Identification Of Olfactory Genes And Functional Analysis Of GOBP2 In Clostera Restitura

Clostera restitura Walker(Lepidoptera:Notodontidae)is one of the most destructive defoliators of poplars in China.It has a high oviposision efficiency and hatchability so that can reproduce fastly,with a lacking research background,forecasting is difficult to achieve high accuracy and efficiency,prevention and control strategies are also lack of pertinence,thus,it will be hard to control if it happens.Therefore,it is of great significance to develop new control methods.We carried out the research on the olfactory recognition system of the moth to uncover the survival strategy of the insect which would lay a solid foundation for the effective control of the moth by chemical ecological control.In this research,We constructed an antennal transcriptome using Illumina Hiseq 2000?sequencing and identified large amount of olfactory genes.Characterized the expression profiles of odorant binding proteins and chemosensory proteins using real time fluorescence quantitative method for better understanding of the olfactory receptive system and the role of putative olfactory proteins in C.restitura.Obtained full length of GOBP and PBP genes using RACE amplification.Got recombinant protein by prokaryotic expression and construct functional analysis by fluorescence competitive binding assay,using homology modeling and molecular docking method predicting the ligand binding of GOBPs,further defined the combination characteristic.Finally,EAG analysis was constructed to find out plant volatiles components which have stimulation activity to adults.The main results are as follows:A total of 165 transcripts were identified using Illumina Hiseq 2000?sequencing,including 43 OBPs,13 CSPs,78 ORs,15 IRs,13 GRs,and 3 SNMPs.Sequence analysis of 43OBP genes showed that 27 of them have complete open reading frame with 15?30 signal peptides;Accord with Blastx results and phylogenetic analysis,motif elicitation and enrichment showed that Cres101130,Cres9587,Cres96897 and Cres98953 were confirmed to be CresGOBP1,CresGOBP2,Cres PBP1 and Cres PBP3.All 13 CSP genes identified in the antennae transcriptome contains complete open reading frames and had 4 conserved cysteine sites,which were consistent with the general structural formula of C1-X_6-8-C2-X_18-19-C3-X₂-C4,sequence alignment results showed that the similarity of the CSP gene sequence between C.restitura and other lepidoptera insects were higher than 55%;Motif analysis showed that only the number of motifs showed difference between groups,while the position of the motif was more conservative than that of the OBPs,indicating the conservatism of CSP gene in the evolutionary process.Within the 78 OR identified here,28 of them have completer coding region contain 5?8transmembrane domain;Combined with Blastx alignment and phylogenetic tree analysis,Cres102169 was identified as an Orco,Cres102537?Cres106862?and Cres98888 was identified as PRs.We identified the full length of GOBPs and ORF of other 6 OBP genes which showed gender differences.Sequence analysis results showed that the N-terminal of 8 OBP genes contains signal peptide sequences composed of 17?23 amino acids,and have 6 conserved cysteine sites that accord with general structural formula.8 kinds of OBP showed various expression patterns in different age groups and tissues in adults.The relative expression levels of CresGOBP1?CresGOBP2?Cres OBP9?Cres OBP 10?Cres OBP 16 and Cres OBP21 were significantly higher expressed in antennae than in heads(antennae excluded),wings and legs suggesting their essential role in antennal recognition processes.Cres OBP10 was abundantly expressed in female and male legs which might be involved in the recognition of volatile or low-volatile chemicals emitted from plants.In addition,Cres OBP10 and Cres PBP1 were significantly high expressed in the head of female than that of male,suggesting that Cres OBP10may be involved in host finding activity during female adults oviposition.PBPs had extremely significantly higher expression levels in male antennae indicating that PBPs probably play an important role in male moth perception of sex pheromone components released by female moths while CresGOBP2 in female antennae was higher expressed in males which suggested that GOBPs are involved in the detections of both general odorants and sex pheromones.We identified the full length of six Cres CSP genes,sequence analysis results showed that six Cres CSPs all accord with C1-X6-C2-X18-C3-X2-C4 general formula,q RT-PCR was carried out to identify gene expression profiles of Cres CSP,results showed that six CSP have different expression profiles,Cres CSP4 and Cres CSP5 are in high expression in first instar larvae,Cres CSP1 and Cres CSP2 have high expression level during the 1?3 instar larvae,Cres CSP3expressed higher during 3?4 instar,since 3?4 instar larvae start to disperse for enough food,these Cres CSPs are likely to be involved in insect host recognition.Cres CSP6 expressed abundantly during egg stage,suggested that it may be involved in non-olfactory behaviors such as growth and development.In adults,except for Cres CSP6,the expression levels of all 5 CSP genes reached the peak during 3?4 days after eclosion,indicated that Cres CSP gene might be involved in sex pheromone recognition during calling behavior or mating.The Cres CSP gene expression spectrum in tissues showed that Cres CSP genes were mainly expressed in the antennae,and some Cres CSP genes were also expressed in head,wings and legs.The expression levels of Cres CSP1?Cres CSP4?Cres CSP5 and Cres CSP6 in the antennae of the female were significantly higher than in males,Cres CSP2 and Cres CSP4 highly expressed in male wings,further illustrated the functional diversity of CSPs.High-quality purified recombinant CresGOBP2 was obtained,Western Blot analyses showed single protein bands at 15?18 k Da which determine the target protein to CresGOBP2.Fluorescent competition binding experiment showed that CresGOBP2 has a narrow binding spectrum,it could combine with(E)-2-heptene aldehyde,trans-2,4-sebacic olefine aldehyde,benzoic acid,beta ionone,dibutyl phthalate and diisobutyl phthalate within 29 volatiles,and the binding ability with dibutyl phthalate and diisobutyl phthalate is strongest(Ki=10.92?M,9.75?M).We predicted the structure of CresGOBPs by homology modeling,analyzed the interaction of CresGOBPs with 6 ligands by molecular docking.Results showed that both CresGOBP1 and CresGOBP2 shared high similarity with GOBP2 from Bombyx mori,assessment results indicated that two GOBP structure is reasonable.Docking results showed that both two CresGOBPs could bind 1-NPN(Total score>4.0)and showed highest docking scores with dibutyl phthalate and diisobutyl phthalate(Total score>6.5);in addition,CresGOBP1?Dibutyl phthalate?CresGOBP1?Diisobutyl phthalate and CresGOBP1?Trans-2-Heptenal showed highest CScore(CScore=5),CresGOBP2?Beta-ionone had the highest CScores(Cscore=5);During the docking process,both two CresGOBPs formed hydrogen bonds by THR 9,TRP 37 and SER 56.Therefore,it can be preliminarily concluded that the above three amino residues are the key amino acid sites for CresGOBP to interact with odor molecules.We tested the electro-antennogram responses(EAG)for male and female adults to 14 host plant volatiles,results showed that both male and female antennae reacted to(E)-2-Heptenal?Cis-3-Hexen-1-ol?hexanal?Cis-3-Hexenyl acetate?2-Hydroxybenzaldehyde and Trans-2,4-Decadienal strongly,that was to say C.restitura adults tend to react to aldehydes?alcohols and esters with small molecular weight within host volatiles,indicated these compounds involved in host recognition.Under 0.2?g/?L concentration stimulation,females response stronger to most of the volatiles than males,especially to dibutyl phthalate and diisobutyl phthalate,which further verified the binding capacity of CresGOBP2 at the level of electrophysiological.