The Study Of Protein Expression In Pygol And Nulp1 Gene

Heart development is an extremely complicated process which requires precise regulation of a series of genes. The mutations and abnormal spatio-temporal expressions of these genes will impact the cardiomyocyte differentiation, the diversity and the occurrence of cardiac cell, resulting in mortality in pregnancies and cardiac disease. In recent years, the research on heart disease has made a great progress, however, the mechanisms underlying the diseases occurs remain 9Pygol gene was previously cloned from a human embryonic heart cDNA library in our lab and the function of this gene in heart development was investigated through a cell line suggesting that Pygol plays important roles in cardiomyocyte differentiation that may activate the Wnt/β-catenin pathway. Here, the hydrophilic antigen regions of the Zebrafish gene Pygol selected by bioinformation analysis were obtained by PCR amplification, and then were inserted into pGEX-4T-1 vector to generate the prokaryotic expressing system. The recombinant plasmids were transformed to E. coil. Expression of the target proteins were induced by IPTG and assayed by SDS-PAGE, respectively. After purified, the fused proteins were injected into New Zealand big white rabbits to produce polyclonal antibody, respectively. Western blotting revealed that the Pygol protein was expressed in brain, heart, muscle and eye. Its protein expression started in embryo at 2 cells period. After knocking-down the Pygol expression in zebrafish embryos with Pygol-MO, Western blotting showed that the protein expression of a series of transcription factors such as GATA4, Nkx2.5 and dHand was reduced, indicating that Pygol associated with the early development of heart.Also, expression of the genes such as Nodal, Lefty1, and Pitx2 which mediate the left-right asymmetry development of heart was reduced, indicating that Pygo1 might be associated with the left-right asymmetry development of heart.In previous work, a human heart development gene Nulp1 was identified and the heart specific expression gene FHL2 was identified as an interacting protein candidate. It maybe play an important role in cardiomyocyte differentiation by inhibiting the Wnt/β-catenin pathway. In order to help our understanding the function of Nulp1, its knockdown was performed in zebrafish embryos with a morpholino directed against Nulp1. Western blotting showed that the expression of a series of transcription factors such as GATA4 and Nkx2.5was increased. The expression vectors for five deletion mutants of Nulp1 and FHL2were also constructed, respectively. The results by co-immunoprecipitation analyses showed that the basic region in bHLH domain of Nulp1 was the only region that interacted with FHL2and the LIM1 domain of FHL2 interacted with Nulp1.Taken together, the present studies demonstrate that Pygol and Nulp1 might have important function in the heart development. Clarifying the mechanisms underlying these genes founction will lay the foundation for heart disease research.