Construction Of β-Mannanase Gene Integration Inducible Vector And Expression In Wild Yeast

Expressionβ-Mannanase gene in wild type yeast. Cloned 25S rDNA fragment of JYS4 from wild type yeast in pig intestinal, then ligage it into vector YIP5 by enzyme EcoRⅠand BamHⅠ,and get a new vector YIP5-rDNA. Ligaged rDNA from YIP5-rDNA digested by BamHⅠand SalⅠ,ManⅠfrom vector pGEM-ManⅠdigested by EcoRⅠand SalⅠ,and promoter AOX1 from pPIC9K digested by BglⅡand EcoRⅠat sane tine. A high clone integrated expression vector YIP5-rDNA-AOX1-ManⅠwas finished. Conversed JSY4 whth DNA of YIP5-rDNA-AOX1-ManⅠand pAX15 as the proportion of 3:1 . Transformants were screened in YEPD flat panel of 300μg/ml G418 by PCR. Tnducing by 2% methyl alcohol, the engineering yeast YIP5-rDNA-AOX1-ManⅠsuccessfully expression.Successfully expressed theβ-Mannanase that specific activity is 0.90 IU/ml. furthermore, It can examine ManⅠgene occuring after genesis ten times and achieveβ-Mannanase expressing. Successful expressionβ-Mannanase gene in wide type yeast and it can stability hereditary.