论文部分内容阅读
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 of eukaryotic 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 of miR-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.
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 of eukaryotic miRNA expression vector was described. 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. Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested for 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression o f miR-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.