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目的:研究小鼠CDC25B蛋白S149位点对小鼠1-细胞期受精卵发育的影响及S149位点与S321位点的关系。方法:构建pBSK-CDC25B-S149A和pBSK-CDC25B-S149A/S321A突变体,体外转录成mRNA;采用小鼠超排卵技术取G1期受精卵,显微注射CDC25B-WT-mRNA和突变CDC25B-S149A-mRNA、CDC25B-S149A/S321A-mRNA,观察其对受精卵发育、M期促进因子(MPF)活性及CDC2-pTyr15磷酸化状态的影响。结果:CDC25B-S149A-mRNA注射组受精卵卵裂率明显高于CDC25B-WT-mRNA注射组,MPF活性提前1 h达到高峰;而联合突变体CDC25B-S149A/S321A-mRNA注射组卵裂率又显著高于CDC25B-S149A-mRNA注射组。结论:在小鼠1-细胞期受精卵有丝分裂过程中,蛋白激酶A(PKA)对小鼠CDC25B蛋白S149位点的磷酸化修饰是控制受精卵G2/M转换的重要方式,并且S149位点与S321位点可能具有协同作用。
Objective: To investigate the effect of mouse CDC25B protein S149 on the development of mouse 1-cell stage fertilized eggs and the relationship between S149 and S321. Methods: The pBSK-CDC25B-S149A and pBSK-CDC25B-S149A / S321A mutants were constructed and transcribed into mRNA in vitro. The G1 phase fertilized eggs, CDC25B-WT-mRNA and CDC25B-S149A- mRNA, CDC25B-S149A / S321A-mRNA, and their effects on the development of fertilized eggs, M-phase promoting factor (MPF) activity and CDC2-pTyr15 phosphorylation were observed. Results: The cleavage rate of fertilized egg in CDC25B-S149A-mRNA injection group was significantly higher than that in CDC25B-WT-mRNA injection group, and the MPF activity peaked at 1 hour ahead of time. However, cleavage rate of CDC25B-S149A / S321A- Significantly higher than the CDC25B-S149A-mRNA injection group. CONCLUSION: Phosphorylation of protein kinase A (PKA) on murine CDC25B protein S149 during mitosis of mouse 1-cell stage zygotes is an important way to control the G2 / M transition of zygotes, and the interaction of S149 with S321 sites may have synergistic effects.