Study On Transformation Of Linearized PhyA Ⅱ Gene Into Maize Inbred Lines By Pollen-tube Pathway

The paper aims to obtain phytase transgenic maize by genetic engineering technique, which makes transgenic maize become a kind of high add-ons value animal feedstuffs by degrading phytic acid and releasing phosphorus in maize. At the same time, it can enhance utilization rate of phosphorus for monogastric animals and solve environmental problems as well.Three linearized DNA fragments without selectable marker gene and vector backbone were constructed containing phyA gene with regulation sequences(UPN) or T-DNA borders(T-UPN) or the plasmid borders(V-UPN). These three linearized genes were transformed respectively into maize via pollen-tube pathway.PCR amplification for three transformants T₁ seeds showed different positive rate: UPN was 0.44%, T-UPN was 2.2% and V-UPN was 1.8%. The results also showed that the ratio of successful transformant by pollen-tube pathway was related to the location of seeds and the time after pollination but not to the line of maize.Southern-blot analysis indicated that the foreign gene has been integrated into the genomes. Positive results of RT-PCR and phytase activity assays further demonstrated that the phyA gene has been expressed at RNA and protein levels in the transgenic maize.The results proved that by pollen-tube pathway technique, transformation of maize with linearized gene without selectable marker and vector backbone is possible and doable, so that the hidden trouble inducing by selectable marker gene and vector backbone is eliminated. Moreover, obtained phytase transgenic maizes are.valuble materials for new maiz line breeding.