In silico cloning and bioinformatic analysis of PEPCK gene in Fusarium oxysporum

Phosphoenolpyruvate carboxykinase (PEPCK), a critical gluconeogenic enzyme, catalyzes the first committed step in the diversion of tricarboxylic acid cycle intermediates toward gluconeogenesis. According to the relative conservation of homologous gene, a bioinformatics strategy was applied to clone Fusarium oxysporum phosphoenolpyruvate carboxykinase gene (PEPCK) by blasting search of EST database with homologous gene cDNA of Neurospora crassa and identified. Some characters of the PEPCK that were analyzed and predicted by the tools of bioinformatics in the following aspects include the composition of amino acid sequences, physical and chemical properties, O-glycosylation site, hydrophobicity or hydrophilicity, secondary and tertiary structure of the protein and function. These results showed that the full-length of PEPCK was 1771 bp and it contained a complete ORF (1575 bp), encoded 524 amino acids, which is much conserved in ascomycetes. The calculated molecular weight of PEPCK was 58358.2 Da, theoretical pl of 6.84. It has 20 a-helices, 37 sheets, and 12 glycosylation sites. It was a hydrophilic and stable protein with active site, ATP - binding site, metal-binding site and substrate-binding site.