Identification And Expression System Construction Of Carposina Sasakii Odorant Receptor 45 Gene

Carposina sasakii is a serious fruit-boring pest and harmful to many fruit trees,including apple,jujube,and pear,hawthorn,peach,plum,apricot,sour jujube and other fruit trees,causing great economic losses.Traditional chemical control has resulted in high residue levels due to insecticide abuse,affecting the environment and fruit safety.The technical solution of applying specific semi-chemicals to regulate insect behavior and control insect damage has the advantages of being specific and efficient,residue-free,environmentally friendly and safe for fruit,which is an important development trend of integrated pest management.The recognition of specific semi-chemicals by insects mainly relies on the olfactory system,in which the odorant receptors play core role in the process of compound recognition.The authors found that the OR45 of C.sasakii has high protein sequence similarity with the OR3 of Epiphyas postvittana,which may be related to the specific recognition of plant-derived volatile,monoterpene citral.A series of studies on OR45 were carried out in order to lay a foundation for the analysis of host recognition mechanism and development of behavioral regulators in C.sasakii.The main findings are as follows:The specific primers were designed and synthesized refer to or45 from the Gen Bank database.The total c DNA was obtained by reverse transcription of the total RNA of adult C.sasakii antennae,and the full-length of or45 was cloned using c DNA as a template.After sequencing the cloned vector containing or45,the gene sequence showed 99.8% similarity to the C.sasakii or45 nucleotide sequence existed in the NCBI database.Bioinformatics analysis revealed that the OR45 protein contains 425 amino acid residues and has a relative molecular mass of 49.6 k D;the theoretical p I(isoelectric point)is 8.63,which is a basic protein;the GRAVY(Grand average of hydropathicity)is 0.577,which is a hydrophobic protein;there is a N-glycosylation site and no O-glycosylation site;and there are 27 potential phosphorylation sites.The OR45 protein has 7 transmembrane domains,which is typical of It is a typical odorant receptor.The qPCR primers were designed,the qPCR technology was applied to study the differential expression of or45 in antennae of C.sasakii adult of different sexes and different developmental stages,using β-actin as the internal reference gene.And the results showed that the or45 was differentially expressed in antennae of different sexes and different developmental stages after emerging.The highest expression of or45 was found in the antennae of mated female adults after emerging for 24 h.After analysis of the structure of the OR45 receptor,the peptide,CLLGKPDQIWDKRLP,of C.sasakii OR45 was selected for synthesis and used as an antigen to prepare a polyclonal antibody,which titer is higher than 1:512000 measured by ELISA.Immunohistochemical technique was applied to localize OR45 in antennae among different sexes and developmental stages of C.sasakii.The result shows that OR45 is expressed at the base of sensilla coeloconica.In this study,the gene of or45 was cloned,and we verified its gene expression pattern and tissue expression site,which provides a theoretical foundation for the next study on the biological function of OR45 and the application of specific semi-chemicals to regulate the behavior of C.sasakii to control its damage.