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

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,%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.