A Preliminary Study On The Role Of PARD6A In The First Meiotic Division Of Porcine Oocytes

The PAR6 protein encoded by partitioning defectivegene PAR6 is an important polar molecule,discovered in Caenorhabditis elegans originally,mainly including PARD6 A,PARD6B and PARD6 G,and exists the homolog in Drosophila,Xenopus and mouse.In order to explore the effect of PAR6 onporcine oocytesin vitro maturation,the PARD6 A and PARD6 B gene cloned by RT-PCR from porcine ovarian tissue,constructed pPARD6A-Venus and pPARD6B-Venus eukaryotic expression vector,thentransfectedthe successfully constructed vector intoHela cells for validation;Secondly,pPARD6A-Venus and pPARD6B-Venus were transcribed into RNA by transcription in vitro,and injected into porcine oocytes by microinjection technique,and the first polar body excretion rate was counted after maturation in vitro;Thirdly,the GV stage,GVBD and mature stage of porcine oocytes were stained by immunofluorescence staining to investigate the localization of PARD6 A in the maturation of porcine oocytes;Finally,PARD6 A antibody was injected into porcine oocytes,and the first polar body excretion rate was counted after maturation in vitro.The results are as follows:1.The CDS sequence of PARD6 A and PARD6 Bwere cloned byRT-PCR from porcine ovaries,and connected to the eukaryotic expression vector of p-Venus,construction of eukaryotic expression vector for pPARD6A-Venus and pPARD6B-Venus,after double enzyme digestion and sequenced by company,it showed that the vector was constructed correctly.The vector was transfected into Hela cells,and its expression in Hela cells was found,the products showed unconspicuous polar localization.2.Transfer thepPARD6A-Venus and pPARD6B-Venus vectorinto RNA in vitro transcription,two RNA microinjected into GV stage porcine oocytesrespectively,culture in vitro.The results showed that both of them had no significant effect on the excretion rate of the first polar body of porcine oocytes.3.Through the staining of porcine oocytes in GV stage,GVBD stage and MII immunofluorescence,PARD6 A protein showed no obvious polarity orientation in porcine oocytes,and its location is also not with porcine oocytes in vitro maturation changes.4.After the microinjection of monoclonal antibodies of PARD6 A into porcine oocytes,the excretion rate of the first polar body was markedly decreased.Conclusion:We successfully constructed the eukaryotic expression vector of pPARD6A-Venus and pPARD6B-Venus;overexpression of PARD6 A or PARD6 B of porcine oocytes had no significant effect on theexcretion rate of the first polar body.PARD6 A showed no polar localization in the process of porcine oocyte maturation in vitro;interfering with PARD6 A protein decreased the first polar body excretion rate of porcine oocytes in vitro maturation culture.