Transformation Of The Aspergillus Awamori Phy Gene In Tobacco

The paper aims to purify and characterize phytase produced by Aspergillus awamori, clone the gene of phytase, construct the plant expression vector, and transfer it into tobacco, and analyze the expression of the phytase gene in tobacco and the characterization of the recombinant enzyme.Extracellular phytase produced by Aspergillus awamori using technique of semi-solid cultivation was purified to homogeneity by employing an initial ultrafiltration step, followed by chromatography using anion exchange, gel filtration and chromatofocusing steps. The purified enzyme was an 118kDa protein including 40.6% glycosylation. It possessed a temperature optimum of 55℃, and a pH optimum of 5.5. The K_m value of the phytase for dodecasodium phytate at 37℃ was 1.03 nM with a V_max2.13 μM/min. Phytase activity was not significantly affected by EDTA. Activity was moderately inhibited by Ca²⁺, Mg²⁺, Mn²⁺ and was evidently inhibited by Fe²⁺, Zn²⁺. The enzyme displayed higher thermostability at the high temperature than the commercial phytase product.The phytase gene and its ORF ware cloned by the way of PCR, and a plant expression vector pBI121-SP/phy was constructed. The resultant plasmids pBI121-SP/phy was transferred into Agrobacterium tumefaciens (LBA4404) by the liquid nitrogen freezing thaw method.The phytase gene was transferred into tobacco via Agrobacterium mediation. The results of PCR and Southern Blotting showed that the phytase gene had been transferred into tobacco. The phytase activity assays of tobacco leaves demonstrated that the recombinant phytase gene could be expressed in tobacco.The primary purification and characterization of the recombinant enzyme expressed in tobacco had been done. The recombinant enzyme possessed the same temperature and pH optimum with the phytase produced by Aspergillus awamori, although it displayed a little lower thermostability. All the results showed that the recombinant phytase gene could be expressed in tobacco exactly.