Ectopic expression of RNA-dependent RNA polymerase in Drosophila melanogaster and microRNAs identification in Helicoverpa armigera

Abstract

Drosophila melanogaster, along with all insects and the vertebrates, lacks an RNA-dependent RNA polymerase (RdRp) gene. We therefore asked whether the C. elegans RdRp genes could be introduced into Drosophila in order to study the effects on RNAi. This system also allowed us to ask whether there were any effects on development, possibly due to interference with microRNA (miRNA) regulation of genes. To address these questions, we introduced the two C. elegans RdRp genes, rrf-1 and ego-1, into D. melanogaster through microinjection. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. Activation of the RdRp genes did not elicit any observable phenotypic changes in the eyes, wing discs, or whole body of adult flies. RT-PCR confirmed the successful RdRp gene expression. We also asked whether these RdRps were capable of enhancing RNAi of a specific, known target gene, triggered by a dsRNA corresponding to that gene. In order to test this, we selected pebble as the candidate endogenous and eGFP as an exogenous gene. Expression of RdRps in RNAi results in differential silencing activities: rrf-1 enhances transitive RNAi and ego-1 silences transgenes by a non-RNAi pathway, the silencing by ego-1 is independent of dsRNA-dependent RNAi, appearing to be transcriptional and restricted to transgenes. We therefore postulate that ego-1 transcriptionally silences transgenes through mitotic unpaired gene silencing mechanism in Drosophila. For identification of miRNAs in Helicoverpa armigera, initially, next generation Illumina sequencings were performed with three Helicoverpa total RNA samples from different tissues and different stages, i.e, neonate, gut, and pupae. about 1.26M unique sequences from 19-24 nts long were harvested. To identify candidate miRNAs, sequence alignment with miRNAs retrieved from miRBase from similar species and the pipeline miRDeep analysis were used. Sequence comparison matched 76 distinct miRNAs while miRDeep prediction gave 164 predicted miRNAs, this gave final 204 miRNAs in total, among which 76 miRNAs are conserved and 128 are novel. 15 miRNAs from above are uniquely expressed in the gut; three of them were confirmed with Northern blot and Hybridization.