siRNA对多头绒泡菌丝氨酸/精氨酸蛋白激酶表达的沉默

EXPRESSION SILENCE OF PHYSARUM POLYCEPHALUM SERINE/ ARGININE PROTEIN KINASE BY SMALL INTERFERING RNA

SR蛋白激酶(serine/arginine protein-specific kinase,SRPK)是一类特异磷酸化剪接因子SR蛋白的激酶家族,可调节SR蛋白的选择型剪接,影响SR蛋白在核内的分布与定位,在pre-mRNA剪接调控上具有重要的作用。目前对于多头绒泡菌SRPK的功能仍不清楚。为了进一步研究PSRPK的功能,设计可转录小干扰RNA(siRNA)的寡核苷酸序列含U6启动子的载体pSIREN-RetroQ定向连接,构建了真核表达质粒pSIREN-PSRPK-1,pSIREN-PSRPK-2,pSIREN-PSRPK-3,pSIREN-PSRPK-4,pSIREN-PSRPK-5及对照质粒pSIREN-PSRPK-Neg。把编码PSRPK基因的cDNA与红色荧光蛋白表达载体pDsRed-N1重组,构建了表达PSRPK-红色荧光融合蛋白的表达质粒pP-SRPK-DsRed。将真核表达质粒和质粒pPSRPK-DsRed共转染HEK293细胞。转染后72h通过倒置荧光显微镜观察,结果表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5可以有效沉默红色荧光融合蛋白的表达;进一步采用RT-PCR和Northern点杂交分析表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5对红色荧光融合蛋白mRNA表达具有明显的抑制作用与倒置荧光显微镜下观察到的结果一致。这为进一步研究多头绒泡菌SRPK的功能奠定了实验基础。

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-Nl 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-transfectiono 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.