Screening Of Candidate Molecules Interact With Wee1 By Yeast Two-hybrid And The Effect Of Wee1 On Early Development Of Mouse Embryos

ObjectiveStudying on the expression of protein kinase Wee1 during the one-cell-stage mouse embryos. Screening of new candidate molecules which interact with protein kinase Wee1 by yeast two-hybrid and confirming the interaction between jun B and Wee1.Laying the foundation for further exploring the mechanism of Wee1 in early developmental regulation mammalian embryos.Methods1. The expression of Wee1 m RNA and protein was detected by Real-time PCR and Western Blot in the each period of the one-cell-stage mouse embryos.2. Molecular cloning methods was used for construction of recombinant yeast two-hybrid plasmid p GBKT7-Wee1,and verified the toxicity,self activation and expression of it in the yeast.3. Yeast two-hybrid system was used for screening of the candidate molecules that interact with protein kinase Wee1 from human ovary c DNA library and verified the interaction in the yeast.4 Co-IP was used for verifying the interaction between jun B and Wee1 in mammalian cell.Results1. The protein level and m RNA level of Wee1 in mouse one-cell stage embryos changed along with the progress of cell cycle,decreased significantly in M period.2. The recombinant bait plasmid p GBKT7-Wee1 was constructed successfully,and the bait plasmid has been certified non-toxic and without self activated ability,also the bait plasmid could be expressed in the yeast.Therefore the screening of yeast two-hybrid can be made.3. The yeast two-hybrid system has been done,and 30 interacting proteins were screened out which interacted with protein kinase Wee1,the interaction was also certified in the yeast.4. The transcription factor jun B was interacted with protein kinase Wee1 in the cells.Conclusions30 interacting proteins which interacted with protein kinase Wee1 were screened by yeast two-hybrid.The transcription factor jun B was indeed interacted with protein kinase Wee1.The dynamic expression of Wee1 could indicate that Wee1 may regulate the early development of mouse embryos by the interaction with transcription factor Jun B.