Functional Analysis Of Two MiRNA In Silkworm Hemolymph Induced By BmNPV

MicroRNA is a class of endogenous single stranded non coding RNA molecules of about 21-23 nt,and through the complementarity of the target gene with post-transcriptional regulation.With the discovery of more and more miRNAs and the deepening of related research,their production and the way of action are gradually clear.MiRNAs not only participate in the growth and development of insects,but also play an important role in the immune defense of insects interacting with pathogens.However,the study of silkworm mi RNAs is relatively late.Existing studies have shown that silkworms express antiviral mi RNAs after infection with Bombyx mori nuclear polyhedrosis virus(BmNPV)to respond to viral proliferation.MiRNAs play an important role in the molecular mechanism of the interaction between host and pathogens,not only the host expresses defensively miRNAs after infection by the virus,but also the virus invades the host and expresses miRNAs to destroy the host’s defense and promote their own proliferation.In this paper,we studied the regulation of BmNPV gene by miRNAs in silkworm,which enriched the basic information of the molecular mechanism of innate immunity.The main results are as follows:1.Target gene prediction and expression analysis of bmo-miR-217 and bmo-miR-3901)Two different miRNAs(bmo-mi R-217/bmo-mi R-390)of Bombyx mori were obtained by high-throughput sequencing.2)The target gene of bmo-miR-217 in BmNPV was predicted by using the online mi RNA target gene prediction software.The results showed that there were 14 target genes of bmo-miR-217.A total of 10 target genes of bmo-miR-390 were predicted by bom-miR-390.3)The results showed that bmo-miR-217 and bmo-miR-390 were both expressed in the experimental group and the control group,and the expression of bmo-miR-217 in the experimental group was higher than that in the control group.The expression of bmo-miR-390 in the experimental group was lower than that in the control group.4)The relative expression levels of bmo-miR-217 and BmNPV-lef-1 in haemolymph of BmNPV were analyzed by qRT-PCR.The expression of bmo-miR-217 increased gradually and the expression of BmNPV-lef-1 gene decreased gradually in the time period of 0h-24 h infection.The expression trend of bmo-miR-217 and BmNPV-lef-1 gene was similar in 24h-120 h time.The 24h-48 h expression of BmNPV-lef-1 gene increased sharply but the amount of bmo-miR-217 increased slowly,48h-96 h bmo-miR-217 decreased no BmNPV-lef-1 gene expression decreased rapidly;expression of BmNPV-lef-1 gene in 120h-144 h period increased,while the expression of bmo-miR-217 sag.5)The expression of bmo-miR-390 and BmNPV-cg30 gene in silkworm hemolymph tissue of silkworm infected BmNPV at different time showed that the expression of BmNPV-cg30 gene was smooth and the expression of bmo-mi R-390 was rapid Rise.The expression of BmNPV-cg30 gene in 24h-48 h increased,while the expression of bmo-miR-390 began to decrease.In 48h-96 h time,the expression of BmNPV-cg30 tended to decline gradually,but the expression of bmo-miR-390 decreased gradually.The expression levels of bmo-miR-390 and BmNPV-cg30 were both increased in the 96h-144 h infection time,but the increase of bmo-miR-390 was earlier than that of BmNPV-cg30 gene expression.2.Preliminary study on the function of bmo-miR-217 and bmo-miR-3901)The cloned gene fragments of bmo-mir-217,bmo-mir-390,and expression vectors pcDNA3.0(IE-1-egfp-bmo-mir-217-SV40)/pcDNA3.0(IE-1-egfp-bmo-mir-217-SV40)were constructed.MiRNAs target gene expression vector pGL3(A3-luc-lef-1 3’UTR-SV40)and pGL3(A3-luc-BmNPV-cg30 3’UTR-SV40)were constructed.2)The effect of bmo-mi R-217 on BmNPV-lef-1 was studied by double luciferase activity assay.The results showed that bmo-miR-217 could down-regulate lef-1 expression.3)The effect of bmo-mi R-217 on BmNPV-lef-1 gene expression was inhibited by detecting double luciferase activity.Subsequently,we transfected the bmo-miR-390 plasmid and bmo-mi R-390 mimic into BmN cells infected with BmNPV to detect the expression of BmNPV-cg30 gene,the results showed that the expression of cg30 gene was down-regulated by bmo-miR-390 and decreased its expression.