| Complex chemosensory systems,including olfactory and gustatory systems,were helpful for insects to perceive and distinguish all kinds of chemical information from the environment,and were crucial in the daily behaviors of insects,such as foraging,oviposition,mating,and escaping predators.Odorant-binding proteins(OBPs),as the first proteins interacted with the recognition of external stimulants,were responsible for the transport of stimulants to chemoreceptor neurons,and thought to play an important role in the chemical communication from outside world.As a dominant indigenous crop pollinator,Apis cerana cerana Fabricisus was superior to Apis mellifera L.in some biology characteristics such as sensitive olfactory,and full advantage of sporadic nectar and pollen,which was an irreplaceable species in most parts of China and inhabited a variety of habitat types,especially mountains and forests.In the previous sequencing study,we obtained 1 significantly up-regulated OBP genes-Acer OBP15,from the antennal transcriptome in the different developmental stages of worker honeybees.The present research clarified the role of Acer OBP15 in the process of olfaction and gustatory perception at molecular level,and deeply revealed the chemosensory mechanism of A.cerana cerana,which provided a fundamental foundation for the developing of attractant for problem crops.The main results were as follows:(1)The expression levels of Acer OBP15 in different tissues(antenna,head,thorax,abdomen,legs,and wings)at three different periods(overwintering,beginning of reproduction,and development)were detected by q RT-PCR.The results showed that Acer OBP15 transcripts in antenna and legs were significantly higher than that in heads,thorax,abdomen,and wings(P < 0.01).According to the expression profiles of Acer OBP15 in antenna and legs at different periods,the legs of foragers had the highest expression level at development period which was significantly higher than other periods(P < 0.01);there was no difference in the expression profiles at the other two periods(P > 0.05),and the lowest expression level was found at spring reproduction period.m RNA expression pattern in antennae were basically consistent with that in legs,but there was no difference in the expression profiles at three periods(P > 0.05).Polyclonal antisera made from purified Acer OBP15 was used for the Western blot assay.Acer OBP15 was differently expressed at 1-,5-,10-,15-,20-,25-,and 30-day-old in antenna and legs,and was highly expressed in foragers.These results suggested that Acer OBP15 was involved in not only the olfactory recognition,but also gustatory perception.(2)Fluorescence competitive binding assay results indicated that the dissociation constants of Acer OBP15 with 1-NPN was 3.318 ?M.Acer OBP15 displayed high binding affinities for alarm pheromone(2-heptanone and isoamyl acetate)and nasonov pheromone(nerol and geraniol),but had low binding affinity for queen pheromone(9-ODA and HVA)and brood pheromone(methyl palmitate,methyl oleate,ethyl linoleate and ethyl oleate).In addition,Acer OBP15 had high binding affinities with most floral volatiles,among which its affinity for myrcene was the strongest(Ki = 6.50 ± 0.07 ?M),which provided further evidence that Acer OBP15 played a key role in the chemical communication outside nest and in the search and collection of nectar and pollen.(3)Electroantennogram(EAG)bioassay results revealed that EAG values of A.cerana cerana to myrcene,2-heptanone,and isoamyl acetate were relatively high,which were significantly higher than that to other compounds(P < 0.05).The silence efficiency of Acer OBP15 m RNA was up to 94.53% at 72 h after bees were fed with ds Acer OBP15(the length of 311 bp).In this case,the EAG response to myrcene was found to be significantly reduced after the ingestion of ds Acer OBP15(P < 0.05),while there was no significant decrease in response to 2-heptanone and isoamyl acetate(P > 0.05).These results indicated that Acer OBP15 was required for the recognition of floral volatile myrcene.Based on the docking simulations,Phe91 of Acer OBP15 was a key site involved in the binding of myrcene with H-? conjugation.(4)Using Illumina sequencing platform and differential expression analysis,we obtained one significantly up-regulated OR genes—Acer OR46 in A.cerana cerana head(with antenna)transcriptome.It was suggested that Acer OR46 was the downstream gene of Acer OBP15,and might be a candidate receptor for myrcene or other ligands.The c DNA sequence of the olfactory receptor gene was cloned from the antenna of A.cerana cerana foragers.Acer OR46 c DNA contains a 1 221 bp open-reading frame(ORF)encoding a putative protein of 406 amino acids.The encoding protein has no signal peptide and four transmembrane domains which N-terminal is located inside of cell membrane.The expression profiles of Acer OR46 in antenna both of nurses and foragers were significantly higher than that in head,throax,abdomen,legs and wings(P < 0.01);the expression levels of Acer OR46 in different tissues of foragers were higher than that in the corresponding tissues of nurses,suggesting that Acer OR46 was essential in the process of searching and locating nectar and pollen sources.(5)RNA-seq was performed with RNA samples extracted from the legs of 1-,10-,and 20-day-old workers of A.cerana cerana.A total of 124 candidate chemosensory proteins,including 15 odorant binding proteins(OBPs),five chemosensory proteins(CSPs),seven gustatory receptors(GRs),two sensory neuron membrane proteins(SNMPs),and 95 odorant receptors(ORs),were identified from the assembled leg transcriptome.In silico analysis of expression showed that 36 of them were differentially expressed among the three different ages of A.cerana cerana workers.Analysis of FPKM values showed that the genes encoding ORs,GRs,and SNMPs(except SNMP1)were expressed at low levels,whereas the genes encoding OBPs and CSPs had great but extremely variable FPKM values and thus were highly expressed in the legs of workers.Among 15 Acer OBPs,Acer OBP15 had the highest expression level.Tissue-specific expression patterns indicated that Acer OBP15 was predominately expressed in the legs of foragers,suggesting it may play an essential role in gustatory recognition and selection of plant nectars and pollens.This study provided a foundation for further research of the feeding preferences of honeybees.(6)The interaction proteins for Acer OBP15 were identified by GST pull-down coupled with LC-MS/MS mass spectrometry.A total of 40 potential interaction proteins were screened from the tissue protein lysates of A.cerana cerana.GO and KEGG enrichment analysis showed that interacting proteins of Acer OBP15 were mainly involved in the metabolism of carbohydrates,proteins,and some small molecular substances.It was speculated that Acer OBP15 might be a carrier protein of various nutrients and participate in many biological functions in A.cerana cerana.Interestingly,we also found three signal transduction pathways: TGF-beta,Fox O and Wnt,which might provide a new clue for further study of honeybee chemosensory mechanism.In this study,Acer OBP15 was found to play a dual role in foraging activities of A.cerana cerana for the first time.Acer OBP15 was not only involved in the olfactory recognition to floral scents,but also participated in the gustatory perception system on legs to detect the quality of nectar and pollen sources,laying a theoretical foundation for the study of chemical sensing mechanism of A.cerana cerana. |
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