Study On Identification And Function Of Genes Related To Royal Jelly Secreted By Workers Of Apis Mellifera L.

Royal jelly(RJ),one of the important bee products,is secreted from the tissue of hypopharyngeal glands(HGs)and mandibular glands(MGs).Meanwhile,the ability of workers to secrete RJ is related to the high-level expression of genes,proteins,neuropeptides in the brain.The transcriptome and proteome of HGs,MGs and brains were extensively reported in recent years,which has greatly promoted the molecular basis of RJ secretion.However,the molecular mechanism of RJ secretion was still unclear.In order to exclude the influence of age on the mechanism underlying the RJ secrection,here we constructed artificial colonies to collect 3-day-old workers,10-day-old nurses,10-day-old foragers,21-day-old nurses,and 21-day-old foragers,and compared the morphological and physiological differences of the HGs and MGs between nurses and foragers with same age.Moreover,differentially expressed genes(DEGs)associated with synthesis and secretion of RJ were screened in the tissue of HGs,MGs and brains using transcriptome.Also the expression patterns of some DEGs were verified by qRT-PCR.The main results for this study were as follows:1.Age-matched nurses and foragers(10-/21-day-old)were obtained by constructing artificial colonies.Acinus size and total protein content of HGs from age-matched nurses and foragers were initially determined,and then genes associated with RJ production were identified using RNA-Seq.It showed that the HGs of 10-/21-day-old nurses had larger acinus areas and higher total protein content than the age-matched foragers,indicating that HGs of nurses have a stronger protein secretory activity.In addition,RNA-Seq analysis exhibited that 510 DEGs were both up-regulated in the HGs of 10-day-old compared with 3-day-old workers,10-day-old foragers,and also the expression of those DEGs in the HGs of 21-day-old nurses was significantly higher than that of 21-day-old foragers,suggesting that these up-regulated DEGs in HGs was closely related to the synthesis and secretion of RJ.GO and KEGG enrichment analysis showed that up-regulated DEGs were mainly concentrated in translation(157 DEGs),transcription(20 DEGs),and DNA replication(7 DEGs),energy metabolism(13 DEGs).Meanwhile,Mrjp1,Mrjp4,Mrjp5,and another Mrjp1(LOC551813),the major component of RJ protein,were significantly higher in the HGs of the nurses than that of foragers with same age.These results implied that HGs of nurses might employ many strategies described above to stimulate highly efficient RJ protein synthesis.2.To detect the content difference of 10-hydroxy-2(E)-decenoic acid(10-HDA)secreted by MGs from age-matched nurses and foragers,it was measured in the MGs of 3-day-old workers,10-day-old nurses and foragers,21-day-old nurses and foragers by high performance liquid chromatography.Besides,RNA-Seq was also employed to screen DEGs that are closely related to RJ synthesis and secretion in the MGs.The results showed that the content of 10-HDA in the MGs of workers increased gradually with age(3-day-old workers,10-day-old nurses and 21-day-old foragers),but it was higher in the MGs of foragers than that of nurses with corresponding age.The expression of 46 DEGs was significantly higher in the MGs of 10-day-old nurses than that of 3-day-old workers,10-day-old foragers,and also those DEGs in the MGs of 21-day-old nurses was significantly higher than that of 21-day-old foragers,suggesting that these up-regulated DEGs in MGs was closely related to the synthesis and secretion of RJ.GO and KEGG enrichment analysis showed that up-regulated DEGs were mainly enriched in fatty acid metabolism and energy metabolism,indicating that the MGs of nurses may adopt the above-mentioned multiple strategies to stimulate the synthesis of related fatty acids in RJ.In addition,Mrjp1,Mrjp2,Mrjp3,Mrjp4,Mrjp7 and genes(ACSF2,LOC408567,LOC409150,LOC725274,LOC408291)involved in process of fatty acids β-oxidation,were significantly higher in the up-regulated DEGs in MGs of the nurses than those foragers with the corresponding age.3.In order to screen the genes involved in brain regulation of nursing behavior,RNA-Seq were used to comprehensively analyse the gene expression of profile in the brains from same age-matched nurses and foragers(10-/21-day-old).The expression of 32 DEGs in the brain of 10-day-old nurses was significantly higher than 3-day-old workers,10-day-old foragers,and also those DEGs in the brain of 21-day-old nurses were significantly higher than that of 21-day-old foragers,suggesting these up-regulated DEGs were closely associated with nursing behavior.GO and KEGG enrichment analysis showed that these genes were mainly involved in the nursing behavior through regulating signal transduction and energy metabolism,which may play an important role in the brain for regulation of RJ synthesis and secretion.Based on the above results and previous researches,we speculated that nurses can sense the larval pheromones and transmit chemicals signals to the brain through chemoreceptor organs,and then central nervous organ(brain)regulate the HGs and MGs to promote the synthesis of major RJ proteins and fatty acids,which induce nurse bees exhibiting nursing behavior,causing that nurses can secrect RJ to feed young larvae in the first three days after hatching and queen.