Protein Kinase Pkmyt1 Interact With CAMK1G And To Study The Influence Of Early Development Of Mouse Embryos

ObjectiveScreening the CAMK1 G which can interact with protein kinase Pkmyt1 through the system of yeast two-hybrid and confirming the interaction and mechanism of the CAMK1 G and Pkmyt1, and to further explore how it affects the early development of mouse embryos and laying the foundation for Pkmyt1 protein kinase in the signal transduction pathway research of the Mammalian embryo development.Methods 1. According to the sequence of mouse Pkmyt1（Genebank ID:NM₀23058）, the synthetic primer was designed to construct p GBKT7-Pkmyt1 bait plasmid. 2. Recovering the yeast and preparing the competent cells, then transfecting the p GBKT7-Pkmyt1 bait plasmid into yeast cells to detect its toxicity, activation and expression in yeast. 3. Mixing the human ovarian c DNA library with the yeast cells containing the p GBKT7-Pkmyt1, and the positive clones were extracted and transfected into E.coli, and sequenced for identification. 4.Constructing the plasmid of pc DNA3.1-flag-Pkmyt1 and pc DNA3.1-Myc-CAMK1 G, verifying the interaction between the CAMK1 G and Pkmyt1 protein kinase by co-immunoprecipitation. 5. Detecting the Pkmyt1 m RNA expression in the process of mouse embryos mitosis by real-time PCR. 6.Observing the situation of Pkmyt1 and CAMK1G during the early development of mouse embryos through immunofluorescence.Results 1. p GBKT7-Pkmyt1 bait plasmid was constructed effectively. 2.p GBKT7-Pkmyt1 plasmid is no toxic and no activation to yeast cell,and p GBKT7-Pkmyt1 is expressed in yeast cells. 3. 46 interacting proteins were screened by using yeast two hybrid system. 4. The interaction between the Pkmyt1 protein kinase and CAMK1 G was confirmed by co-immunoprecipitation. 5. Pkmyt1 protein kinase was expressed in mouse embryos detected by real-time PCR,the level is increase first and then reduce. 6. Pkmyt1 and CAMK1 G protein kinase can be co-localized in mouse embryos by immunofluorescence.ConclusionsBy using yeast two hybrid system, we screened 46 interacting proteins which can interact with Pkmyt1. The interaction between Pkmyt1 and CAMK1 G was confirmed by co-immunoprecipitation. Detected by real-time PCR, Pkmyt1 was expressed in each time of the process of mouse embryos mitosis. Pkmyt1 and CAMK1 G protein kinase can be co-localized in mouse embryos by immunofluorescence.The results above suggested that CAMK1 G may regulated the mouse embryos early development through the interaction with Pkmyt1.