| Royal jelly(RJ)is one of the main bee products.Compared to Italian honeybees,the high royal jelly producing strain of bees(RJBs)can now produce 10-fold RJ.RJ is a composite product of fatty acids produced in mandibular glands(MGs)and proteins secreted by HPGs.The HPGs is the main exocrine gland to synthesize and secrete RJ protein.In other words,the RJ production is strongly relied on the developmental and secretion activity of HPGs.But the molecular regulation mechanism of the development of HPGs is still unclear.In this study,we investigated the molecular regulation mechanism of the effects of microRNA(miRNA)on the HPGs development using histocytological analysis,molecular biology and bioinformatics methods.The main research contents and results are as follows:1.The development of HPGsIn the experiment,five healthy colonies,from sister-queen with similar population were chosen.Newly emerged workers(NE),3,6,9,12,15,18,21,24,27,and 36-day old workers were collected.The HPGs of 20 workers were dissected to make sections.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to observe the surface morphology and ultrastructure of the HPGs of workers at different days.The main results were as follows:1)The diameter of the acinars were generally increased with the age of the workers before decreased.The diameter of the acinars of the 9-day old workers were the largest,and the diameter of the acinars began to decrease after 12 days(P<0.05).2)The results of TEM showed that the rough endoplasmic reticulum(RER)of the acinar secretory cells was the most obvious organelle with increasing age.The RER of the secretory cells of the HPGs showed a long tube shape at 6 days.There are fewer ribosomes attached to the outer surface.The RER of the HPGs is densely packed around the nucleus at the age of 9 days,and a large number of ribosomes are attached to the outer surface.The number of RER in HPGs began to decrease after 15 days.2.Expression of genes related to hypopharyngeal gland development at different agesNE,3,6,9,12,15,18,21,24,27,and 36-day old workers were collected,and the HPGs of60 workers were dissected at each age.The results are as follows:The relative expression level of Major royal jelly protein 1(MRJP1),Major royal jelly protein 3(MRJP3),and eukaryotic translation initiation factor 4 gamma(Eif4g)of HPGs showed the highest levels in the 9 days(P<0.05).The expression of Caspase 1(Cas1),Caspase 8(Cas8),P53 and TOR reached the highest levels at 18 or 21 days(P<0.05);the expression level of the P70 S6kinase(P70S6K)was highest at 21 days and lowest at 3 days(P<0.05).3.Analysis of the expression profile of miRNA in HPGs from different agesIllumina HiSeq~TMM 4000 paired end sequencing technology was used to test the miRNA expression of HPGs of 6,9,and 12 days old worker.The results showed that:1)115.89 M clean reads were obtained.322 miRNAs were detected,of which 159 were known miRNAs and 163 were novel miRNAs.2)Through differential expressed genes screen(DEGs):27difference expressions of miRNAs were screened(6 d vs 9 d);16 difference expressions of miRNAs were screened(6 d vs 12 d);11 difference expressions of miRNAs were screened(9d vs 12 d).3)The annotation of the targeted genes of differentially expressed miRNAs was performed using Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.KEGG analysis showed that the targeted genes of differentially expressed miRNAs were annotated to the circadian rhythm,Hippo signaling pathway,FoxO signaling pathway,and Phagosome,Purine metabolism pathway and Ribosome biogenesis in eukaryotes(P<0.05).4)GO analysis shows that the miRNA target genes are mainly involved in organic substance transport,transport,and vesicle-mediated transport,ion transmembrane transport,growth and so on.4.Effects of ame-miR-184 on the development of HPGs in the pupaeOne-day-old larvae were transferred to a 48-well cell culture plate.The pupae were divided into four equal groups,each group consisting of three replicates,each with 48 workers.The experimental groups were injected with 0.5μL/mimics,mimics NC,inhibitor or inhibitor NC reagent,respectively.After 3 hours of injection,workers was collected.The results are as follows:1)ame-miR-184 had a significant effect on the emergence rate,and the emergence rate of the inhibitor and mimics group were significantly reduced(P<0.05).2)The expressions of the target genes insulin-like receptor(InR2)and cyclin dependent kinase 12(CDK12)were significantly decreased when ame-miR-184 was overexpression(P<0.05),and the expressions of MRJP1,MRJP3 and 2-oxoglutarate dehydrogenase(Ogdh)and Polycomblike(Pcl)were also inhibited(P<0.05).3)When the expression of ame-miR-184was interfered,the expression of Ogdh,Pcl and MRJP3 genes were increased significantly(P<0.05),and the expression of target genes InR2 and CDK12 were also significantly increased(P<0.05).5.Effects of ame-miR-184 on the development of HPGs in workersNewly emergence workers were marked for sample collecting,and 9-day old marked workers were collected in wooden cages.A total of 720 new worker bees were collected and randomly divided into 4 groups(mimics group,mimics NC group,inhibitor group and inhibitor NC group).Each group consisted of 3 repetitions,each with 60 workers.The treatment groups were injected with 0.5μL/mimics,mimics NC,inhibitor or inhibitor NC between the antennae of the head,respectively.The results are as follows:1)The survival rate of mimics was significantly lower than that of other groups(P<0.05),and the survival rate of inhibitors was no significantly change compared with the control group.2)When ame-miR-184 is overexpressed,the expression level of ame-miR-184 is significantly increased(P<0.05),the expression of target genes InR2 and CDK12 was inhibited(P<0.05),and the expression of MRJP1,MRJP3,TOR and Eif4g genes were also inhibited(P<0.05).3)When the expression of ame-miR-184 was interfered,the expression of target genes InR2 and CDK12 were increaced(P<0.05),the expression of MRJP3,TOR and Eif4g were also significantly increased(P<0.05).The results indicate that,ame-miR-184 promotes the production of RJ protein by activating the expression of key genes of mitochondrial metabolism and key genes of the TOR signaling pathway. |
