Gene Cloning, Tissue Distribution And Ligand-Binding Affinity Analysis Of Odorant Binding Proteins In The Beet Army Worm, Spodoptera Exigua(Lepidoptera:Noctuidae)

In the long-term evolutionary process, insects evolved a highly sensitive olfactory system to percept volatile compounds in the environments, and accordingly respond with physiological behavioral responses such as finding mates, food, and habitat. In the process of olfaction, odorant binding proteins (OBPs) function to bind and transport odorants to odorant receptors, which is thought as the first biochemical step of olfaction. Spodoptera exigua (Hiibner) is a worldwide important agricultural pest, and the study on OBPs of S. exigua is not only helpful for the efficient use of behavioral manipulation technique, but also can provide potential targets for the development of new behavioral control techniques. Previously, S. exigua pheromone binding proteins (PBPs) and general odorant binding proteins (GOBPs) have been cloned and analysed in our laboratory. In this study,8new OBP genes from S. exigua were further obtained by transcriptome data analysis and molecular cloning, their tissue expression patterns were determined, and finally the physiological functions were preliminarily explored by fluorescence competitive binding experiments. The main results are as follows:1Gene cloning and sequence analysis of SexigOBPsBy transcriptomic analysis and RACE technology,8full-length cDNAs of OBPs from S. exigua was identified, which were named as SexigOBPl, SexigOBP2, SexigOBP3, SexigOBP4, SexigOBP5, SexigOBP6, SexigOBP7and SexigABP2, respectively. Sequence analysis showed that all OBPs beared the characteristics of typical OBPs, including six conserved cysteine sites except for SexigOBP2(only four conserved cysteine sites). The amino acid sequences of SexigOBP1, SexigOBP2, SexigOBP4, SexigOBP5and SexigABP2had high similarities of88%,70%,82%,88%,72%, respectively, with reported OBPs of other Lepidopteran insects, while SexigOBP3, SexigOBP6and SexigOBP7shared low similarities with other OBPs, of44%,54%and52%, respectively.2Expression patterns of SexigOBPs at different development stage and in different adult tissuesThe expression levels of SexigOBP1, SexigOBP2, SexigOBP7and SexigABP2in different tissue of the larvae and adults were examined by RT-qPCR. The results showed that in the larval stage, four OBPs expressed mainly in the head. Among four genes, SexigOBP2expressed in the highest level, followed by SexigOBPl, SexigOBP7and SexigABP2were the lowest. In the adult stage, SexigOBP7and SexigABP2mainly expressed in antennae, with little or no expression in other tissues; but SexigOBP1and SexigOBP2expressed in various tissues, especially expressed at highly levels in the legs and wings. The highly expression in antennae and other chemosensory tissues suggests roles of these OBPs in the olfaction and gustation of S. exigua.3Prokaryotic expression and purification of SexigOBPsAs above results showed, SexigOBP1and SexigOBP2were highly express in larval stage, and therefor may play role in the larval olfaction. To provide evidence, the two OBPs and SexigABP2were expressed and purified using a prokaryotic expression system. Three genes were firstly built into the pET-30a(+) expression vector, then expressed in Escherichia coli with induction by IPTG. SDS-PAGE analysis showed that there was obvious bold band of expected size of about19kDa,18kDa and22kDa of pET/SexigOBPl, pET/SexigOBP2and pET/SexigABP2, respectively. Recombinant proteins were purified mainly through nickel ion affinity chromatography and enterokinase hydrolysis, from which the His-tag was removed.4Ligand-binding affinity analyses of SexigOBPsThe binding affinities of three SexigOBPs with odorant compounds were tested by fluorescence competitive binding experiments. The results showed that SexigOBP1and SexigOBP2which were highly expressed in larvae stage diplayed strong binding affinities with compounds of long carbon chain (8C) with functional group of hydroxyl, carbonyl and ester. SexigOBP1was suggested to play role in percepption of β-caryophyllene, acetophenone and other plant volatiles in the larvae stage. SexigABP2which was highly expressed in adult, exhibited the higher binding affinities with long carbon-chain compounds with unsaturated bond than short carbon-chain compounds without unsaturated bond, and might be involved in adult perception of farnesol and other plant volatile compounds.