A new strategy for identification of highly conserved microRNAs in non-model insect, Spodoptera litura.
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.
Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China.
Lu
L
Gao
Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China.
Hongliang
H
Zuo
Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China.
Keling
K
Liu
Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China.
Haiyi
H
Li
Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China.
Guohua
G
Zhong
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA; D018411:Spodoptera / Q000235:genetics*
D035683:MicroRNAs