Cloning And Prokaryotic Expression Of Phytase Gene Of Trichoderma Pleuroticola

As an important kind of feed additive, phytase has a broad application prospect. But at present, different microbial source have phytase activity differently. Although it has been on a variety of microbial strains whose phytase activity are studied but it can meet that the production requirements of strains are still hardly to see. Trichoderma is a kind of widespread fungus. Trichoderma, and literatures show that Trichoderma generally have phytase activity and show great potential in the prediction of protein structure in biological activity and thermal stability. Therefore this research in view of the nature and role of Trichoderma phytase, basically did the following work:Fristly, design primer on21Trichoderma strains'phytase sequence which produce phytase for PCR amplification, at last obtained1100bp size of the phytase sequences in1lof17strains of Trichoderma, then select500bp fragments which are conservative, then using ITS4and ITS5to amplify of the area between ITS1-5.8s-ITS2of Trichoderma. Use of software to analyse Trichoderma phylogenetic relationships and construct evolutionary trees, it shows that the phytase sequence is put in apparent difference except conservative areas.after comparing with the phylogenetic tree bases on ITS sequences, they are nearly the same except longibrachiatum, suggest that Trichoderma phytase gene has diversity characteristics, and has potential to be phylogenetic marker.Secondly, select Trichoderma Pleurotus T2-1, which is preserved in our lab, and finally get the full-length phytase gene by overlap extension PCR method, which is defined as phyAT2-1, and it gets ready for protein expression of phytase in the next step.Finaly, use EcoRI and HindIII to digest phyAT2-1and construct plasmid by connecting phyAT2-1and plasmid pET-His together and then transform it in to BL21(DE3). Induced by IPTG the phytase gene was expressed to be a protein of Inclusion body by SDS-PAGE analysis.the Inclusion body is refolded through stepwise dialysis strategies and on-column refolding after wash and solution.and then study the sample through SDS-PAGE and detection of phytase activity. The results show that phytase activity of sample refolded through dialysis renaturationr is7.44U/mg, the one that refolding on-column get the phytase activity for14.871U/mg, and The latter has more single and a higher concentration SDS-PAGE band.The optimal reaction temperature is45-55℃.This result lay the foundation for research of phytase of Trichoderma further.