| Honey bees(Apis mellifera ligustica)are the most widely used pollinator for greenhouse crops,they can significantly increase the fruit setting rate and yield of greenhouse crops such as tomatoes and melons,as well as improve fruit quality.Compared to greenhouse melons,honey bees have showed poor flower-visitation rates and low pollination efficiency in greenhouse tomatoes.Floral scents play a key role in the pollination process of insects,they interact with multiple olfactory proteins in antennae of honey bees in completing their pollination behavior.The odorant binding proteins present in the antennae lymph fluid are key proteins for insects to perceive the odorants in the external environment.In this study,GC-MS was used to identify the volatile components of floral scents in tomatoes(4805 Dahong)and muskmelons(Xingtian muskmelon).By comparing the differences in the composition of floral scents between them,we have analyzed the differences in the selection of greenhouse tomato/muskmelon floral scents in honey bees.Electrophysiological techniques were used to measure the EAG responses of honey bees to different floral scent substances in tomatoes/muskmelons.In addition,the three-arm olfactometer was used to further observe behavioral and olfactory responses in the honey bees.The Illumina RNA Denovo high-throughput sequencing technology was used to establish a honey bee olfactory molecular regulatory network,and to explore the mechanism of OBPs in the pollination response process of greenhouse tomatoes/muskmelons in gene and protein expression levels,respectively.The PET30a/OBP1,PET30a/OBP5,and PET30a/OBP16 prokaryotic expression vectors were successfully constructed,and obtained recombinant target proteins through prokaryotic protein expression,and prepared three kinds of polyclonal antibodies.The binding ability of OBP1,OBP5,and OBP16 to different odorant ligands was identified by fluorescence competition binding assay,and the role of OBPs in the phenotypic changes of bee antennae was analyzed by combining RNAi and EAG techniques.The main results obtained in this study were as follows:(1)The volatile compounds of the male and female floral scents of tomato and muskmelons were measured by headspace solid-phase microextraction gas chromatography-mass spectrometry(SPME-GC-MS).A total of 124 volatile components belonging to 13 chemotypes were identified for the three floral scents.The main components of tomato floral scents was terpenoid;the main components of both female and male muskmelon floral odors was aldehyde,and terpenoid was not detected in muskmelon flowers.The common compound to three floral scents was the n-nonenal.(2)The antennal electrophysiological responses of honey bees to the standard floral scents of the tomato/muskmelon were measured by the antenna positioning system,and the results showed that the relative EAG responses of honey bee antennae to n-nonenal,trans-2-hexenal,E-2-octenal,benzaldehyde,phenylethylaldehyde,toluene,and(+)-dihydrocarvone,were significantly higher than those of other floral scent standard compounds.And antennae of honey bees were most sensitive to aldehydes,and less sensitive to terpenes.It was speculated that terpene and aldehyde compounds were important factors affecting the foraging activity of honey bees to greenhouse tomatoes/muskmelons.A three-arm olfactometer was used to screen the behavioral responses of honey bees to five standard floral scents.The results showed that honey bees had exhibited a certain repellency to n-nonenal,E-2-octenal,and dihydrocarvone;their responses to toluene and trans-2hexenal were not obvious.This indicated that a single floral scent standard had no attractiveness to honey bees,and it also revealed that the recognition of floral scents of honey bees was a behavioral response to the interaction of multiple floral scents in greenhouse crops.(3)Transcriptomic analysis showed that a total of 23 candidate olfactory-related genes were identified in the antennae transcriptome of the greenhouse honey bees,including 12 OBPs,5 CSPs and 6 ORs.The results of GO and KEGG analysis showed that the differentially expressed genes were mainly enriched in the functions of substance synthesis,energy metabolism regulation,substance transport,and gating channel activity regulation,and they were mainly involved in the metabolic processes of protein,tyrosine,fructose,mannose,nitrogen,phenylalanine and fatty acid.This indicated that the pollination behavior of honey bees in greenhouse tomatoes/muskmelons was carried out under the premise of multiple substance transport and energy metabolism.It also revealed that pollination in honey bees was a behavioral response coordinated by various olfactory-related proteins in vivo under external stimulation.(5)The prokaryotic expression vectors were successfully constructed,PET30a(+)-OBP1,PET30a(+)-OBP5,and PET30a(+)-OBP16,induced expression,and obtained high-purity(> 85%by SDS-PAGE)recombinant target proteins,OBP1,OBP5,and OBP16.We have also prepared three polyclonal antibodies against these OBPs.The results of q RT-PCR and WB showed that the expression patterns of OBP1,OBP5 and OBP16 in the antennae of honey bees in greenhouse tomato/muskmelon were similar at the gene and protein levels,indicating that these genes were mainly related to the recognition of floral odors in greenhouse tomatoes/muskmelons by foraging bees.OBP16 and OBP21 were highly expressed in the feet of the honey bees,suggesting that these two genes were related to the use of feet by the bees in perceiving non-volatile chemical substances through taste sensation at close proximity.(4)The results of the fluorescence competitive binding assay showed that the ligand binding spectra and affinities of the three OBPs were different.OBP1 showed a strong binding affinity to most of the tested odor standards,and showed a very strong affinity to benzaldehyde and toluene.OBP5 showed strong binding affinity to toluene and isoeugenol methyl ether,and showed none or weak binding affinity to other tested odor standards.OBP16 showed strong binding affinity to all tested odor standards,with extremely high binding affinity to nonanal and(E)-2-decenal.After RNAi was used to silence the target OBPs,the relative values of EAG of the tested odor standards in the antennae of the honey bees were significantly reduced.The results showed that these three OBPs had certain selection preferences in the process of identifying and binding floral scents in greenhouse crops,with OBP1 and OBP5 having higher specificity and OBP16 being more versatile.All three OBPs had affected the olfactory perception ability of the antennae of honey bees.In conclusion,this study measured and analyzed the external and internal factors that may affect the pollination behavior of Honey bees in facility crops.The aim of this study was to elucidate the molecular mechanisms regulating the responses of honey bees to floral scents of the tomato/muskmelon,which provided a theoretical basis for understanding the olfactory recognition of different crops by honey bees,and screening of suitable pollinators for the crops.It also provided insights for the use of plant volatiles in regulating the flower-visitation behavior of insects. |
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