Cloning,Expression And Functional Analysis Of The Olfactory Co-receptor Orco In Drosophila Suzukii

Drosophila suzukii?Matsumura,1931?is a new polyphagous pest devastating approximate 60 diverse fruit hosts distributed among 18 different plant families..D.suzukii females possess a prominent serrated ovipositor that allow them to lay eggs inside unblemished ripening fruit like cherry,blueberry,strawberry,persimmon etc.The visible physical damage directly caused by external piercing,internal larval feeding and oviposition wounds rendering contaminated fruits unmarketable.The olfactory system is an important characteristic in the life activities of D.suzukii including foraging,courting and spawning behaviors.Moreover,the Orco receptor can transport the traditional odour receptor to the surface of dendritic membrane of olfactory neurons and maintain its conformation stability.Exploring the expression and function of Orco receptor in D.suzukii is important to develop new pest management strategies from the view of olfactory system.In this paper,PCR and RACE techniques were used to clone the Orco gene of D.suzukii and its bioinformatics analysis,then real-time fluorescence quantitative PCR technique was used to analyze the expression levels in different stages and tissues of D.suzukii.The function of Orco gene in D.suzukii was studied by RNA interference technique.The main results are as follows:1.The cloned Orco gene of D.suzukii was 1461 bp in length,which was the same as that obtained by NCBI?accession No:NM₀01328601?.Bioinformatics analysis showed that the gene encoding a protein of including 478 amino acids in length,molecular weight with 54271.14 Da and an isoelectric point of 8.69;the subcellular of the protein may distribute mainly in the endoplasmic reticulum;there is a seven-segment transmembrane structure and no shear site in the amino acid sequence of this gene;this protein belongs to hydrophobic protein,but does not belong to sretory protein.Protein conserved domain analysis showed that at the 70-476 position of the Orco gene of D.suzukii of the amino acid residues had a Pfam 7tm₆ domain.The sondary structure of protein mainly included a helix,and the tertiary structure is mainly composed of a helix with random winding folding.2.Fluorescence quantitative PCR was used to analyze the expression of different tissues and developmental stages of D.suzukii.The results showed that the D.suzukii Orco is mainly expressed in antenna,and the male was higher than that of female.In addition,there is also a small amount of expression in the foot,abdomen and ovipositors.The results of different developmental stages showed that Orco of the D.suzukii was expressed in eggs,3 instar larvae,pupae and adults,but the expression level at adult stage was significantly higher than that at other periods,which was 12 times than that at the egg stage.3.Microinjection approach has been applied to introduce dsRNA molecules for Dsuz\Orco into the abdomen of D.suzukii that have emerged for 24h.Fluorescence quantitative PCR was used to monitor the expression of Orco in D.suzukii after 12 h,24 h and 36 h intervals.The results showed that the expression of Orco gene was significantly decreased during 12 h to 24 h,indicating that dsRNA was silent on the expression of Orco gene in the D.suzukii,In addition,behavioral experiments also showed that the ability of finding food source in D.suzukii was greatly reduced,indicating that it had an effect on its search host.It was proved that this gene was related to olfactory sense of D.suzukii,and silencing could damage the olfactory ability of D.suzukii.