| FAK, AKT, TSC2 is the important proteins in cell signaling pathways.In human and some other animals, FAK, TSC2 and AKT have been researched for many years, but there is not enough attention have been paid on in the goat. Even the sequences of these three genes are still unknown till now. Therefore, the research of FAK, AKT, TSC2 genes and encoded proteins can distinguish genetic differences of species and evolutionary features, and it is fundamental to the study of cell growth and development.RT-PCR was performed first, and the CDS fragment of FAK, TSC2, and AKT gene cDNA were got by sequencing. Secondly, the expression patterns of these genes were analyzed among different organs of cashmere goat by semi-quantitative PCR. Thirdly, yeast two hybrid (YTH) was carried out to elucidate how FAK interacts with TSC2 and AKT.The sequencing results showed that, in Inner Mongolia Cashmere Goat, FAK gene cDNA is 3159bp, TSC2 gene cDNA is 5184bp (HQ684023.1), AKT gene cDNA is 1443bp(HM130679); and their encoding proteins are 1052 amino acids,1727 amino acids, and 480 amino acids, respectively.According to the results of gene cloning, the recombinating bait vectors (pGBKT7-AKT,pGBKT7-FAK1,pGBKT7-FAK2 pGBKT7-FAK3,pGBKT7-TSC2) and prey vectors (pGADT7-AKT,pGADT7-TSC2) were got. After confirmation that the recombinating bait vectors and prey vectors have been transformed into two different yeast strains, the yeast were mating in flasks. The screening of yeast mating showed that it was the C-terminal of FAK (aa649-1076) that interacted with TSC2 in its middle region-from amino acid 542 to 1061. And this C-terminal of FAK also interacted with the AKT (aa1-474). The result of Co-Immunoprecipitation showed there were the interactions between FAK and TSC2.In sum, these results have enriched the fundamental data of Gene sequences in Cashmere goat. The molecular mechanism in series between FAK signaling pathway and AKT-/TSC2 signaling pathway has been further clarified.
|
PDF Full Text Request