سرکوب مسیر microRNA در کرم غوزه پنبه، Helicoverpa armigera

MicroRNAs (miRNAs) are small non-coding RNAs (18 to 25 nt) that are produced by all animals and plants as well as some viruses. Their roles have been revealed in many physiological processes including development, cancer, immunity, apoptosis and host–micro-organism interactions through post-transcriptional regulation of gene expression. MiRNA genes are transcribed mainly by RNA polymerase II in the nucleus forming the primary miRNA (pri-miRNA) and cleaved by Drosha to a 60–80 nt precursor miRNA (pre-miRNA), which is then exported to the cytoplasm and matured by the action of Dicer-1. Mature miRNA strand is subsequently incorporated into the RNA induced silencing complex (RISC) containing Argonaute (Ago1) to guide the silencing complex to the target transcripts. Considering the essential role of Dicer-1 in this pathway, we used RNAi to silence the expression of Dicer-1 in the cotton bollworm Helicoverpa armigera. To do this, a specific dsRNAs targeting coding region of Dicer-1 was synthesized and injected into the hemolymph of the fifth instar larva while dsGFP was used injected into the control larvae. Total RNA samples were extracted from whole body of the dsDicer-1-injected larvae and the controls at 48 hours post injection and cDNA libraries were synthesized by using RT-PCR and oligo dT primer. Expression levels of Dicer-1 were analyzed by RT-qPCR utilizing RPL27 as the reference to normalize gene expressions. The results revealed specific silencing of Dicer-1 in H. armigera following the dsRNADicer-1 injection. To assess if silencing of Dicer-1 affected miRNA in the insect, the expression levels of Let-7 and miR-184 were analyzed using stem-loop RT-PCR. The cDNA samples were synthesized using the specific stem-loop primers and the expression levels of the miRNAs were assessed by qPCR using miRNA specific forward and universal reverse primes. The results showed that silencing of Dicer-1 decreased the levels of cellular miRNAs, Let-7 and miR-184. Therefore, RNAi of the Dicer-1 resulted in miRNA pathway blockage and miRNA contents depletion in the fifth instar larvae of H. armigera.