Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada.
Curr Genet. 2011 Feb;57(1):63-73. doi: 10.1007/s00294-010-0324-0. Epub 2010 Oct 10.
Genetic transformation of organisms with large genome fragments containing complete genes, with regulatory elements or clusters of genes, can contribute to the functional analysis of such genes. However, large inserts, such as those found on bacterial artificial chromosome (BAC) clones, are often not easy to transfer. We exploited an existing technique to convert BAC clones, containing genomic DNA fragments from the barley-covered smut fungus Ustilago hordei to binary BACs (BIBACs) to make them transferable by the Agrobacterium tumefaciens T-DNA transfer machinery. Genetic transformation of U. hordei with BAC clones using polyethylene glycol or electroporation is difficult. As a proof of concept, two BAC clones were successfully converted into BIBAC vectors and transferred by A. tumefaciens into U. hordei and U. maydis, the related corn smut fungi. Molecular analysis of the transformants showed that the T-DNA containing the BAC clones with their inserts was stably integrated into the U. hordei genome. A transformation frequency of approximately 10⁻⁴ was achieved both for U. hordei sporidia and protoplasts; the efficiencies were 25-30 times higher for U. maydis. The combination of in vivo recombineering technology for BAC clones and A. tumefaciens-mediated transformation of Ustilago species should pave the way for functional genomics studies.
利用含有完整基因、调控元件或基因簇的大片段基因组的生物遗传转化，可以促进对这些基因的功能分析。然而，像细菌人工染色体 (BAC) 克隆中发现的大插入片段通常不容易转移。我们利用现有的技术将大麦黑粉菌 Ustilago hordei 的基因组 DNA 片段的 BAC 克隆转化为二元 BAC（BIBAC），使其能够通过根癌农杆菌 T-DNA 转移机制进行转移。使用聚乙二醇或电穿孔将 BAC 克隆遗传转化到 U. hordei 中非常困难。作为概念验证，两个 BAC 克隆成功转化为 BIBAC 载体，并通过根癌农杆菌转移到 U. hordei 和 U. maydis，即相关的玉米黑粉菌中。转化体的分子分析表明，含有 BAC 克隆及其插入物的 T-DNA 已稳定整合到 U. hordei 基因组中。U. hordei 分生孢子和原生质体的转化频率约为 10⁻⁴；对于 U. maydis，效率提高了 25-30 倍。体内重组酶技术与根癌农杆菌介导的 Ustilago 物种转化的结合应该为功能基因组学研究铺平道路。
