以amdS为筛选标记的黑曲霉遗传转化体系的建立

黑曲霉是一种重要的发酵工业微生物，也是用于研究丝状真菌生长、发育、代谢、基因功能等的模式微生物。将构巢曲霉来源的乙酰胺酶编码基因amdS在黑曲霉中异源表达，成功得到黑曲霉amdS转化子。经表型分析，amdS赋予了黑曲霉在乙酰胺为唯一氮源的培养基上生长的能力，而野生型菌株则生长微弱。构建了以amdS为筛选标记的基因敲除载体，以草酰乙酸水解酶基因oahA上下游同源序列构建oahA敲除质粒，转化黑曲霉后成功筛选到oahA敲除菌株，草酸合成水平明显降低。结果表明，利用amdS作为营养筛选标记能够很好地应用于黑曲霉遗传转化操作中。

Transformation System of Aspergillus niger Using amdSas a Dominant Selective Marker

Aspergillus niger is an important fermentation industrial microorganism and also a model microorganism for the study of the growth, development, metabolism, and gene function of filamentous fungi. amdS of Aspergillus nidulans was heterologously expressed in A. niger, and the transformants of A. niger were obtained. By phenotypic analysis, amdS made A. niger grow on medium with acetamide as the sole nitrogen source, whereas wild-type strains growed weakly. A knockout vector with amdS as a selective marker was then constructed. Finally, the oxaloacetate hydrolase gene oahA knockout plasmid was constructed by ligation of the upstream and downstream homologous sequences, and the oahA knockout strain was successfully screened after transformation into A. niger. Oxalic acid production of ΔoahA strain was significantly decreased. The results showed that amdS could be well applied to the genetic manipulation of A. niger as a nutrient screening marker.