Construction Of Maize Recombination Expression Vector With PhyA Gene Encoding Region From An Aspergillus Niger N25

According to the sequence of phytase phyA gene (GenBank Accession No. AF218813) that we registered in GenBank, A pair of primers were designed and synthesized. The upstream primer started with signal cleavage site (XbaI site was linked), and the downstream primer ended at stop codon (Kpn I was linked). By using a high-fidelity polyerase (pfu), an encoding region (1.4kb) was amplified by PCR. The fragment was inserted into Xbal and Kpn I sites of pBPC47 vector, and transformed into JM109 strain. As result, white colonies were screened on LB plate (100ug /ml amp added). The result of enzyme digestion of vector proved that the recombination vector (named pBA) was obtained . pBA vector was digested by EcoR I and HindIII, and the EGH5-phyA-nos gene was taken out, inserted into the multi-cloning site of pCAMBIA1300 vector, transformed into E.coli JM109 strain finally. Positive colonies was selected by situ-hybridization with photo-biotin labeled phyA gene. The result of enzyme digestion of vector proved that recombination vector was obtained (named pBCA).The successful construction of pBCA vector is the key foundation of phyA gene expressed in maize. It is the first time that phytase maize expression vector is tissue-specifically expressed in endosperm and regarding maize seed as plant biology reactor within the country and abroad. The recombination plasmids have been transformed into maize callus, and the differentiation seedlings have been transplanted in farmland.