Austin B, Hall R M, Tyler B M
Research School of Biological Sciences, Australian National University, Canberra ACT.
Gene. 1990 Sep 1;93(1):157-62. doi: 10.1016/0378-1119(90)90152-h.
To provide a dominant selectable marker for transformation of Neurospora crassa strains lacking specific auxotrophic mutations, we have engineered the bleomycin (Bm) resistance-encoding gene (ble) from the bacterial transposon Tn5 for expression in N. crassa. The coding region of the ble gene was fused to the promoter and terminator regions of the N. crassa am gene. In some vectors, multiple cloning sites were placed flanking the ble gene to provide a versatile ble cassette. When introduced into N. crassa, the hybrid ble gene conferred resistance to greater than 15 micrograms Bm/ml. Under optimal conditions, the levels of Bm required (2.5 micrograms/ml) make even large-scale transformation experiments very economical. Aspergillus nidulans could also be efficiently transformed to Bm resistance using the N. crassa ble gene fusion. Since the ble gene functions in both N. crassa and A. nidulans, the gene should be useful as a transformation marker for the many other filamentous fungi which are sensitive to Bm.
为了给缺乏特定营养缺陷型突变的粗糙脉孢菌菌株的转化提供一个显性选择标记，我们对来自细菌转座子Tn5的博来霉素（Bm）抗性编码基因（ble）进行了改造，使其在粗糙脉孢菌中表达。ble基因的编码区与粗糙脉孢菌am基因的启动子和终止子区域融合。在一些载体中，多个克隆位点位于ble基因两侧，以提供一个通用的ble盒。当导入粗糙脉孢菌时，杂交ble基因赋予了对大于15微克/毫升Bm的抗性。在最佳条件下，所需的Bm水平（2.5微克/毫升）使得即使是大规模转化实验也非常经济。使用粗糙脉孢菌ble基因融合体也可以有效地将构巢曲霉转化为对Bm具有抗性。由于ble基因在粗糙脉孢菌和构巢曲霉中均起作用，该基因应该可作为对Bm敏感的许多其他丝状真菌的转化标记。
