Pieterse C M, van't Klooster J, van den Berg-Velthuis G C, Govers F
Department of Phytopathology, Wageningen Agricultural University, The Netherlands.
Curr Genet. 1995 Mar;27(4):359-66. doi: 10.1007/BF00352105.
The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.
通过异源杂交从基因文库中筛选出卵菌致病疫霉的硝酸还原酶（NR）基因niaA。NiaA以单拷贝基因形式存在，其表达受氮源调控。测定了niaA的核苷酸序列，并将推导的902个氨基酸残基的氨基酸序列与高等真菌和植物的NR进行比较，发现有显著的同源性，尤其是在钼、血红素和黄素腺嘌呤二核苷酸这三个辅因子结合结构域内。致病疫霉的niaA基因被用作模型基因，以测试卵菌基因在构巢曲霉（一种易于进行分子遗传学研究的真菌）中是否具有功能。完整的niaA基因被稳定整合到构巢曲霉nia缺失突变体的基因组中。然而，含有一个或多个niaA基因拷贝的转化体无法互补nia突变体。这表明导入的niaA基因在构巢曲霉中没有功能性表达。此外，在瞬时表达试验中分析了另外两个卵菌基因启动子的活性。将含有与致病疫霉泛素基因ubi3R或莴苣盘梗霉ham34基因启动子融合到大肠杆菌β-葡萄糖醛酸酶（GUS）报告基因编码序列的嵌合基因的质粒转移到构巢曲霉原生质体中。未检测到显著的GUS活性，表明ubi3R和ham34启动子在构巢曲霉中无活性。显然，在卵菌中足以激活基因的调控序列在子囊菌构巢曲霉中没有功能。
