Identification,expression And Functional Analysis Of MiRNAs Target Genes In Fat Body Of Bombyx Mori

Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs and play important roles in the development of insect metamorphosis.Research on miRNAs and their target genes related to the development of B.mori will help to elucidate the molecular mechanisms of miRNAs in the development of B.mori metamorphosis.Based on the Previous work of high-throughput sequencing and bioinformatics analysis,differentially expressed miRNAs in B.mori fat body treated with 20 E were identified.Bmo-miR-2769 was significantly down-regulated,while Bmo-miR-2763 was significantly up-regulated.In order to further study the functions of Bmo-miR-2769 and Bmo-miR-2763,the target genes of the two miRNAs were predicted by bioinformatics methods,and the expression of the target genes in20E-treated fat body were analyzed.The dual luciferase reporter system was used to detect the interaction between miRNA and the predicted target genes in vitro,and real-time fluorescence quantitative PCR was applied to detect their expression characteristics in different tissues and developmental stages of B.mori.Bm E75 B,an alternative spliceosome of nuclear hormone receptor gene Bm E75,is a predicted target gene of Bmo-miR-2769.Bmo-miR-2769 could inhibit the expression of Bm E75 B at the translational level by interacting with its 3′ UTR region.In addition,since Bm E75 A and Bm E75 C have the same 3′ UTR sequence with Bm E75 B,we speculated that Bmo-miR-2769 could also regulate the expression of Bm E75 A and Bm E75 C.The expression of Bmo-miR-2769 in the fat body treated with20 E showed opposite trend with Bm E75 A and Bm E75 B,and Bm E75 A and Bm E75 B could be rapidly activated by 20 E.The temporal and spatial expression characteristics of Bmo-miR-2769 and different spliceosomes of Bm E75 in the B.mori showed that the expression of Bmo-miR-2769 was negatively correlated with the expression of Bm E75 A and Bm E75 B from the 4th instar larva to the 3rd day adult,and was also opposite to that of Bm E75 C at the end of the 5th instar and the early pupal stage.In addition,Bmo-miR-2769 showed tissue specificity in the regulation of the expression of different spliceosomes of its target gene Bm E75.Therefore,we speculated that Bmo-miR-2769 may play a role in the metamorphic development of the B.mori by regulating the temporal and spatial expression of target genes Bm E75 A,Bm E75 B and Bm E75 C.Bm GRF,germ cell nuclear receptor factor,was a predicted target gene of Bmo-miR-2763,which is an early and late gene in the 20 E signaling pathway.Bmo-miR-2763 could also inhibit the expression of Bm GRF at the translational level by interacting with the 3′ UTR region of Bm GRF.Quantitative PCR results showed that the expression of Bmo-miR-2763 and Bm GRF in the fat body were consistently up-regulated by 20 E.In the different developmental stages,the expression trend of Bmo-miR-2763 and Bm GRF were different,however,Bm GRF was highly increased at the end of pupa,which was opposite to Bmo-miR-2763.Additionally,Bmo-miR-2763 and Bm GRF are specifically and highly expressed in the testis of the B.mori,while Bmo-miR-2763 also exhibited high expression in ovary and silkgland.It is speculated that Bmo-miR-2763 may have different regulatory mechanism in different tissues and development stages of B.mori,and it plays an important role in the development of genital organs of B.mori by interacting with the target gene Bm GRF.In summary,Bmo-miR-2769 and Bmo-miR-2763 could regulate the expression of their target genes at translational level by interacting with 3′UTR separately.Bmo-miR-2769 may play a role in the metamorphosis and development of B.mori by regulating the spatial and temporal expression of different spliceosomes of Bm E75,and Bmo-miR-2763 may be involved in the development of reproductive organs of B.mori by regulating the target gene Bm GRF.This research not only enriches and expands the functional research of miRNAs involved in the regulation of B.mori metamorphic development,but also lays a foundation for further research on the molecular mechanisms of miRNAs and target genes in insect metamorphosis and development.