Research On The Function Of Aurora A/bora And PLK1 In Mitosis Of Mouse Zygotes

Objective: The molecular mechanism of mitosis has been a hot topic in molecular biology research.In recent years,research on mitotic-related kinases has also made some progress.In somatic cell mitosis,the conserved serine-threonine kinases Aurora A and PLK1 play an important role in the G2/M transition and cell cycle regulation.Bora is a mitotic regulator found in recent years.One of its functions is as a binding protein of Aurora A,which indirectly plays a role in regulating mitosis progression.The first cleavage of the fertilized egg marks the beginning of life,but the molecular mechanism is not yet clear.The first cleavage of the fertilized egg is not exactly the same as the normal mitotic process.The function of Aurora A,Bora and PLK1 in the first cleavage of fertilized eggs has not been reported to be systematically studied.Mice are mammalian models with a very high degree of similarity to human genes.In addition,the long duration of the first cleavage of the mouse fertilized eggs also provides us with good conditions for the first mitosis study of fertilized eggs.Therefore,this study investigated the expression and localization characteristics of Aurora A,Bora and PLK1 in fertilized egg 1-cell to 2-cell stage and their possible interactions.In order to further study the function and the mode of action of Aurora A,Bora and PLK1 in fertilized eggs,the interactions among them and the related signal pathways provide the basis for the study.Methods: In this study,mouse fertilized eggs were used as a research model to introduce siRNA into mouse fertilized eggs by microinjection to knock down the expression levels of Aurora A,Bora and PLK1 respectively,and then explored by Real-time PCR and Western-Blot techniques.The expressions of Aurora A,Bora and PLK1 in fertilized eggs of normal and knock-down groups were different.Their location and co-localization were detected by indirect immunofluorescence and confocal laser scanning microscopy.The expression of one gene was down-regulated by SPSS12.0 software.The effect on the process of cleavage from the 1-cell stage to the 2-cell stage was explored to investigate its role in the first mitosis of mouse fertilized eggs.Results: 1.The mRNA and protein expression levels of Aurora A and Bora in the fertilized eggs of the normal group showed an upward trend and peaked in the M phase.2.Both PLK1 and Aurora A were located in the cleavage furrow in M phase.3.The knockdown effect of cytoplasmic siRNA on Aurora A,Bora and PLK1 was verified by comparing the mRNA and protein expression levels of Aurora A,Bora and PLK1 in the normal group.4.Knocking down the expression levels of one of Aurora A,Bora,and PLK1 genes will affect the expression levels of the other two.5.Aberrant accumulation of Bora in the cytoplasm occurred in the Aurora A and PLK1 knockdown group after entering the M phase,forming a filamentous bright band that connects the two daughter nuclei.6.Aurora A,which was originally localized in the nucleus of Bora knockdown,was located in a ring around the cell membrane.There was no distribution in the nuclear area and its surroundings.PLK1 lost its nuclear entry in G2.7.After PLK1 knockdown,Aurora A was largely distributed in the nucleus during G2 phase,and its content was significantly higher than that of the cytoplasm.8.The splitting of fertilized eggs in the knock-down group was delayed and the abnormal splitting rate was greatly increased.Conclusion: Aurora A,Bora,and PLK1 expressed most significantly in the M phase of the fertilized egg 1/2 cell cycle in mice.They interacted and interacted with each other,and were involved in the cleavage and spindle of the mouse fertilized egg.Body formation and other functions jointly regulate the early division of mouse fertilized eggs.