| microRNAs(miRNAs) are a class of endogenous small non-coding RNAs about 22nt long that present extensively in many species such as Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Homo sapiens and Arabidopsis thaliana et a, which play important roles in many biological processes as posttranscriptional regulators of gene expression. Although several hundred miRNAs have been predicted and identified by computational and experimentally methods both in animals and plants, search for new miRNAs is still a hot academic domain because miRNAs identified so far are far from saturated based on theoretical prediction. Recently, identification of miRNAs using bioinformation is gradually popular with the completion of genomic sequencing in human and many model organisms. miRNA in mammalian is highly conversative during evolution, not only in highly relative species, but also in widely divergent species. So we can use identified seed sequences to search silkworm miRNA and their targets by computational analysis. And this may help us to understand the impacts and functions of miRNA to the growth in silkworm more clearly and deeply, and at the same time, will be propitious to improve the breed and quality of silkworm by genetics, in this way high yield breed could be selected and more benefits will get from it.We apply comutational RNomics and experiment Rnomics to predict and validate miRNA in silkworm based on sequence conservation of mature miRNAs and their precursor displaying hairpin structure, and perform RT-PCR assay to verify expression of mature miRNAs. The following is the summarization of our result:1. We identified 62 potential miRNAs in silkworm. Computational analysis showed that a majority of miRNA identified in this work can be folded to the typical stem-loop secondary structure of the miRNA family. Our results indicate that the majority of potential miRNAs have homologues in previously known Drosophila melanogaster.2. Our results indicate that several miRNAs are conserved among families of Arthropods, such as miR-965, miR-993 and miR-1000. And we found that both of these mature miRNAs and their precursors are highly conserved in Arthropods.3. Analysis show that the 62 homologue of Drosophila melanogaster miRNA belong to 40 families, and miR-2, miR-9, miR-25 and miR-263 family have 5, 3, 2 and 2 members, respectively. Most of families only have one member, which is different from plant. Members of animal family display multi-copy, closely related or homology.4. Through sequence analysis, we found that of the 62 miRNA identified in this work, 20 are grouped into 8 clusters, and the largest cluster is composed of 5 miRNAs. Further analysis indicates that these clusters are all composed of homologous miRNAs and they can also be found in Drosophila melanogaster.5. We predict target of identified miRNAs using 3-UTR. Further analysis of miRNAs targets prediction indicates that the 10 targets participate in some important life activities in silkworm, for example, metamorphic development, apoptosis and signal transduction. These target genes involved those of hormone-regulated pathways(such as juvenile hormone esterase/Jhe), cell cycle control (such as inhibitor of apoptosis protein/lap), signal transduction (such as achaete-scute-like protein/ASE). The diversity of targets show that miRNAs paly a vital role in cellular processes.6. Performing RT-PCR assay of 4 miRNAs, we found that they have different expression level among different organs, such as miR-iab-4as. These results suggest that the examined miRNAs have diverse expression patterns during development.
|
PDF Full Text Request