Expression And Functional Analysis Of Dnmt3 And Wing Development Associated Genes In The Brown Planthopper

The brown planthopper(BPH),Nilaparvata lugens,is an important agricultural insect,causing huge yield loss in rice production.In this work,we cloned the full-length cDNAs of Dnmt3 by RACE.The gene structure and domain of Nlu-DNMT3 and its orthologous were analyzed.Developmental expression pattern of Nlu-Dnmt3 in BPH was detected by quantitative real-time PCR(qRT-PCR).RNA interference(RNAi)of Dnmt1 and Dnmt3 was conducted by injecting dsRNA.Knockdown of Nlu-Dnmt3 led to reduced number of offsprings,suggesting that DNA methylation plays an important role in the fecundity.1.Molecular cloning and gene structure analysis of Dnmt3 in N.lugensThe sequence of Dnmt3 was obtained from the brown planthopper transcriptome.After the verification by RT-PCR,the full length transcript of Nlu-Dnmt3 was successfully cloned by rapid amplification of cDNA ends(RACE).The cDNA sequence of Nlu-Dnmt3 had been submitted to NCBI and we got the GenBank accession number:KP890855.Then amino acids of DNMT3 were used to calculate molecular weights(Mw)and isoelectric points(pl).Domain analysis,gene structures and phylogenetic analysis were carried out.Nlu-DNMT3 possessed one catalytic domain—DNA methylase domain,which was conserved in other species.The catalytic domain shared high sequence similarity.There was a big difference in length of Dnmt3 between different species.Gene length of Nlu-Dnmt3 was similar to that of two types of bees in Hymenoptera.Compared with vertebrates counterparts,insect Dnmt3 had long exons,but less number.Phylogenetic analysis showed that Nlu-Dnmt3 had a closely evolutionary relationship with Hymenoptera insects.2.Expression analysis of Nlu-Dnmt3 in different development stagesDevelopmental expression profile of Dnml3 were analyzed in the brown planthopper.The nymphs samples from the 1st to 5th instars,macropterous and brachypterous females at egg-laying stage and virgin females,macropterous and brachypterous males were chosen to study expression changes.The results indicated that Dnmt3 mRNA level was low and simliar in nymph stages.But there was a big difference in different types of adults.The expression level was higher in brachypterous than in macropterous regardless of male and female.The expression level was considerably higher in females at egg-laying stage than that in virgin females and nymphs.Males had the lowest expression level of Nlu-Dnmt3.In order to further examine the dynamic change of Nlu-Dnmt3 in the adult stage,we selected 1 to 7 day's adult of four types and studied the gene expression patterns,yielding similar results.3.RNAi of Nlu-Dnmtl and Nlu-Dnmt3 in N.lugensRNAi was conducted with dsRNAs designed based on the sequences of Nlu-Dnmtl and Nlu-Dnmt3.Brachypterous females with eggs were selected for RNAi and the back of the chest was chosen as the injection site.The injection volume was 200 nl and the concentration of dsRNA was 2,500 ng/?l.Thirty females and twenty males was put in the beaker.Three separate repeated experiments were carried out.Compared with the control,the levels of Nlu-Dnmt1 and Nlu-Dnmt3 mRNA on the 24h?48h?72h after dsRNA injection were significantly low,indicating that Nlu-Dnmt1 and Nlu-Dnmt3 dsRNAs had a good effect.The number of the offsprings in RNAi-treated group was reduced significantly after the interference of Nlu-Dnmt1 and Nlu-Dnmt3.4.RNAi of wing developemnt related genes in N.lugensThree wing development related genes in Drosophila were obtained from Flybase,then a homologous search was conducted in the genome of N.lugens,finding the orthoulogous genes.Feeding-based RNAi was carried out with dsRNAs designed based on the sequences of the three genes.Second instar nymphs were selected for RNAi and the final concentration of dsRNA was 200 ng/?l.The level of mRNA at 24h,48h and 72h after feeding were detected by qRT-PCR.The mRNA level of the three genes were significantly declined.However,there is no apparent effect on the wing development and dimorphism.