Molecular Cloning And Functional Analysis Of Pheromone Binding Proteins In Carposina Sasakii(Carposinidae)

The peach fruit moth,Carposina sasakii Matsumura(Lepidoptera:Carposinidae),is one of the most destructive and common fruit-boring pest in orchards in north China.It is difficult for prevention due to the characteristic of wide distribution,strong concealment and lack of natural enemies.Chemical pesticides are mainly used tocontrol or reduce the pests to occur effectively in production.Sex attractants and other attractants are widely used because of their high efficiency,specificity,security to beneficial insects,and environmental friendly control in integrated pest management.Sex pheromone and host volatiles play an important role in attractingpests.Insects using their olfactory system locate host for food and shelters,seek spouses and avoid predators,and the combination of host volatiles and sex pheromones can significantly enhance the effect of attractants.Therefore it is an important study for the olfactory proteins to peach fruit moth,in addition,we investigates the differential genes between male and female,so as to provide the basic theory for the development of effective attractant and the population control.Firstly,transcriptome sequencing for male and female antennae were conducted using second-generation sequencing,then a large gene family in olfactory system were annotated and identified by bioinformatics.Lastly,we primarily studied the functions of 3 PBPs and 2 GOBPs genes,and investigated the expression file of them.The primary results are as follows:1.Antennal transcriptome analysis and verification of the chemosensory gene families in Carposina sasakiiIn this study,the Illumina HiSeq4000 was used for high-throughput sequencing,and we identified several kinds of putative olfactory genes including 29 OBPs,13CSPs,520Rs in C.sasakii antennae by bioinformatics,and investigated the expression patterns of these genes via fluorescence quantitative real-time PCR.The alignment of amino acid sequence showed that 19 OBPs belong to the classical OBPs subgroup with the motif "C1-X15-39-C2-X3-C3-X21-44-C4-X7-12-C5-Xs-C6".In addition,5 OBPs(OBP1,OBP4,OBP9,OBP20,OBP22)have no signal peptides.Surprisingly,the phylogenetic analysis demonstrated GOBP3 was clustered into GOBP1 subgroup,and further analysis of homology matrix indicated that sequence identity among each GOBP1 shared an average of 65.2%identity.This means GOBP3 may play a similar function with GOBP1.Fluorescent quantitative analysis showed that OBP7 was specifically expressed in both female and male antennae,suggesting that it may play an important role in the recognition of pheromones,finding host plants and locating oviposition sites.Other OBPs(OBP7,OBP9,OBP12,OBP15,OBP19,GOBP3,GOBP1,PBP2)were expressed in other tissues except for high expression in antennae.All of 13 CSPs,with complete ORF and signal peptide,have four highly conserved cysteine residues and fit the "CSP sequence motif'.Only CSP5 were specifically expressed in the antennae of both sexes,which indicated that CSP5 may play a key role in the process of recognition.However,there was a more or less expression existing in other tissues.It was worth noting that CSP1 and CSP12 were highly expressed in the abdomen,indicating that it might be involved in the growth and development for C.sasakii.All of 52 ORs were predicted in view of ORs sequence characteristics.The results indicated that all of them owned 2-7 transmembrane domains and no signal peptide in the N terminal.We detected 14 ORs(OR3,OR4,OR8,OR17,OR21,OR24,OR30,OR31,OR33,OR34,OR41,OR46,OR48,OR49)successfully by quantitative RT-PCR.The results showed that some candidate ORs were expressed not only in the olfactory organ,but also in the non-olfactory organs.The expression levels of 9 ORs(OR4,OR17,OR21,OR24,OR34,OR46,OR48,OR49,GR8)were significantly higher in female antennae than that in male antennae,which showed that these genes may be involved in the locating the host and oviposition sites;In addition,4ORs(0R8,OR30,OR31 and OR33)were significantly overexpressed in the male antenna,indicating that these genes may play a vital role in the recognition of pheromones.2.Molecular cloning and functional analysis of PBPs and GOBPsFirst of all,RACE-PCR was used to clone PBPs and GOBPs gene,and the results of qRT-PCR and western blot showed that the expression of PBPs and GOBPs genes in maleantennae was significantly higher than females.Fluorescence competitive binding experiment indicated that all of these proteins(except for GOBP2)showed stronger binding affinities to Z-7-eicosene-11-one and Z-7-nonadecene-11-one,while only PBP3 exhibited specific affinity towards the two sex pheromone components.CsasPBP1-2 exhibited strong affinity towards to a number of apple volatiles;GOBP1-2 can specifically bind to host volatiles.It is noteworthy that they have strong affinity with esters and alkenes compounds.Further analysis showed that most of them exhibited weaker binding affinities to linear aliphatic alcohols and ketones of approximately 6-carbon chain length.This indicates that the existence of unsaturated double bonds in carbon chains and the length of carbon chains may significantly affect the binding ability between proteins and compounds.