Rikkerink E H, Solon S L, Crowhurst R N, Templeton M D
Molecular Genetics Group, Horticulture and Food Research Institute of New Zealand Ltd., Mount Albert Research Centre, Auckland.
Curr Genet. 1994 Mar;25(3):202-8. doi: 10.1007/BF00357163.
An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA promoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.
已为植物病原真菌围小丛壳菌（炭疽菌盘长孢状刺盘孢）开发了一种同源转化系统。构建了一个含有与潮霉素磷酸转移酶基因融合的围小丛壳菌gpdA启动子的转化载体。Southern分析表明，该载体在大多数转化体中以单一位点整合。一种跨越重组连接点的新型PCR扩增方法表明，超过95%的转化体中整合事件是通过同源重组发生的。缺失研究表明，505 bp（仍具有启动子功能的分析的同源启动子DNA的最小长度）足以靶向整合事件。载体的同源整合导致gdpA启动子区域重复。当转化体在无选择压力下生长时，通过重复区域之间的重组进行载体切除的发生率很高。参照进行基因破坏实验的可行性讨论了这些重组特性的意义。
