Marek E T, Schardl C L, Smith D A
Department of Plant Pathology, University of Kentucky, Lexington 40546-0091.
Curr Genet. 1989 Jun;15(6):421-8. doi: 10.1007/BF00376799.
A vector was constructed for transformation of the plant pathogenic fungus Fusarium solani. The promoter 35Sp, from cauliflower mosaic virus, was fused to the bacterial gene APH(3')II, which confers resistance to the aminoglycoside antibiotic G418. Two transformation procedures were developed: one using isolated fungal protoplasts, the other using germinated fungal spores. A transformation frequency of 3.3 G418-resistant colonies were obtained per microgram DNA. Of 14 colonies analyzed, 12 had vector sequences integrated into their high molecular weight DNA, and 2 were untransformed. Integration was sometimes accompanied by rearrangements of both the vector and flanking fungal DNAs. Primer-extension analysis of the mRNA from one transformant revealed two putative transcription initiation sites in the chimeric APH(3')II gene. Both sites differed from the normal initiation site in plants. This vector will be useful in transformation systems in which integration by non-homologous recombination is desired.
构建了一种用于转化植物病原真菌茄类镰刀菌的载体。来自花椰菜花叶病毒的启动子35Sp与细菌基因APH(3')II融合，该基因赋予对氨基糖苷类抗生素G418的抗性。开发了两种转化方法：一种使用分离的真菌原生质体，另一种使用萌发的真菌孢子。每微克DNA获得了3.3个对G418有抗性的菌落的转化频率。在分析的14个菌落中，12个菌落的高分子量DNA中整合了载体序列，2个未转化。整合有时伴随着载体和侧翼真菌DNA的重排。对一个转化体的mRNA进行引物延伸分析，发现在嵌合APH(3')II基因中有两个推定的转录起始位点。这两个位点都与植物中的正常起始位点不同。该载体将用于需要通过非同源重组进行整合的转化系统。
