Penicillium Q-7 Phya Gene Cloning And Structural Analysis

Phytic acid and phytates are the principle storage forms of phosphorus and myo-inositol in cereal grains, soybeans and oilseeds, which play an important role in keeping the balance of physical phosphorus. Due to lack of phytase, monogastric animals are incapable of hydrolyze phytic acid and phytates, resulting in the decreased usage of phosphorus in feeds. Phytases are a special class of phosphatases that catalyze thesequential hydrolysis of phytates so as to enhancing the utilization of phosphorus in feeds and improving the problems caused by the waste of phosphorus resource.In this study, morphologic observation and homologous comparison of 18SrDNA of filamentus fungi were employed to indentify the isolation acquired from soil, the results shows that the targeted stain was Penicillium. Tardum Thom. We name it Penicillium Q-7.Genomic DNA of Penicillium Q-7 was isolated and paritially digested by Sau3A I . To construct genomic library, the desired 2-5Kb fragments were reclaimed and linked with pUC19 vector cutted by BamH I . PCR technology was used to screen the genomic library. The targetted fragment was subcloned into pMD18-T vector and recombinant plasmid pMD18-T-phyA was successfully constructed. The heterologous phyA was sequenced and analyzed, the result indicated that phyA consists of 1521 nucleosides and encodes 472 amino acid residues, which is approximately the same with phyA reported. Submitted the sequence to Genbank and an Accession Number AY640235 was obtained. Homology modelling shows that this phytase belongs to the family of histidine acidity phosphatase.The phyA gene was subcloned to Pichia pastoris shuttle plasmid pPIC9. Recombinant plasmid pPIC9-phyA was constructed and transformed intoPichia pastorisSMD1168 by electroporation. Recombinants were obtained and identified by PCR, showing that phyA has been inserted in the chromosome of Pichia pastoris.