Construction Of β-Mannanase Integration Constitutive Expression Vector And Screening Of Integrants In Yeasts

The GAP gene promoter(PGAP ) which is about 500 bp is amplified from P.pastoris GS115 genome and used to replace the AOX1 promoter(PAOX1) on pPIC9K, resulting in constitutive vector pGAP. The 25S rDNA sequence which is about 1700bp is amplified from wild JSY4 strain genome and used to replace the HIS4 sequence on the pGAP, resulting in integration constitutive vector pGAP-rDNA. The ManⅠgene which is encoding the mature mannanase protein is cutted from recombinant plasmid pT002-ManⅠand inserted into the MCS of vector pGAP-rDNA, downstream ofα-factor singal peptide sequence.The integration constitutive expression vector pGAP-rDNA-ManⅠis constructed and linearized with NcoⅠand then electrotransformed into JSY4 wild strain.G418 and PCR analysis showed that ManⅠhas been integrated into JSY4 genome.The recombinant strain JSY4(pGAP-rDNA-ManⅠ)that expresses the secretoryβ-Mannanase is detected by double-antibody sandwich ELISA and DNS method.