| Aphids belong to the Insecta Hemiptera,which live mainly by sap of plant phloem,and reproduce fast and are widely distributed throughout the world.Previous studies revealed that many microRNAs(miRNAs)showed differential expression in the winged and wingless individuals of Sitobion avenae.In order to further study the regulatory mechanism of these miRNAs in aphid wing differentiation,pea aphid Acyrthosiphon pisum was choosed as the research object,five miRNAs(Let-7,miR-92 a,miR-92 b,miR-92a-1-p5 and miR-277)with significantly differential expression levels between winged and wingless individuals of S.avenae and whose target genes were related to ecdysone,insulin signaling pathway and wing development were selected.miRanda and RNAhybrid software were used to predict the target genes of these miRNAs in A.pisum.The expression profiles of miRNAs and their target genes in the 3rd,the 4th instar nymphs and the adults of A.pisum were studied in winged and wingless individuals by qPCR.The interaction of miRNAs and their target genes were studied in HEK293 cell line by dual luciferase activity assay.Finally,ecdysone(20E)was used to treat the 2nd instar aphids of A.pisum,and expression changes of 5 miRNAs and their target genes were detected to explore the roles of ecdysone played in aphid wing differentiation through miRNAs.The main findings are as follows:1.Targets prediction of miRNAs.To predict the target genes of miRNAs in A.pisum,the software of miRanda and RNAhybrid were used.Results showed that the seed sequences of Let-7 and PC-3p-94006-17 matched the 3'UTR of abrupt;miR-9a matched the 5'UTR of flightin;miR-92 a matched the CDS of wingless gene;miR-92 b and miR-996-p5 matched the CDS region of Foxo better;miR-277 matched the 5'UTR region of Uba1;miR-92a-1-p5 matched the CDS region of flightin and the CDS region of ultraspiracle(Usp);miR-3020-p3 matched the 3'UTR of Usp,indicating that these genes might be the real targets of miRNAs.2.Expression profiles of miRNAs and their target genes in different developmental stages of A.pisum.RNA of A.pisum at different developmental stages were extracted,the expression profiles of the miRNAs and their target genes at different developmental stages were detected by real-time quantitative PCR.Results showed that the expression level of the five miRNAs in adults of A.pisum were higher than those in nymphs,but the expression level of the predicted target genes in the 4th instar nymphs were higher than those in adults,which is opposite to the miRNA expression trend,suggesting that miRNAs might play regulatory roles on their target genes at the adult stage.The expression level of five miRNAs in winged individuals were higher than those in wingless ones of adults,especially for the miR-277,whose expression level in winged individuals was 7.5 times as high as that in wingless individuals of adults,then followed by Let-7 with three-fold expression difference.And miR-92a-1-p5,whose expression level in winged individuals was 1.6 times as high as that in wingless individuals of adults,Moreover,the expression difference of Let-7 between winged and wingless individuals of the 3rd instar nymphs was the most significant,its expression level in the winged individuals being 37.8 times as high as that in wingless ones,then followed by miR-277 with 7.6-fold expression difference.The expression patterns of predicted target genes abrupt and Foxo in the 3rd and the4 th instar nymphs in winged and wingless individuals of adults of A.pisum were opposite to those of miRNA Let-7 and miR-92 b,respectively.The target gene flightin predicted by miR-92a-1-p5 was significantly higher than that of wingless individuals in the 3rd and the4 th instar A.pisum and winged individuals of young aphid adults.The target genes Uba1 and wingless were significantly higher in the 3rd instar wingless individuals of A.pisum than in the winged individuals,which was opposite to the expression of miR-92 a and miR-277 at this age,but at the 4th and adult ages,there was no strong correlation between the expression profiles of the two miRNAs and the predicted target genes.3.miRNA target gene verification.Overexpression vector carrying the CDS region of flightin,pmirGLO-flightin-CDS and the overexpression vector carrying the 3'UTR of abrupt,pmirGLO-abrupt 3'UTR were successfully constructed.Dual luciferase reporterassay was used to detect the interaction between miRNAs and their target genes in an exogenous HEK293 cell line.Results showed that the luciferase activity decreased by 40%when co-transfected with the miR-92a-1-p5 mimic and pmirGLO-flightin-CDS compared with co-transfected with the negative control mimic and pmirGLO-flightin-CDS,reaching a significant level.This shows that the true target of miR-92a-1-p5 is flightin.Meanwhile,the luciferase activity decreased by 48% when co-transfected with the Let-7 mimic and pmirGLO-abrupt 3'UTR compared with co-transfected with the negative control mimic and pmirGLO-abrupt 3'UTR,reaching a significant level,which indicate that the real targets of miR-92a-1-p5 and Let-7 is flightin and abrupt,respectively.The interaction of other miRNAs and target genes were not significant.4.Effect of ecdysone on the expression of miRNAs and their target genes.The ecdysone mimics 20-E was used to treat the 2nd instar aphids,the expression profiles of miRNAs and their target genes were detected by real-time quantitative PCR after ecdysone treatment.Results showed that the expression levels of the five miRNAs were all increased to different degrees after 20 E treatment for 30 minutes,especially Let-7,whose expression level increased by 54% and 1.94 times after 10 minutes and 30 minutes.Secondly,miR-92a-1-p5 was increased by 1.14 times after 20 E treatment for 30 min.The expression levels of the target genes of the five miRNAs were all decreased after 20 E treatment for 10 min.Among them,the expression of flightin was significantly decreased,which was reduced by 37% after 20 E treatment for 10 min,and 39% after 30 min.The expression of abrupt also decreased significantly,which reached by 33% after 20 E treatment for 10 min.It is suggested that 20 E may inhibit the expression of target genes.After 20 E treatment,the expression of miR-92a-1-p5 increased,which led to the down-regulation of the target gene flightin expression.flightin plays important roles in the wing differentiation of pea aphid,reduction of flightin after 20 E treatment might lead to the winged individual of pea aphid decrease,but this need more evidence.This study clarify the exact stage,during which these miRNAs play roles,the preliminary function of miRNAs and ecdysone in the wing dimorphism of the parthenogenetic pea aphid A.pisum were also studied,which may lay foundations forfurther study of the mechanism of miRNAs participating in the wing dimorphism of parthenogenetic aphids. |
PDF Full Text Request