purified;fluorescence competition binding assay was used to analyze the binding ability of SlitOBP11 and SlitOBP23 with plant odorants and sex pheromone components;the double selection method was used to preliminarily screen the active substances that can cause the larval behavioral response of S.litura;the binding site of SlitOBP11 and ligand was analyzed through computer homology modeling and molecular docking.The main findings are as follows:1.Sequence analysis and expression profile analysis of SlitOBP11 and SlitOBP23 of S.lituraThe open reading frames of SlitOBP11 and SlitOBP23 are 465 bp and 438 bp,encoding 155 and 146 amino acid,respectively.SlitOBP11 has a signal peptide of 23 amino acid residues,and SlitOBP23 has a signal peptide of 24 amino acid residues.Fluorescence quantitative results showed that the two OBPs genes were expressed in each instar of S.litura larvae,and in various tissues of adult male and female,and the expression levels were also different.From the perspective of the expression level of the antennae,both SlitOBP11 and SlitOBP23 are highly expressed in the larval antennae;from the location point of view,SlitOBP11 has a higher expression level in the head of the male,while the expression of SlitOBP23 is higher in the antennae and foot parts of the female and male.From the perspective of age,SlitOBP11 has the highest expression in the first age,and the expression in other ages is similar.The expression level of SlitOBP23 increased at the 3rd age,and the highest at the 4th and 5th ages,and began to decrease at the 6th age.2.Expression of SlitOBP11 and SlitOBP23 proteins and screening of active substances in S.lituraThe prokaryotic expression system was used to successfully obtain SlitOBP11 and SlitOBP23 soluble proteins,and the results of competitive binding experiments showed that SlitOBP11 sex pheromone has a strong binding ability,and the binding constant values are Z9,E11-14: Ac Ki=13.88±1.4412 μM,Z9,E12-14: Ac Ki=14.40±0.3173 μM,Z9-14: Ac Ki=9.97±0.4923 μM,E11-14: Ac Ki=13.53±0.4789 μM.The binding constant Ki of SlitOBP23 and the tested odorants were both more than 50 μM,and no binding ability was detected.In order to further verify the attracting function of sex pheromone to S.litura larvae,we added sex pheromone Z9,E11-14: Ac,Z9,E12-14: Ac,Z9-14: Ac,E11-14:Ac to the food,it is found that the food added with four sex pheromone alone having obvious attracting effect on the first instar larvae.3.Analysis of the binding sites of SlitOBP11 and ligands of S.lituraSlitOBP11 has 6 α-helices and three disulfide bonds,which is a typical OBPs structure.The molecular docking results showed that the six α-helices of OBP11 formed an open hydrophobic binding cavity,and there was a possibility that the ligand containing polar atoms could form hydrogen bonds with the binding cavity.After removing the signal peptide,the 5th glutamine Gln5,the 9th asparagine Asn99 and the123 rd asparagine Asn123 may be potential sites for the formation of hydrogen bonds,especially the amino acid residue Asn99 located deep in the cavity,which may be the most critical amino acid in ligand binding.