A GENE-TRANSFER SYSTEM BASED ON THE HOMOLOGOUS PYRG GENE AND EFFICIENT EXPRESSION OF BACTERIAL GENES IN ASPERGILLUS-ORYZAE
A GENE-TRANSFER SYSTEM BASED ON THE HOMOLOGOUS PYRG GENE AND EFFICIENT EXPRESSION OF BACTERIAL GENES IN ASPERGILLUS-ORYZAE
复制标题
DOI：
10.1007/bf00391472
复制
发表时间：
1989-09-01
期刊：
影响因子：
2.5
通讯作者：
VANDENHONDEL, CAMJJ
中科院分区：
文献类型：
--
作者：
DERUITERJACOBS, YMJT;BROEKHUIJSEN, M;VANDENHONDEL, CAMJJ
本文介绍了一种米曲霉同源转化系统。该系统基于A.将缺失乳清酸核苷-5 ′-磷酸脱羧酶（pyrG）的水稻菌株与含有功能性A.选择标记为pyrG基因。A.用环状PA 04 -2的pyrG突变体导致Pyr+转化体的出现，频率高达20个/μg DNA，而用线性PA 04 -2获得高达200个转化体/μg DNA。在75%的Pyr+转化体中，在pyrG位点发生了重组事件，其中大部分（90%）导致插入一个或两个拷贝的载体，而其他（10%）导致突变等位基因被野生型等位基因取代。载体pA 04 -2也能转化相应的尼日尔曲霉突变株。利用该转化系统将A.水稻异源非选择性细菌基因lacZ和duidA分别编码β-半乳糖苷酶和β-葡萄糖醛酸酶。利用构巢曲霉gpdA启动子驱动细菌基因在A.通过蛋白质印迹分析判断，获得了相对高水平的活性以及蛋白质本身。
A homologous transformation system forAspergillus oryzaeis described. The system is based on anA. oryzaestrain deficient in orotidine-5′-phosphate decarboxylase (pyrG) and the vector pA04-2, which contains a functionalA. oryzae pyrGgene as selection marker. Transformation of theA. oryzae pyrGmutant with circular PA04-2 resulted in the appearance of Pyr+transformants at a frequency of up to 20 per μg of DNA, whereas with linear pA04-2 up to 200 transformants per μg DNA were obtained. In 75 % of the Pyr+transformants recombination events had occurred at thepyrGlocus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pA04-2 is also capable of transforming a corresponding mutant ofAspergillus niger. This transformation system was used to introduce intoA. oryzaethe heterologous and non-selectable bacterial geneslacZ, encoding β-galactosidase, anduidA, encoding β-glucuronidase. Using theAspergillus nidulans gpdApromoter to drive bacterial gene expression inA. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.