Screening And Cloning Of Phytase Gene And (?)-Glucosidase Gene From Medical Fungi

Those producing phytase medical fungi were screened from Coriolus versicolor, Hericium erinaceus et al sixteen medical fungi .A series of primers were designed from the consensus sequences of phytase gene and a-glucosidase gene. And then phytase gene fragements were cloned from above four medical fungi;and at that time the a-gucosidase gene fragements were cloned from above sixteen medical fungi. One is having screen phytase fungi,the others is having screen primers.Hign homologus phytase gene have been cloned from Coriolus versicolor which has hign enzyme activity and assimilaritt in this study, phytase gene.phytase gene fragement was isolated by primer phyR1, phyF3,then designed the nested special primer physpl, physp2, physp3 and physp 1, physp2, physp3' on the 5' -flanking and 3' -flanking on the known sequence, and then the flangking sequence of known fragement isolated by TAIL-PCR. Blastn, ORF and Primer5.0 softwares analysised these sequence,we could design special primer phyP3, phyP4 on the flanking sequence,finally,we cloned whole phytase gene by the primer phyP3, phyP4 from Coriolus versicolor.A pair of special primers bgsR1, bgsF3 was used, thea-glucosidase gene segment (H1) was amplified with the genomic DNA of mycelia of Hericium erinaceus ,.And then two terminus H2 and H3 of H1, flanking sequences were amplified by TAIL-PCR. The special primers on the flanking sequence H2 and H3 were designed according to these sequences that were analyzed by using the softwares of Blastn, ORF and Primer5.0.Then the full a-glucosidase gene (H4) sequence about 2228bp was amplified with the special primers from the genomic DNA of Hericium erinaceus.The results of screening were that among them eight fungi can produce transparent cycle but others not can. Enzyme activity measuring was done among the four fungi that the ratio of transparent cycle to velum diameter was larger,and the results indicated these fungi can really produce phytase.According to sequences and analysis by TAIL-PCR amplified and registered in genebank,desigened the RT-PCR primers of phytase and a-glucosidase,and then amplfyed these two genes by RT-PCR. The phytase gene cloned by TAIL-PCR and RT-PCR...