摘要
MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction ofeukaryotic miRNA expression vector was described. The cytoplasmic actin 3 （A3） promoter and flanked sequences of miRNA-9a （miR-9a） precursor were amplified from genomic DNA of the silkworm （Bombyx mori） and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein （EGFP） gene was used as reporter gene. The Bombyx mori N （BmN） cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression ofmiR-9a was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR） and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.
