| Apis cerana cerana is also known as the Chinese bee,is a unique bee species in my country.Compared with Apis mellifera ligustica,the A.cerana cerana has strong resistance ability to disease and mite resistance,strong environmental adaptability and good at collecting sporadic honey powder and other advantages,it is very suitable for fixed-site breeding in my country’s mountain and forest areas.The sense of smell plays an important role in the foraging,mating,and gathering behaviors of A.cerana cerana.The main olfactory organ of insects is the antennae,and the Odorant Binding Proteins(OBPs)present in the lymph fluid of the antennae are the key proteins for insects to perceive odor substances in the external environment and peripheral olfactory signals.At present,the research on OBPs of A.cerana cerana is not comprehensive enough,and the function is not yet fully understood.In this study,the odorant-binding proteins AcerOBP6,AcerOBP7 and AcerOBP14 identified in the research of transcriptome sequencing were used as the research objects,and their relative expression levels of m RNA in different developmental stages and different tissues of foragers were detected by q RT-PCR;The Pet28a/AcerOBPs recombinant expression vector was constructed,and the recombinant protein was obtained by prokaryotic expression,purification,renaturation and concentration.The fluorescence competition binding assay was used to detect the binding ability of AcerOBP6,AcerOBP7 and AcerOBP14 with different odorant ligands;Synthesize ds RNA,then use RNAi and EAG technology to further verify the odorants that specifically bind to the three proteins.The main results obtained in this study are as follows:1.Obtain the spatiotemporal expression profile of AcerOBP6,AcerOBP7 and AcerOBP14 odorant binding protein genes.Three object genes were expressed in the antennae of different developmental stages of A.cerana cerana.AcerOBP6 m RNA had the highest relative expression at 25 day-old,AcerOBP7,AcerOBP14 The m RNA expression was highest at 20 day-old,and all three genes were specifically expressed in the antennae of the foragers.2.Successfully constructed Pet28a/AcerOBP6,Pet28a/AcerOBP7 and Pet28a/AcerOBP14 prokaryotic expression vectors,and induced the expression of recombinant proteins in E.coli and expressed them in inclusion bodies.Purified by Ni2+ Sepharose column and dialyzed,the recombinant protein with biological activity was obtained.3.The results of titration of recombinant proteins showed that: AcerOBP6,AcerOBP7 and AcerOBP14 can bind well to N-phenyl-1-naphthylamine(1-NPN),confirming that 1-NPN can be used as fluorescence competitive probes for binding experiments.Fluorescence competition assay results showed that the ligand binding spectrum and binding ability of the three AcerOBPs are different.They can all bind to a variety of pheromones and plant volatiles.Among them,AcerOBP6 has the strongest binding ability with linolenic acid,followed by eugenol,Ethyl trans-cinnamate and(+)-3 carene;AcerOBP7 has the strongest binding ability with 9-ODA and 1-nonanol,followed by(+)-limonene and 1-octene-3-ol,Linalool;AcerOBP14 combines with queen bee pheromone,alarm pheromone,Nash’s pheromone and a variety of plant volatile compounds.The strongest binding ability is plant volatiles β-ocimene,followed by α-farnesene.AcerOBP6 and AcerOBP7 have no binding ability with the 7 larval pheromone components.AcerOBP14 has some binding ability with larval pheromone components that methyl oleate and methyl stearate,but has no binding ability with the other five components.4.We synthesized two ds RNAs for AcerOBP6,AcerOBP7 and AcerOBP14,respectively,and successfully silenced the expression of each target gene of A.cerana cerana by feeding method.The EAG test was carried out in combination with the odorants with strong binding ability screened out by the fluorescence competition binding experiment.The results showed that: compared with 30% sugar water control group,the EAG response values of the antennae after feeding dsAcerOBPs to different odor substances were all decreased,and the specific performance was that the EAG response value of the antennae A.cerana cerana to 2-heptanone,hexyl acetate,eucalyptol,β-ocimene,1-nonanol,myrcene,(+)-limonene and linalool were extremely significantly decreased after feeding ds AcerOBP6(P < 0.01);After feeding ds AcerOBP7,the EAG response value of antennae to eucalyptol,1-octen-3-ol and 9-ODA decreased extremely significantly(P < 0.01),and the EAG response value of nonanal decreased significantly(P < 0.05);After feeding ds AcerOBP14,the EAG response values of 2-heptanone,α-farnesene,β-ocimene and citral of A.cerana cerana antennae were extremely significantly reduced(P < 0.01),and nerol and piperitone were significantly reduced(P < 0.05).This study conducted a preliminary analysis on the functions of AcerOBP6,AcerOBP7 and AcerOBP14.The results of the experiment of great significance for exploring the functions of A.cerana cerana odorant binding proteins and have significance to analyze the molecular mechanism of A.cerana cerana olfactory behavior regulation.Provide a reference for the further development inducers with different odor componentoriented and improve the pollination ability of A.cerana cerana to crops. |
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