Cloning And Function Identification Of The Neurospora Sp. Phytase Gene

Phytic acid is the main storage form of the phosphorus in a variety of plant tissue, which is 50%- 82%of total phosphorus content, with strong complexation ability, easy to plant 2(3) the price of ions and proteins to generate insoluble compounds,phytic acid salts cannot be directlyabsorbed by the plant, so reduce the utilization of all kinds of ions for the plant.Phytase is a special kind of monoester phosphate hydrolase, it can be decomposed into a lower inositol phosphate derivatives and inorganic phosphate, increase the efficiency of phosphorus, remove the anti nutritional function of phytic acid and reduce the phosphorus pollution to the environment. This study isolateda newproduction phytasestrain of Neurosporafrom soil, the production condition was optimized, purification it and research its enzymology properties.And then adopt the method of PCR amplification, obtained part the genesequences of the phytase,constructed the prokaryotic expression vector, and determined the enzyme activityof the express product;And constructed the plant expression vector, maked the phytase genes transferred into the tobacco by agrobacterium mediated method, detected the activity of phytase in transgenic tobacco.The main results were as follows: 1. Screened the strain ofproduction phytase and studyed of the enzyme production conditionsThis study used to the phytase strain can be formed the transparent circleon on the culture medium that containing phytic acid calcium hydrolysis to screen, obtained theproduce phytase strain.Through comprehensive assessment what observated the morphology of the strain and 18 s ribosomal RNA PCR amplification, the strain was preliminary identified as Neurospora.The studied for the enzyme production conditions of the strain, the results showed that the best carbon source for soluble starch;nitrogen source was yeast powder, the bacteria culture 4 d, reached the highest enzyme activity.By using the orthogonal experiment on the important factor in fermentation medium optimization, it is concluded that the composition of culture medium for: 0.01 g/m L phytic acid calcium;Soluble starch yeast powder 0.018 g/m L, 0.0125 g/m L;Mandel fluid A 0.1 m L/m L;Mandel liquid B 0.0001 m L/m L. 2. Purification and properties of phytase in researchAfterliquid fermentation cultured of the strains, centrifugal obtained the rough enzyme fluid, Through acetone precipitation, SDS and Native PAGE purification steps of phytase, using sds-page detection for a single strip, its molecular weight is about 35 kd.The optimum temperature for the enzyme is 55 ℃;The optimum p H for 5;Phytase in 55 ℃ 25 and 200 min after the treatment, the residual enzyme activity were 80.3% and 80.3%. 3. Cloned the phytase gene and expred the vectorExtracting total RNA of phytase strain, used 3 ’RACE kit for purposesegments‘s amplificated.Got part sequence which can encoding phytase gene, the sequence length of 750 bp, coded 485 amino acids. Constructed the prokaryotic expression vector p GEX4t-3-xphy A, used the IPTG to induce expression, and at 40 kd successfullyexpressed.Buildeda plant expression vector CPB-xphy A, and maked its plasmid into agrobacterium EHA105, obtained contains phy A of agrobacterium-mediated gene bacterium fluidcan whichi can be used to transform the plant. 4. Agrobacterium mediated genetic transformation of tobaccoBy agrobacterium mediated method maked thephy A genes transferred into tobacco, after 1% of Bar screening and PCR detection,obtained the transgenic tobacco plants, proved that the phytase gene has been successfully transferredintotobaccogenome.The determination of phytase activity in transgenic tobacco plant, preliminarily proved phy A gene encoding in the tobacco seed phytase can be expressed correctly, and expressed in transgenic tobacco seed activity of phytase has good.