The Screening Of Funguses With Producing Phytase And Cloning Its Phytase Gene

Phytase was a kind of enzyme for hydrolyzing phytate to release myoinositol and inorganic phosphate, which would promote the utilization of phosphate, reduce the drainage of phosphate in the dejecta and reduce the environmental pollution. The more important thing was to promote the utilization of protein for combined protein, reduce the combination of phytate and microelement and increase the nutrient value of feedstuff. In addition, phytase had great applicable foreground for the addition of phytase to feedstuffs because it had a widespread application in food and medicament et al. For all of these reasons, researching of phytase was one of the most heated fields in the world nowadays.Although it had ten years since the engineering study of phytase, there was not great development in the feedstuff industry at the present, the main reasons were included the low expressed level of phytase gene in its germ, the capability of enduring high temperature in the industrial procedure of making granule and the low pH value in the alimentary tract of animals. In order to solve these problems and satisfy the need of producing phytase in the industry, one of methods was to look for the funguses with excellent characters by the way of gene egineering . The phytase gene was cloned in this study would not only provided excellent gene resource which could express in the microbe and plant but also remedied the vacancy of phytase gene cloned in the funguses.Three strains that largely produced phytase were screened from the twenty-one strains of funguses. Three fragments with the length of 789bp, 919bp and 622bp respectively were cloned by PCR amplification from chromosomal DNA of No.1 Flammulina velutipes, No.1 Pleurotus ostreatus and No.2Pleurotus ostreatus by seven specific primers designed according to the conserved sequence of phytase genes in GenBank, and named Flal phy, Plel phy and Ple2phy respectively. The results of Blastx showed that the homologue of Flal phy With phytase gene of Agrocybe pediades (GenBank Accession: CAC48160) was 50%, and the homologue of Plelphy and Ple2phy with phytase gene of Trametes pubescens (GenBank Accession: CAC48234.1) respectively was 64% and 47%.The flank unknown regions of Plelphy were cloned by the way of TAIL-PCR with the nested primers being designed according to the known phytase gene region in Ple2,...