Effect Of The DNA Methylation Inhibitor 5-Azacytidine On The Expression Of Porcine GDF11 And Expression Of Porcine GDF11 In E. Coli

Growth/differentiation factor 11 (GDF11),also known as bone morphogenetic protein 11 (BMP11) is a secreted signaling molecule in transforming growth factor-β(TGF-β) superfamily. Since GDF11 gene was first identified in 1999, studies have been mainly concentrated on its regulative function in early stages of embryonic development, especially in anterior-posterior patterning of the axial skeleton and kidney organogenesis. Carcass length is an important economical trait in pig breeding,and it has been shown to be closely related with the number of thoracic and lumbar vertebrae. In this study,GDF11 is selected as a candidate for studying the effect of DNA demethylation on its expression and elucidating the epigenetic mechanism of variations in porcine caracass length. The result will be helpful for improving carcass trait by using epigenetic methods.The present study contains two parts:PartⅠ: Porcine kidney proximal tubular epithelium cell line LLC-PK1 was treated with different concentrations of 5-azacytidine, a DNA methylation inhibitor, and for different time. The effect of 5-azaC on the expression of porcine GDF11 gene was analyzed by RT-RCR and semi-quantitative PCR using the total RNA extracted from treated and control LLC-PK1 cells. The results indicated that the relative expression level of GDF11 gene among 0.5, 1 and 3μM 5-azaC treatments differ significantly for 24h (P<0.05), but not for 48. And it differ significantly among 0, 0.05, 0.1, 0.3, 0.5, 1 and 3μM 5-azaC treatments for 72h (P<0.05) as well. According to each concentration there are also highly significant changes among 24, 48 and 72 h treatment (P<0.01).PartⅡ: Primers were designed according to porcine GDF11 gene. By RT-PCR, a cDNA sequence encoding 327 amino acids from the C terminal of porcine GDF11 protein was amplified from pig embryo kidney. The target DNA fragment was inserted into the prokaryotic expression vector pGEX-6p-1 and sequenced, and the recombinant plasmid pGEX-6p-1-GDF11 was obtained. The competent E.coli cell strain BL21(DE3) were transformed by the recombinant plasmid pGEX-6p-1-GDF11 and induced by IPTG. The target fusion protein GST-GDF11 was detected by SDS-PAGE and purified by electroeluting. Finally, the purity of the expressed GST-GDF11 fusion protein was examined by SDS-PAGE as well.