Identification And Characterization Of Three Pheromone Binding Proteins In The Legume Pod Borer, Maruca Vitrata Fabricius (Lepidoptera: Crambidae)

Insects can continuously thrive in the complicated natural environment, the key factor is they have a sense of olfaction system. They perceive the phemones which released by interspecies or enermies to locate spouse or avoid predators; they can accept plant volatiles to locate host, find food or oviposition sites. The odorant binding protein family plays a key role in the olfaction perceviton. According to the characteristics of OBPs from Lepidoptera are divided into GOBPs and PBPs. PBPs become the best solvation in the process of locate spouse.Maruca vitrata is the worldwide agriculture pests which main make large damage to leguminous plants. It mainly distributed in tropical and subtropical regions. It is famous for its larvae drill decay behavior, mainly harms leguminous plants flowers and pods. According to the data of transcriptome sequencing from antenna, we get three PBPs genes by bioinformatics and molecular biology method, analyse the genes expression level of tissues and whole body, then induced and purified target proteins in vitro to explore its function. The main results are as follows:1. Gene Cloning, sequence and phylogenetic analysis of MvitPBPsWe clone three PBPs, which named as MvitPBP1, MvitPBP2, MvitPBP3, the lengh of ORF are 495,501,519 bp respectively. Multiple sequence alignment shows MvitPBP1 is similar to BmorPBP1 with identity values of 47.27%, MvitPBP2 is similar to AtraPBP2 with identity values of 48.82%, MvitPBP3 is similar to ApolPBP3 with identity values of 43.03%. The neighbor-jointing tree of these PBP sequences revealed that MvitPBP1 was clustered with Chilo suppressalis CsupPBP1, MvitPBP2 was clustered with Ostrinia nubilalis OnubPBP4, MvitPBP3 was clustered with Cnaphalocrocis medinalis CmedPBP2.2. Expression patterns of MvitPBPsWe use qRT-PCR method to measure the transcription abundance of three MvitPBP genes (MvitPBP1, MvitPBP2 and MvitPBP3) in adult tissues and whole body. The express profile prove that all of three genes are highly expressed in antennae than in other tissues and the expression level of three genes are also sex-biased. MvitPBP1 is specially expressed in male body, this indicate that the gene may have a importance role for locating spouse; MvitPBP2 and MvitPBP3 are female-biased, suggests have other functions in the olfaction of M.vitrata.3. Prokaryotic expression and purification of MvitPBPsFor explore MvitPBPs function, we express target proteins in vitro. Firstly we built recombinant expression vector pET32a-PBPs, transformed into competent E. coli BL21, then induced by IPTG, obtained the supernatant, purified with Ni ion affinity chromatography, removed His-tag by enterokinase. SDS-PAGE analysis showed that there was obvious bold band of expected size of about 27 kDa for pET32a-PBPs.4. Fluorescence displacement binding assayFirst the binding affinities of three MvitPBPs with four sex pheromone were tested, the results showded that all MvitPBPs have robust affinities to pheromone components, MvitPBPl and MvitPBP3 strongly binding E10E12-16:Aid, MvitPBP2 have most affinities to E10-16:A1d. This indicated that those two components are the impotant element and MvitPBPs may have highly affinities to aldehyde.Then we also tested the the binding affinities to nineteen floral volatiles.This suggested that MvitPBP1 and MvitPBP2 can weakly bind with volatile, showed no obvious difference, while MvitPBP3 have robust affinities to Butanoic acid octyl ester, which have consistency with the previous concequence that MvitGOBP2 have strongly affinities to Butanoic acid octyl ester, this indicated that MvitPBP3 may plays a key role not only in the recognition of pheromone, but also the location of host,In conclusion, function of MvitPBPs from Maruca vitrata is similar to other insects of Lepidopteron. They all have a key role in sex communication, especially for MvitPBP1. MvitPBP3 have strongest affinity to Butanoic acid octyl ester, which is the commonents of cowpea flowers volatiles, indicats that it can help finding host or oviposition for Maruca vitrata.