α-半乳糖苷酶基因Mel1在毕赤酵母中的组成型表达  

Constitutive Expression of α-galactosidase Mel1 Gene in Pichia pastoris

作　　者：张学文[1] 唐香山[1] 张金谌[1] 章怀云[2] 

机构地区：[1]湖南农业大学生物科学技术学院,长沙410128 [2]中南林业科技大学生命科学与技术学院,长沙412006

出　　处：《农业生物技术学报》2006年第2期269-272,共4页Journal of Agricultural Biotechnology

基　　金：湖南省中青年基金项目(No.01JZY2099)资助

摘　　要：用PCR方法从酵母(Saccharomycescerevisiae)AH109中扩增出α-半乳糖苷酶的Mel1基因,将其克隆至整合型载体pGAPZαA中构建成组成型分泌表达酶产物的重组质粒pGAPZα-Mel1。将线性化的重组质粒pGAPZα-Mel1电击转化至毕赤酵母(Pichiapastoris)KM71,在含有100mg/mLzeocin和预先涂布有X-α-gal的YPDS平板上选择蓝色阳性菌落。发酵培养酵母的上清经SDS-PAGE分析,在53kD处有特异带;经非变性PAGE凝胶电泳,与显色底物的反应,检测到α-半乳糖苷酶活性带。重组菌pGAPZα-Mel1/KM71摇瓶发酵6d后,培养液α-半乳糖苷酶粗酶活性为12U/mL。Mell gene was amplified from yeast（Saccharomyces cerevisiae ） AH109 with PCR, and cloned into integrated vector pGAPZαA, constitutive and secreted expression α-galactosidase plasmid pGAPZα-Mell was constructed. The linearized recombinant plasmid pGAPZ α-Mell was transformed into Pichia pastoris KM71 by electropotation, and the blue positive colonies were screened out on YPDS plate with X-α-gal and 100 mg/mL zeocin. There was a special 53 kD band by SDS-PAGE from supematant of yeast culture and a color band was observed by soak the native PAGE gel in X-α-gal to illustrate the α-galactosidase activity; The α -galactosidase enzyme activity was 12 U/mL after fermenting pGAPZα-Mell/KM71 for 6 days.

关 键 词：Α-半乳糖苷酶 Mell基因 重组质粒pGAPZα-Mell 组成型表达 

分 类 号：S188[农业科学—农业基础科学]