[Expression silence of Physarum polycephalum serine/arginine protein kinase by small interfering RNA].

Abstract

Serine/arginine protein kinases are specific kinase family for phosphorylating SR protein regulating alternative splicing of SR protein and its distribution, localization in the nucleus. However, it is unclear how Physarum Polycephalum Serine/Arginine Protein Kinase(PSRPK) functions in the cells. In order to study its function, Oligonucleotides for transcribing siRNAs were designed and inserted into pSIREN-RetroQ vector to construct pSIREN-PSRPK-1, pSIREN-PSRPK-2, pSIREN-PSRPK-3, pSIREN-PSRPK-4, pSIREN-PSRPK-5 for expressing siRNAs targeting at PSRPK, as well as the negative control pSIREN-PSRPK-Neg. The PSRPK cDNA amplified by PCR was inserted into the pDsRed-N1 vector to construct a pPSRPK-DsRed plasmid. After the pPSRPK-DsRed was co-transfected into HEK293 cell with recombinant siRNA expression plasmids respectively, the PSRPK-DsRed fusion fluorescent protein was observed under fluorescent microscope after 72 hours co-transfection. The results indicated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 were able to inhibit the expression of PSRPK-DsRed fusion fluorescent protein efficiently. RT-PCR and Northern dot blot analysis further demonstrated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 can effectively inhibit PSRPK expression, which accorded with the results under the fluorescent microscope.