Royer J C, Dewar K, Hubbes M, Horgen P A
Department of Botany, University of Toronto, Mississauga, Ontario, Canada.
Mol Gen Genet. 1991 Jan;225(1):168-76. doi: 10.1007/BF00282655.
A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 x 10(3) transformants/micrograms DNA per 10(7) protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.
开发并分析了一种榆长喙壳菌（Ophiostoma ulmi (Buism.) Nannf.）的转化系统。在用2-巯基乙醇预处理后，通过在MgSO4中用诺维信234消化从活跃出芽的酵母样细胞中产生原生质体。当使用0.6 M蔗糖作为渗透压稳定剂时，原生质体再生效率最高。几个含有真菌启动子与潮霉素磷酸转移酶细菌基因融合体的质粒成功地将榆长喙壳菌转化为对潮霉素具有抗性。这些载体之一pPS57，其含有来自产黄青霉的异青霉素N合成酶启动子，始终赋予对潮霉素最大的抗性。载体的线性化以及在转化反应中加入2-巯基乙醇导致转化效率提高。使用优化程序，每10^7个原生质体每微克DNA获得约4×10^3个转化体。交变电场和标准电泳后的Southern杂交表明串联重复序列（有些大于250 kb）随机插入真菌染色体。抗生素抗性在有丝分裂过程中是稳定的。然而，减数分裂后转化DNA的表达高度可变。
