The Annotation Of Odorant Binding Proteins And The Function Research Of Bdor BdorOBP56f-2 In Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel)(Diptera,Trypetidae)is one of the most destructive fruit attacking pests,which causes huge economic losses to the world fruit and vegetable industry.It is a great challenge to prevent and control this pest,because of their high fecundity,strong adaptability and high level of insecticide resistance.At present,luring and killing is the most effective control method against B.dorsalis.So far,the main component of the widely used commercial attractant is methyl eugenol(ME).Although ME has been used as the attractant for B.dorsalis for nearly 70 years,the molecular mechanism on the perception is still unknown.Odorant binding proteins(OBPs)are important proteins in olfactory perception in insect which bind odorants and transport them to olfactory neurons.Although OBPs play an important role in the olfactory physiological processes,the information of OBPs in terms of quantity,diversity and expression pattern is still fragmented in B.dorsalis.In this study,we used genome and transcriptome information to identify and annotate the OBPs of the oriental fruit fly.As a result,we expanded the number of Bdor OBPs annotated to 49,and renamed these BdorOBPs following Drosophila melanogaster orthologs.Based on the phylogenetic analysis and multiple sequence alignment,BdorOBPs were divided into four subfamilies.The expression profiles of BdorOBPs gene in different tissues(including antennae,feet,wings,cuticles of the head,thorax,and abdomen,the fat body,midgut,Malpighian tubule,testis,and ovary)were analyzed by qPCR.Then,BdorOBP56f-2 protein which has been reported to be associated with precepting ME was heterologously expressed in Escherichia coli.The binding ability was measured in vitro to prove that BdorOBP56f-2 could combine with ME and other odorants.In addition,CRISPR/Cas9 was applied to knock out BdorOBP56f-2.After the deletion of this gene,the the mutant flies showed decreased response to ME.In the current study,we conducted a comprehensive identification and annotatin of the OBPs of B.dorsalis at the genomic level.Furthermore,we confirmed that BdorOBP56f-2 is responsible for the perception of ME in this fly by a variety of techniques.Our results will not only enrich the understanding of the physiological functions of the OBPs of B.dorsalis,but also provide a paradigm for the olfactory mechanism study in other species.Moreover,it may provide a theoretical basis and new target for the development of new attractantsagainst B.dorsalis.The main findings are as follows:1 Identification of BdorOBPs and temporal expression profiling.The BdorOBPs were identified based on the genome and transcriptome data.The number of annotated Bdor OBPs was expanded to 49,and renamed these BdorOBPs following D.melanogaster orthologs.They were divided into four subfamilies including classical OBPs,Minus-C OBPs,Plus-C OBPs and Atypical OBPs based on the phylogenetic and multiple sequence alignment analysis.There are 33 Classic OBPs,4 plus-C OBPs,10 Minus-C OBPs and 2 AtypicalOBPs.The expression levels of BdorOBPs in different body segments(antennae,feet,wings,cuticles of the head,thorax,and abdomen)and internal tissues(fat body,midgut,Malpighian tubule,testis,ovary)were analyzed.There were 21 BdorOBPs showed significant high expression in the antennae,head cuticle and legs.Bdorobp56f-1 and bdorobp47a-2 have a high transcriptional level in the abdominal cuticle.The expression of BdorOBPs in external tissues was generally higher than all internal tissues.Among them,19 BdorOBPs expressed significantly highly in fat body,while 4 BdorOBPs showed high expression in the reproductive organs.2 The binding ability of BdorOBP56f-2 with ME and other odorants.The recombinant protein of BdorOBP56f-2 was obtained by the prokaryotic expression system of E.coli.The expression vector PET28 a was utilized to construct the recombinant plasmids.It was transformed into E.coli strain BL21(DE3)and then induced by IPTG.After ultrasonic crushing,protein was extracted.Subsequently,the recombinant protein was purified by a nickel column and examined by an SDS-PAGE electrophoresis.The recombinant protein showed a single band of about 15 kDa on the gel.The specificity of the recombinant protein was further verified by Western-blot.Furthermore,the affinity of BdorOBP56f-2 to odorants was determined by microscale thermophoresis(MST)in vitro.As the result indicated,BdorOBP56f-2 showed strong binding in a dose dependent manner to 3 odorants,including ME,4-Carvomenthenol,and trans-2-henxenal.3 The roles of BdorOBP56f-2 in chemical perception in B.dorsalis.BdorOBP56f-2 was knocked out by using CRISPR/Cas9 system.Based on our genotyping screening,there were 4 mutant individuals in the parent generation(G0).Hybridizing with wild type,we obtained 7 types of heterozygotes mutant in F2.By sequencing,we confirmed that 1-9 bp indel occurring at the target site of BdorOBP56f-2 gene.Among them,the mutant strain m5-m1 with 4 base deletion was adopted for subsequent physiological and behavioral studies.Based on the behavioral assays,the attraction rate of ME decreased 30% after BdorOBP56f-2 knock out.However,the selectivity of 4-Carvomenthenol,trans-2-henxenal did not change significantly.While though the electrophysiological data,the heterozygous individuals showed no significant change compared with wild type flies.Our results confirmed that BdorOBP56f-2 was associated with the perception of ME in B.dorsalis.