ATR-Chk1-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控

ATR-Chkl-Cdc25 involved in the regulation of Dictyostelium discoideum during G2/M cell cycle

显微镜观察盘基网柄菌野生型KAx--3细胞和突变型RNAi-allC细胞,计数结果表明后者的单细胞繁殖速度约为前者的8倍.为探究该突变型盘基网柄菌细胞周期缩短的原因,用荧光定量PCR和western blot研究了ATR-Chk1Cdc25信号通路在其中的可能作用.实验结果表明:RNAi-allC细胞中cdc25基因相对表达量约为KAx-3细胞的8倍,而其Chk1与ATR基因的相对表达量却明显低于KAx-3细胞.突变细胞中Cdc25蛋白含量高于KAx-3细胞,但其Chk1蛋白含量却显著低于KAx-3细胞.这些数据表明,两种类型细胞之间的ATR、Chk1、Cdc25在mRNA水平和蛋白表达上均存在差异,特别是ATR基因表达量的不同明显影响ChK1和Cdc25的表达量,提示ATR-Chk1-Cdc25信号通路应该在一定程度上参与了盘基网柄菌细胞周期G2/M期的调控.

The cell proliferation of wild type KAx-3 and mutant type RNAi-allC observed by light microscope and cell counting, The latter was divided 8 time faste than the former. To eval uate the reason why RNAi-allC cell cycle shortened, the function of ATR-Chkl-Cdc25 signaling pathway were explored by quantitative PCR and western blot techniques. The results showed the differences of ATR, Chkl, Cdc25 in mRNA contents and protein level existed in KAx-3 and RNAi-allC cells, that is, the expression of ATR and Chlel ratio of RNAi-allC to KAx-3 was 0.69：1 and 0.1：1 respectively; the expression of Cdc25 in RNAi allC cells was 8 times that of KAx-3 cells. The data suggested that once the expression of ATR had little change, Chkl andCdc25 expression changed greatly. Western blotting results were consistent with Q-PCR re- ports. The Chkl protein contents were significantly less in mutant type RNAi-allC than that in KAx-3cells; the Cdc25 protein contents were higher in RNAi-aI1C cells. The above mentioned results suggest that ATR-Chk1-Cdc25 signaling pathway involved in the regulation of the G2/M phase in Dictyostelium discoideum.