【作者】 蔺日胜；

【导师】 王和平；

【作者基本信息】 内蒙古农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 从猪肠道内分离到定植野生型酵母菌4株,分别命名为JSY1、JSY2、JSY3、JSY4。进行G418敏感性研究,发现JSY4作为载体共转化菌株最方便筛选。克隆猪肠道野生型酵母菌JSY4的25SrDNA片段,与YIP5经EcoRⅠ与BamHⅠ双酶切连接,构建出载体YIP5-rDNA。BamHⅠ与SalⅠ双酶切YIP5-rDNA,EcoRⅠ与SalⅠ双酶切pGEM-ManⅠ得到ManⅠ基因,BglⅡ与EcoRⅠ双酶切pPIC9K得到AOX1启动子,将三个片段连接,构建出高拷贝整合诱导型表达载体YIP5-rDNA-AOX1-ManⅠ。提取构建出的表达载体DNA用SalⅠ单酶切线性化与pAX15按3:1的比例共转化JSY4,在含300μg/ml G418的YEPD平板上筛选工程菌转化子,并且PCR方法鉴定出共转华的阳性转化子。用2%甲醇诱导共转化工程菌,成功表达出β-甘露聚糖酶,其比活力为0.90 IU/ml,而且传代10次后仍能检测到ManⅠ基因表达产物β-甘露聚糖酶,实现了β-甘露聚糖酶基因随共转化工程菌染色体稳定遗传及表达的目的。对温度、pH、溶氧条件进行优化,确定β-甘露聚糖酶表达最佳发酵条件。更多还原

【Abstract】 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.更多还原