黑曲霉分泌表达载体的构建以及绿色荧光蛋白的表达  

Construction of Secretory Expression Vector of Aspergillus niger and Expression of Enhenced GFP

作　　者：郑红玉[1] 黄毓茂[1] 余希尧[1] 钟泽民[1] 项林盛[1] 唐丽云[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《华南农业大学学报》2013年第4期574-579,共6页Journal of South China Agricultural University

基　　金：广东省现代农业生猪产业技术体系(F10021)

摘　　要：采用PCR扩增得到糖化酶基因启动子(PglaA-g)、色氨酸合成酶基因终止子(TtrpC)和潮霉素抗性筛选标记基因,依次连接这些基因元件,获得黑曲霉Aspergillus niger分泌表达载体pPHT.EGFP基因与黑曲霉表达载体pPHT连接,得到重组表达载体pPHT-EGFP.pPHT-EGFP表达载体通过原生质体转化法转化黑曲霉,经潮霉素抗性筛选、基因组PCR鉴定阳性重组转化子.荧光显微镜观察表明,绿色荧光蛋白成功表达,而且荧光的分布具有一定的规律,主要集中在菌丝顶端、膈膜以及培养基中;固体培养基发出绿色荧光,表明绿色荧光蛋白分泌到培养基中,属于分泌性表达,蛋白分泌的部位主要位于菌丝顶端.After being obtained through PCR amplification , glucoamylase gene promoter and tryptophan hydroxylase gene terminaer were linked together in sequence to construct Aspergillus niger secretory ex-pression vector pPHT .EGFP gene was added to pPHT , forming a recombination expression vector , pPHT-EGFP.pPHT-EGFP was transformed into A.niger protoplasts.Positive transformants were identified through hygromycin resistence and PCR amplification .Positive transformants were cultured in a medium where starch was the sole carbon source .The hyphae were observed under a fluorescence microscope , in-dicating the characteristic of fluorescence distribution , which mainly concentrated on hyphae tips , sep-tum, cell wall and culture medium .Green fluorescence of the solid medium indicated secretion of EGFP to extracellular medium , which belonged to secretory expression .Green fluorescence of other spaces showed positions of secretion .

关 键 词：EGFP基因 黑曲霉 表达 

分 类 号：S855[农业科学—临床兽医学]