β-甘露聚糖酶基因在野生酵母菌中的整合诱导型表达  

Integrated Expression of β-Mannanase Gene in Wild Type Pastoris

作　　者：蔺日胜[1] 王和平[1] 周平[1] 武翠[1] 包晓兰[1] 赵丽霞[1] 

机构地区：[1]内蒙古农业大学生物工程学院,内蒙古呼和浩特010018

出　　处：《生物技术》2007年第2期6-10,共5页Biotechnology

基　　金：国家自然科学基金项目资助(编号:39870558)

摘　　要：目的:实现β-甘露聚糖酶基因在野生酵母菌中的整合诱导型表达。方法:克隆猪肠道野生酵母菌JSY4的25S rDNA片段;并与YIP5经EcoRⅠ与BamHⅠ双酶切连接,构建载体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,构建高拷贝整合诱导型表达载体YIP5-rDNA-AOX1-ManⅠ。提取DNA用SalⅠ单酶切线性化与pAX15按3∶1的比例共转化JSY4,在含300μg/mlG418的YEPD平板上筛选工程菌转化子,PCR方法鉴定。用2%甲醇诱导共转化工程菌以实现表达。结果:成功表达出β-甘露聚糖酶,其比活力为0.90IU/ml。而且传代10次后仍能检测到ManⅠ基因的表达产物β-甘露聚糖酶。结论: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。Objective： Expression β-Mannanase gene in wild type yeast.Methods： 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-ManⅠ digested by EcoRⅠand SalⅠ,and promoter AOX1 from pPIC9K digested by BglⅡand EcoRⅠ at sane tine.A high clone integrated expression vector YIP5-rDNA-AOX1-ManⅠ was finished.Conversed JSY4 with DNA of YIP5-rDNA-AOX1-Man Ⅰ and 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 - Man Ⅰ successlully expression. Results： Successfully expressed the β- Mannanase that specific activity is 0.90 IU/ml. furthermore, It can examine Man Ⅰ gene occuring after genesis ten times and achieve β- Mannanase expressing. Conclusion： Successful expression β- Mannanase gene in wide type yeast and it can stability hereditary.

关 键 词：酵母菌 25SrDNA 整合诱导型表达载体 表达 Β-甘露聚糖酶 

分 类 号：Q786[生物学—分子生物学]