初探lncRNA-Tubb4b的蛋白编码能力及其对微管基因的调控

The Preliminary Research of the Protein Coding Ability of lncRNA-Tubb4b and Its Regulation on Microtubule Genes

旨在探索长链非编码Tubb4b基因(lncRNA-Tubb4b)是否具有蛋白编码能力及其对Tubb4b基因的调控作用。本研究在lncRNA-Tubb4b中添加起始密码子ATG、0/1/2个的T尾和Flag标记,通过构建载体、脂质体转染和Western blot等技术对lncRNA-Tubb4b蛋白编码能力进行分析;随后,构建过表达载体和合成siRNA,通过脂质体转染和实时荧光定量PCR等技术检测lncRNA-Tubb4b对微管基因Tubb4b的影响。结果表明,我们扩增了添加0、1和2个T尾的Flag片段,其与lncRNA-Tubb4b融合后,成功构建到pcDNA3.1(-)载体中;转染小鼠精原细胞后发现,添加0、1和2个T尾的lncRNA-Tubb4b均没有FLAG蛋白条带;以pcDNA 3.1(-)-EGFP载体为对照,在小鼠精原细胞中过表达lncRNA-Tubb4b时,对照组细胞有荧光产生,试验组Tubb4b基因的mRNA表达水平极显著地降低(P<0.01);在小鼠精原细胞中干扰lncRNA-Tubb4b表达后,极显著地提高Tubb4b基因的mRNA表达水平(P<0.01)。这表明,lncRNA-Tubb4b确实不编码蛋白质,过表达或干扰lncRNA-Tubb4b的表达会极显著地降低或升高Tubb4b基因的表达。

The study aimed to explore whether the long non-coding RNA of Tubb4b(lncRNA-Tubb4b)has protein coding capability and the regulatory effect of lncRNA-Tubb4b on the Tubb4b gene.The ATG,0/1/2 T tails and Flag sequence were added to lncRNA-Tubb4b to analyze the coding ability of lncRNA-Tubb4b by constructing vector,liposome transfection and Western blot;Then,overexpression vector of lncRNA-Tubb4b was constructed and the siRNA of lncRNA-Tubb4b was synthesized,while the liposome transfection and real-time fluorescent quantitative PCR technology were used to detect the influence of lncRNA-Tubb4b on the Tubb4b gene.The results showed that the Flag fragments with 0,1,and 2 T-tail,was amplified in this study,were fused with lncRNA-Tubb4b,then were successfully constructed into the pcDNA3.1(-)vector;After transfection of mouse spermatogonia,it was found that lncRNA-Tubb4b with 0,1,and 2 T-tail had no FLAG protein bands.With pcDNA 3.1(-)-EGFP vector as a control,overexpression of lncRNA-Tubb4b significantly reduced the mRNA expression level of Tubb4b in mouse spermatogonia(P<0.01),while there was fluorescence in control cells.And interfering the expression of lncRNA-Tubb4b significantly increased the mRNA expression level of Tubb4b in mouse spermatogonia(P<0.01).It is indicated that lncRNA-Tubb4b does not encode protein,and overexpression or interference with the expression of lncRNA-Tubb4b will extremely significantly reduce or increase the expression of Tubb4b.