CAJ | 학술논문

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．
채용PCR확증득도당화매기인계동자（PglaA-g）、색안산합성매기인종지자（TtrpC）화조매소항성사선표기기인，의차련접저사기인원건，획득흑곡매Aspergillus niger분비표체재체pPHT．EGFP기인여흑곡매표체재체pPHT련접，득도중조표체재체pPHT-EGFP．pPHT-EGFP표체재체통과원생질체전화법전화흑곡매，경조매소항성사선、기인조PCR감정양성중조전화자．형광현미경관찰표명，록색형광단백성공표체，이차형광적분포구유일정적규률，주요집중재균사정단、격막이급배양기중；고체배양기발출록색형광，표명록색형광단백분비도배양기중，속우분비성표체，단백분비적부위주요위우균사정단．
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 .