MicroRNA core machinery genes in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)
عنوان دوره: دومین کنگره بین المللی حشره شناسی ایران
نویسندگان
چکیده
MicroRNAs (miRNAs) are small non-coding RNAs (about 22 nucleotides) that regulate gene expression in post-transcriptional level. MiRNAs are transcribed in nucleus by RNA polymerase II and transported into cytoplasm by Exportin5 (Exp5); miRNAs maturation occur in the cytoplasm by the action of Dicer1 (Dcr1) and 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. In this study, we predicted and experimentally confirmed expression of the miRNA core machinery genes in the cotton bollworm, one of the most polyphagous and cosmopolitan pest species. To identify and characterize putative miRNA core machinery gene candidates in H. armigera, we considered mRNAs from whole transcriptome sequences and ESTs extracted from NCBI as well. By blast search, we identified putative sequences of Dcr1, Ago1, Exp5 from the transcriptome dataset of H. armigera which showed high similarity to the homologous sequences in Bombyx mori and Spodoptera sp. that highlight conserved sequence of these genes in different insects. To find whether these genes are expressed in different developmental stages of H. armigera (i.e. the eggs, first to sixth instars larvae, pre pupa, pupa and adults), total RNA was extracted from each stage and cDNA library was synthesized by using RT-PCR and oligodt primer. Transcript levels of the genes were analyzed by RT-qPCR utilizing 18s rRNA and Actin as the reference genes. Moreover, expression of these genes were measured in different tissues of the forth-instar larvae including gut, fat body and the rest of body. Our results showed that expression of the miRNA core genes in the eggs, first-instar larvae and females were significantly higher than other stages. Exp5 was the most expressed gene in different stages followed by Ago1 and Dcr1, respectively. Tissue specific expression analysis of the genes revealed their expression in the all tissues. In conclusion, we showed that miRNA core machinery genes exist in H. armigera. These genes were expressed during development of the insect particularly in the egg, first instar larvae and adult females. We also showed that these genes were expressed in different tissues including the insect gut and fat body. Widespread expression of miRNA core genes during development of H. armigera highlights the importance of miRNA functions in this insect.
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