Cloning of a phyA from Aspergullus niger and Its over Expression in Escherichia coli

In order to acquire a large sum of highly active phytase and develop a new micro-organism biological agent,the research is conducted to amplify and clone the phytase gene phyA from Aspergillus niger with specific primers designed based on phyA gene full-length coding sequence.After restriction endonuclease reaction analysis and rDNA sequencing,the gene phyA was successfully cloned.The gene phyA fragment was recovered from the pBS-T-phyA and ligated with expression vector pET28a+ to construct the recombined expression plasmid pET28a+-phyA.The gene phyA was expressed in Escherichia coli for high efficiency.The gene analysis protein reading frame with an initiation ATG codon and a termination TGA codon at the two ends,which encoded a mature peptide consisting of 467 amino acids.SDS-PAGE electrophoresis analysis and activity measurement showed that the expression product of the molecules,weight of 51KD and the phytase activity reached maximum 1174 at the condition of 0.4g·L-1 IPTG induction.