毛柄金钱菌gpd启动子驱动福寿螺纤维素酶基因在灰盖鬼伞菌中的表达  

Expression of Ampullarium crossean Cellulase Gene in Corprinus cinereus Driven by gpd Promoter from Flammulina velutipes

作　　者：杨培周[1,2] 郭丽琼[1] 林俊芳[1] 

机构地区：[1]华南农业大学食品学院,广东广州510640 [2]合肥工业大学生物与食品工程学院,安徽合肥230009

出　　处：《食品科学》2011年第15期209-213,共5页Food Science

基　　金：国家"863"计划项目(2006AA10Z301);华南农业大学"211工程"项目(2009C010500001)

摘　　要：为研究毛柄金钱菌gpd启动子驱动福寿螺纤维素酶基因(mfc)在大型丝状真菌中的表达效果,本实验通过构建毛柄金钱菌gpd启动子驱动的mfc基因的真核表达载体,采用PEG(polyethylene glycol)介导法将目的基因重组进色氨酸营养缺陷型灰盖鬼伞菌染色体,对转化子进行PCR、Southern blotting、RT-PCR等分子鉴定,通过测定滤纸酶、CMC酶和木聚糖酶的活力考察mfc的表达效果。结果表明:多功能纤维素酶基因整合入灰盖鬼伞基因组中,毛柄金钱菌gpd启动子能够高效驱动mfc基因的表达,其中酶活力最高的工程菌株为Cfvlm9,其滤纸酶活力、CMC酶活力和木聚糖酶活力分别为21.5、44、235U/mL,分别是对照的1.79倍、1.6倍和2.97倍。In order to investigate the expression of multifunctional cellulase gene of Ampullarium crossean（mfc） in large filamentous fungi driven by glyceradehyde-3-phosphate dehydrogenase（gpd） promoter（Fvl） of Flammulina velutipes,a new eukaryotic expression vector containing F.velutipes gpd promoter（Fvl） and A.crossean cellulase gene（mfc） was constructed.The expression vector was inserted into the chromosome of tryptophan auxotrophic Corprinus cinereus by polyethylene glycol（PEG） mediation.The transformants were selected by PCR and Southern blotting.The transcription of mfc was confirmed by RT-PCR and the expression of mfc was further determined by measuring the activities of filter paper enzyme,carboxymethylcellulase and xylanase.The results showed that mfc driven by F.velutipes gpd promoter exhibited highly effective expression in engineered C.cinereus,and the strain with the highest cellulase activity was Cfvlm9,in which the filter paper enzyme,carboxymethylcellulase and xylanase activities were 21.5,44 U/mL and 235 U/mL,respectively with 1.79,1.6 and 2.97 fold increase as compared to the control strain.

关 键 词：纤维素酶基因 福寿螺 毛柄金钱菌 gpd启动子 

分 类 号：Q789[生物学—分子生物学]