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 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 expression 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, indicating the characteristic of fluorescence distribution, which mainly concentrated on hyphae tips, septum, 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.