Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin, PR China.
J Appl Genet. 2010;51(2):225-32. doi: 10.1007/BF03195734.
The Agrobacterium rhizogenes-mediated transformation procedure was developed by using the hygromycin B phosphotransferase gene (hph) as a selective marker for the oil-producing fungus Umbelopsis isabellina. Different conditions were combined to increase the transformation efficiency. The highest efficiency was obtained by using A. rhizogenes strain R105 and a vector with zygomycete promoter. Southern blot analysis demonstrated that 71% of transformants contained random integrations of T-DNA sequences under optimal conditions. We randomly selected 115 positive transformants resistant to hygromycin to analyze the amount of total fatty acid and gamma-linolenic acid (GLA). Six transformants produced a higher amount of total fatty acids than the wild strain, and one transformant also produced a higher level of GLA than the wild strain in gas chromatography analysis. This is the first report about using A. rhizogenes strain R105 and germinated conidia to transform successfully the recalcitrant zygomycetes and to obtain transformants with a stable phenotype.
发根农杆菌介导的转化程序是利用潮霉素 B 磷酸转移酶基因 (hph) 作为产油真菌伞状枝孢菌的选择标记开发的。结合不同的条件来提高转化效率。使用发根农杆菌菌株 R105 和带有接合菌启动子的载体可获得最高的效率。Southern blot 分析表明，在最佳条件下，71%的转化体含有 T-DNA 序列的随机整合。我们随机选择了 115 个对潮霉素有抗性的阳性转化体来分析总脂肪酸和γ-亚麻酸 (GLA) 的含量。在气相色谱分析中，有 6 个转化体产生的总脂肪酸量高于野生型，有 1 个转化体产生的 GLA 水平也高于野生型。这是首次报道使用发根农杆菌菌株 R105 和萌发的分生孢子成功转化顽固的接合菌，并获得具有稳定表型的转化体。
