Regulation Of Wing Dimorphism By Dnmt And The MiRNA Targeting In Nilaparvata Lugens

The brown planthopper(BPH),Nilaparvata lugens,is a monophagous insect pest and feed only on rice.The BPH has wing dimorphism,with long winged 'macropterous' and short-winged 'brachypterous' forms.The brachypterous forms are responsible for reproduction,and the macropterous forms are potentially migrants and are responsible for colonizing new fields.BPH is an important rice pest in China,Japan,Thailand,Vietnam,Indonesia and other many Asian countries.Here,we knocked out the Dnmt by dsRNA injection,suggesting DNA methylation might have a regulatory role on the wing dimorphism in the brown planthopper.The miRNA targets of both Dnmtl and Dnmt3 were predicted and validated by dual luciferase reporter system,indicated five miRNAs participated in the regulating Dnmt genes.1.The expression patterns and RNA interference of Dnmt geneThe expression pattern of Dnmtl and Dnmt3 were analyzed in tissues of head,thorax,midgut,fat body,ovary and testis.The results showed that the mRNA level of Dnmt1 and Dnmt3 were the highest in ovary,which is significant higher than in the testis,indicated that DNA methylation might involve in regulating the reproductive process and ovary development of BPH female adult.We chosed the two-days newly emerged brachypterous females to knock down both Dnmtl and Dnmt3.The fifty RNAi-treated individuals and fifty males were kept in the beaker.The percentage of brachypterous and macropterous individuals of the offsprings were counted.The results indicated that dsDnmt3-treated group had a significant higher percentange of macropterous adults in the offsprings.However,there was no obvious difference in the dsDnmt1-treated group comparing with the control.In all,the results showed that DNA methylation have some effects on the regulation of wing dimorphism in the BPH.2.Prediction of miRNA targeting Dnmt geneWe predicted the miRNAs targeting Dnmt gene by miranda,targetScan,microTar,PITA,RNAhybrid software.Then the intersection among the five software prediction results were chosed as the cadidates for further anallysis.This strategy would sinificantly improve the miRNA targeting reliability.3.Expression analysis of miRNA targeted on Dnmt geneThe expressions of selected miRNAs(miR-124,miR-263a,miR-263b,miR-980)were analyzed in the nymphs from the 1st to 5th instars of the macropterous and brachypterous using qRT-PCT.The results showed that these miRNAs are differentially expressed in different nymph stages,suggesting that they might have important roles in regulating wing dimorphesim.4.miRNA target validation using Dual luciferase reporter systemTo validate the target relationship between miRNAs and their predicted target genes,the 3'UTR of Dnmtl and Dnmt3 genes were cloned into the pMIR-REPORT plasmid,then co-transfected the HEK293T cell with miRNAs NC mimics.The results showed that miR-263a?miR-263b?miR-279-5p inhibited the expression of Dnmtl,miR-2a?miR-87a inhibited the expression of Dnmt3.These miRNAs might directly participate in the regulation of wing dimorphesim.