Study Of Phytase Gene Being Cloning And Maize Being Transformed

Maize(Zea meays L.) is the third greatest food supplies crops and the one of most important feedstuff material.The output of maize in China is the second greatest in the World. Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds such as Maize, accounting for more than 50% of the total phosphate and even up to 80 % in the main crops. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. The negatively charged phytate is balanced by cations of magnesium, calcium, iron and zinc, preventing efficient utilisation of these minerals which are needed in the daily feed intake. Theoretically, the phosphorus(P) content in corn and soybean meal should be enough to meet the requirement of piglets. However, about two thirds of the P in these feed ingredients are not available to the animal because the element comes in the chemical form of phytate. In recent twenty years, the study have showed the pig can only utilize 10%~20% P in maize and 25%~35% P in bean cake that indicates pig only utilize 15% of total P in maize and bean cake.Due to the lack of phytases in the digestive tract of non-ruminant, monogastric animals such as pigs, poultry (hens, chicken and turkey), phytic acid cannot be utilize dietary and exercted into manure. Thus, phytate is the key nutritional problem regarding phosphate and minerals. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The phytate in foodstuff is degraded by phytase in the alimentary tract of animals and release available P to animal due to the phytase is added into feedstuff or the phytase gene is transferred into maize and expressed. That will improve the utilization of P and alimentation of foodstuff. The use of phytases to degrade seed phytic acid has very important signification forreducing the negative environmental impact of livestock production and improving the benefit of enterprise.Basing on about above the cause, we chose using the fungi Asperillus oryae Cohn which grows rapidly and has been not reported at home and abroad. After extracting and purifying the all DNA of genome in Asperillus oryae Cohn, we designed primers according to sequence published and cloned phytase gene from genome of Asperillus oryae Cohn for the first time by ploymerse chain reaction(PCR), meanwhile, we altered the first base from C to A. We then did phytase gene sequence alignment in GeneBank. The results showed that the phytase gene cloned had 98.7%, 88.8%, 84.5% and 66.4% respectively homology with phytase gene :GI: 22901877, GI: 4185609, GI: 11992744, GI: 17043775 that of reported. The sequence of phytase gene cloned from Asperillus oryae Cohn was registered in GeneBank and the number is GI:47176935.To testify the activity of the gene, the expression vector (pPIC9K-phyA) of Pichia pastoris was constructed. After proving that the phytase gene was cloned correctly into expression vector (pPIC9K-phyA) of Pichia pastoris ,we transferred the expression vector into Pichia pastoris by electroporation and expressed phytase gene in Pichia pastoris. The phytase was produced and the activity of the enzyme was tested. The results showed that the enzyme expressed had very high activity , reaching 25000U/mL at pH5.5 and at 55℃, and that optimum temperature of and optimum pH of the phytase expressed was 55℃and 5.5 respectively. The temperature and the pH of the condition was similar the condition in the stomach of monogastric animal.In order to transfer the phytase gene cloned into Maize, we, in large quantities, cultured and induced the immature embryos of six genotypes of maize on different mediums. We used maize genotypes of maize with high rate of differentiation and induction as receptors after experimenting and analyzing in large quantities. We studied the factors that effect inducement and differentiation immature embryos of maize a great deal and obtained...