Identification Of The Sex Pheromone Component Ratio And Binding Proteins Of Conogethes Punctiferails(Lepidoptera:Crambidae) In Chongqing

Yellow peach moth,Conogethes punctiferalis(Lepidoptera: Crambidae),is an important agricultural insect pest.Larvae of this species are typically ployphagous pests of a number of crops and fruits.Larvae habit of boring feeding makes this species of insect hard to control with traditional ways such as chemical spray.As an alternative,pheromone techniques has been employed in agricultural practice.Within an insect species,sex pheromone has geographical differences which is usually expressed as different ratios of a same series of components.Geographical ratio of the pheromone components for Chongqing population of C.punctiferalis is yet to be identified.Besides,pheromone binding proteins are yet to be investigated as well.In this study,Gas Chromatography(GC),Gas Chromatography and Mass Spectrometry(GC-MS)were employed to analyze sex pheromone components in crude pheromone extracts of the insect.Then a change of pheromone binding proteins(PBP)expression were investigated with q RT-PCR.Finally,a fluorescence competition binding assay was conducted to test the binding ability of PBP.Results are shown as following.1 Identification of the ratio of sex pheromone components in C.punctiferalis.Two components,E/Z 10-16: Ald,were identified from the extracts of sex glands of female C.punctiferalis with GC and GC-MS.Amounts of the components were quantified as E10-16: Ald,5.752 ±1.455 ng per female,and Zl0-16: Ald,0.247±0.023 ng per female.A ratio of E/Z components was approximatetly 95.8:4.2.2 Cloning and sequence analysis of three PBP genes of C.punctiferalis.Based on information of an antennae transcriptome of C.punctiferalis,the full length of the three PBP genes was cloned.The accession numbers were MH006604,KP027486.1,and KP985229.1 respectively.An open reading frame length for three genes ranges from 486 bp to 516 bp,which encodes 161-171 amino acids with a predicted molecular weight of approximately 18 k Da.Amino acid sequences of 3 genes contain approximately 20 signal peptides and belong to the classical OBP(with 6 conserved cysteine ? ? sites between them,and regular appearance of numbers of gapped cysteines).Sequence similarity to other relative lepidopteran insects was ordered as PBP1>PBP2>PBP3.Lipophilic analysis showed that PBP1 and PBP2 have two distinct binding sites.While,no binding site was found in PBP3.Phylogenetic analysis showed that PBP1 and the other two PBP belonged to different branches.Among 3 PBP,PBP1 is closely related to the GOBP of Bombyx mori,and PBP2 is similar to those of C.medinalis.3 Expression Rhythm and Sex Pheromones Induction of PBP genes.No distinct circadian rhythms for 3 genes were found with q RT-PCR.However,after male moth emergency,expression of 3 genes showed increases at the beginning and then decreases with age increase of male moth.Highest expression for PBP1 was 4d after males emergency,and for PBP2 and PBP3,2 days.With pheromone treatments,no obvious changes in expression of 3 genes were found at most time points within 24 hours.While,expression of PBP1 and PBP2 was down regulated by E10-16: Ald treatment,at several time points.And Z10-16: Ald lead to an up expression for PBP1 six hours after the treatment.4 The prokaryotic expression and protein binding properties of PBP.The three PBP genes were constructed into p ET-30a(+)expression vector and successfully expressed in E.coli after induced by IPTG.All genes could be expressed as soluble proteins after sonication.Nickel ion affinity chromatography and concentration were used to obtain purified proteins with a certain concentration.Fluorescence competition binding assays showed that PBP1 was able to strongly bind two sex pheromone components in vitro,and the binding constants was9.267±2.156 and 8.656±1.321 respectively,which indicates that PBP1 was probably involved in binding pheromone components in C.punctiferalis.And the binding constants of PBP2 and PBP3 for both pheromone components were higher than 50,indicating no binding ability.In this study,the ratio of female sex pheromone components and the PBP of males were identified.Results of the study will help to understand the geographical specificity of the sex pheromone of C.punctiferalis in Chongqing.It will also benefits further study on pheromone communication mechanisms in this species.