多头绒泡菌微原质团瞬时表达系统的构建

Transient expression in microplasmodia of Physarum polycephalum

摘要:

真核生物多头绒泡菌的原质团是研究细胞周期的好材料。但尚无合适的表达体系可供选择。本研究用多头绒泡菌ardC actin基因启动子和终止子分别替换哺乳动物细胞表达质粒pDsRed1-N1的CMV IE和SV40 polyA片段, 构建了多头绒泡菌红色荧光蛋白(RFP)表达质粒pXM1; 用PardC-MCS-DsRed1-TardC替换pTB38表达盒PardC-hph-TardC, 构建了多头绒泡菌RFP表达质粒pXM2。将多头绒泡菌转录延伸因子类似蛋白(PELF1)基因与质粒pXM2重组, 构建了PELF1红色荧光融合蛋白(PELF1-RFP)表达质粒pXM2-pelf1。通过荧光显微镜和激光扫描共聚焦显微镜观察RFP表达发现, 电转参数为4 kV/cm(电场)、1 A(电流)、70 ms(电击时间)时, 质粒pXM1和pXM2电转多头绒泡菌微原质团(≤500 mm)后24~48 h内, RFP荧光最显著; 而PELF1-RFP则主要聚集在多头绒泡菌细胞核, 说明本试验建立的表达系统可以用于研究特定蛋白在多头绒泡菌内的瞬时表达。

Abstract:

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRed1-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 ms electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.