A New Strategy for Identification of Highly Conserved microRNAs in Non-Model Insect, Spodoptera litura

Lu Gao,Haiyi Li,Keling Liu,Guohua Zhong,Hongliang Zuo

doi:10.3390/ijms13010612

Lu Gao, Haiyi Li + Show 3 more

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https://doi.org/10.3390/ijms13010612

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Abstract

The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam, were cloned from Spodoptera litura by stem-loop RT-PCR. Two groups of primers were designed, and one of them performed especially well and proved stable. The sequences of two highly conserved miRNAs, sli-miR-14 and sli-miR-2a were identical to those in Drosophila melanogaster. To validate the reliability of this strategy, pre-miR-14 and pre-miR-2a in S. litura as representatives were given as well; this shared high homology with those in D. melanogaster and Bombyx mori, and both mature sequences of sli-miR-14 and sli-miR-2a in their precursors shared 100% identity to the results shown by stem-loop RT-PCR. Moreover, expression patterns of these miRNAs were investigated by real-time quantitative PCR. Sli-miR-14 and sli-miR-2a could be detected successfully and their expression patterns showed similar characteristics with those in model insects, further suggesting stem-loop RT-PCR technology can be used for identification of highly conserved miRNAs in non-model insects. These results provide a simplified and efficient strategy for studying the structure and function of highly conserved miRNAs, especially some critical miRNAs in non-model insects.

Highlights

Introduction3′-untranslated regions (3′-UTR) of messenger RNA (mRNA) of the target genes by incomplete base-pairing and regulate gene expression at post-transcriptional level [1,2,3]
MicroRNAs are small (~22 nucleotides) non-coding RNAs molecules bound to3′-untranslated regions (3′-UTR) of messenger RNA of the target genes by incomplete base-pairing and regulate gene expression at post-transcriptional level [1,2,3]
Mature sequence of sli-miR-2a was underlined by red line, putative sli-miR-13a and sli-miR-13b were underlined by green and blue line, respectively. Both mature sequences of miR-14 and miR-2a in their precursors shared 100% identity to the results shown by stem-loop reverse transcription (RT)-PCR

Summary

Introduction

3′-untranslated regions (3′-UTR) of messenger RNA (mRNA) of the target genes by incomplete base-pairing and regulate gene expression at post-transcriptional level [1,2,3]. The genes encoding miRNAs are usually located in intergenic regions, some in the introns of known genes, and even within expressed sequence tags (ESTs). A stem-loop (hairpin) primary miRNA (pri-miRNA) as a newly-transcribed RNA is processed into a 70~90 nt hairpin long precursor miRNA (pre-miRNA) by Drosha nuclease. MiRNAs have been shown to be ubiquitous in multicellular organisms and many of them are evolutionarily conserved [6,7,8]. MiRNAs were originally isolated by a direct cloning procedure in Drosophila melanogaster and many of them are sequence conserved with those of Caenorhabditis elegans [11,12,13]

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Discussion

Conclusion