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

International Journal of Molecular Sciences

Lu Gao¹  Hongliang Zuo¹  Keling Liu¹  Haiyi Li¹ 

[1] Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China;

关键词: microRNA;    precursor;    identification;    stem-loop RT-PCR;    non-model insect;    sequence conservation;    Spodoptera litura;   

DOI  :  10.3390/ijms13010612

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.