Functional Analysis Of Odorant Receptor Or1 And Or2 From Apis Cerana Cerana

The insect olfactory is a complex chemical signaling process involving multiple protein molecules.It plays a vital role in many physiological activities,such as finding,foraging,mating,spawning,population management and so on.Apis cerana cerana is a unique honeybee species in China.Its highly sensitive olfactory system can identify chemical signals within the group in the complex environment and distinguish the specific smell odors emanating from the food source.The behavior of Apis cerana cerana recognizing odor molecules is closely related to the function of related proteins in the olfactory system.In this study,AcerOrl and AcerOr2 were selected as the research object,and their potential biological functions in the Ca2+/CaM/CaMK? signaling pathway and the regulation of drone sperm motility were explored in vitro and in vivo.The main results of the present study are as follows:(1)The function of AcerOr1 and AcerOr2 in Apis cerana cerana was preliminarily explored by RNAi mediated by feeding specific dsRNA.The results showed that the mRNA and protein expression levels of AcerOrl and AcerOr2 were significantly decreased by dsRNA.The best silencing effects of AcerOrl was 218bp short chain dsRNA,while AcerOr2 silencing effects was 471bp long chain dsRNA.After feeding the dsRNA,on the mRNA expression of the target gene was inhibited on the 4th day,and the inhibition efficiency of RNAi continued to 9th day,and the effect was still obvious.The results of Electroantennogram showed that after disturbing AcerOrl and AcerOr2 also affected the sensitivity of the antennae to odorants such as VUAA1,eugenol and 1-nonanol,indicating that the absence of expression of these two genes could seriously affect the recognition of odorants in Apis cerana cerana.The functions of AcerOrl and AcerOr2 were preliminarily verified.(2)The functions of AcerOrl and AcerOr2 in heterogenous cells were analyzed by constructing eukaryotic expression vectors and transfected into Sf9 cells.Protein sequence prediction revealed that the secondary structure of AcerOr2 protein contains CaM binding sites and Tyr residues which associated with ion channels.However,the protein secondary structure of the AcerOrl protein doe not contain calmodulin binding sites and gated ion channels.After the eukaryotic expression vector was transfected into Sf9 cells,we found AcerOrl binds both Sf9 endogenous Orco and AcerOr2 to form a heterodimer with odorant ligand-gated channels on the Sf9 cell membrane(ORX+Orco heteromeric Complex),which can cause Ca2+influx when cells are stimulated by external odorant compounds.Calcium ion imaging results show that AcerOrl is sensitive to calcium ions in nine compounds:eugenol,lauric acid,ocimene,1-decanol,linolenic acid,hexyl acetate,undecanoic acid,1-octanol,and linolenic acid while AcerOr2 only sensitive to VUAA1.This result indicates on the one hand that the expression of endogenous Orco in Sf9 cells can activate the response of AcerOr 1 to odor molecules,and on the other hand that Orco can play a synergistic role and contribute to the formation of(Orco+AcerOr1)heterodimer and(Orco+Orco)homodimeric complexes The present study provides insight into the mechanism of olfactory discrimination in A.cerana cerana.(3)After site-directed mutagenesis,the predicted AcerOr2 calmodulin(CaM)binding site was mutated and subjected to immunofluorescence and immunoblot detection to detect changes in Ca2+ and intracellular CaMKII expression,and to explore the interaction between AcerOr2 function and its CaM binding site.The results of Ca2+ imaging showed that the cells transfected with AcerOr2 in vitro were stimulated by co-receptor agonist VUAA1 and CaM inhibitor W7,which could significantly affect the concentration of Ca2+ and the expression of CaMKII protein.The activation of CaMKII requires the interaction between CaM and Ca2+.The direct and indirect interaction between AcerOr2 and CaM is proved.The results suggest that AcerOr2 may mediate the Ca2+-CaMK? signaling pathway.It provides direct evidence for a relationship between OR stimulation and CaM production in vitro,and the activation of the CaM downstream target gene CaMK?.This finding provides a basis for the further study about the role of the calcium/calmodulin-signaling pathway in insect olfaction.(4)The results of Real-time PCR and Western blot showed that AcerOr2,CaM,CaMK? and p-CaMKII were expressed in the spermatozoa and testis of Apis cerana cerana.The expression of AcerOr2 agonist VUAA1 and CaM inhibitor W7 were significantly higher in spermatozoa than in testis.Both sperm quality and sperm DNA integrity were affected by the addition of VUAA1 and W7.There is a positive correlation between DNA integrity and semen parameters,which suggests that AcerOr2 may participate in the regulation of spermatozoa through the interaction of Ca2+/CaM/CaMK ? signaling pathway.The present study analyzed the AcerOr1 and AcerOr2 of Apis cerana cerana,and found the potential regulatory function,which can provide reference foundations for the studies on the functions of other olfactory receptors of Apis cerana cerana,and provide scientific basis for the research,development and utilization of bee resources.