Biological Activity And OBP Identification Of Crematogaster Rogenhoferi On The Secretion Of Volatiles From Parasaissetia Nigra Nietner

Crematogaster rogenhoferi is one of the dominant species of predatory natural enemies of the Parasaissetia nigra Nietner.In the field,C.rogenhoferi search for P.nigra Nietner by sense of smell,and prey on its first-age nymphs.It has a good control effect on the population of P.nigra Nietner.Olfaction was proved to plays a vital role in hymenoptera insect behaviors such as mating and foraging.However,the specific function of the odor binding protein in C.rogenhoferi is still unclear.The identification and expression of related protein genes are helpful to the study of olfactory mechanism.In this paper,the antennae transcriptome of acanthopterygii was constructed,using spatiotemporal expression of CrogOBPs gene,behavior research,gene cloning,construction of recombinant expression vector,prokaryotic expression and purification of recombinant protein.The results are as follows:1.Olfactory behavior test and antennal electrophysiology test showed that Hexadecane,pentadecane,pyrene,3,5-ditert-butyl-4-hydroxybenzaldehyde in 18 standard reagents all have attractive effects.Different concentrations of compounds had different attracting effects on C.rogenhoferi.Hexadecane,pentadecane,pyrene had the best attracting effect at 0.10 mg/mL。2.The antennal transcriptome sequencing of the C.rogenhoferi was performed on HiSeq X-ten,the transcripts were clustered and assembled into 53,892 unigenes with a mean length of 904 bp.16,074(99.31%)of which had significant matches in NR database.Unigenes of C.rogenhoferi can bedivided into 26 functional groups according to KOG functional annotations,the cluster for "General function prediction only" was the largest group(383,13.85%),followed by the "Translation,ribosomal structure and biogenesis" group(325,11.75%).For further selecting and blasting,Identified 3 sensory neuron membrane proteins,4 odor binding proteins,3 chemosensory proteins,37 olfactory receptors and 2 ionic receptors.3.Among the identified 4 OBP sequences,4 unigenes had fulllength ORFs,with 6 conserved cysteine residues and n-terminus signal peptide sequences formed 17 to 23 amino acids.4.The expression patterns of CrogOBPs were analyzed by fluorescence quantitative RT-PCR.It was highly expressed in the antennaewhich was significantly higher than other tissues.The antennae-specific or enriched expressed OBPs may play key roles in host location.5.We successfully expressed one CrogOBP proteins in prokaryotic expression systemRecombinant plasmids(pET-30a(+)-CrogOBP3)was successfully Constructed.The expression conditions of pET-30a(+)-CrogOBP3 were:temperature 30℃,IPTG concentration 0.5mmoL/L,time 6h,It was expressed in inclusion bodies.After denaturation and renaturation of the inclusions,and histidine tags was purified by the immobilized metal nickel chelate affinity chromatography.The target protein of CrogOBP3 was obtained at the concentration of 200mmoL/L imidazole.