Max-Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.
Plant J. 2011 Mar;65(5):829-42. doi: 10.1111/j.1365-313X.2010.04462.x. Epub 2011 Jan 14.
Recombineering, permitting precise modification of genes within bacterial artificial chromosomes (BACs) through homologous recombination mediated by lambda phage-encoded Red proteins, is a widely used powerful tool in mouse, Caenorhabditis and Drosophila genetics. As Agrobacterium-mediated transfer of large DNA inserts from binary BACs and TACs into plants occurs at low frequency, recombineering is so far seldom exploited in the analysis of plant gene functions. We have constructed binary plant transformation vectors, which are suitable for gap-repair cloning of genes from BACs using recombineering methods previously developed for other organisms. Here we show that recombineering facilitates PCR-based generation of precise translational fusions between coding sequences of fluorescent reporter and plant proteins using galK-based exchange recombination. The modified target genes alone or as part of a larger gene cluster can be transferred by high-frequency gap-repair into plant transformation vectors, stably maintained in Agrobacterium and transformed without alteration into plants. Versatile application of plant BAC-recombineering is illustrated by the analysis of developmental regulation and cellular localization of interacting AKIN10 catalytic and SNF4 activating subunits of Arabidopsis Snf1-related (SnRK1) protein kinase using in vivo imaging. To validate full functionality and in vivo interaction of tagged SnRK1 subunits, it is demonstrated that immunoprecipitated SNF4-YFP is bound to a kinase that phosphorylates SnRK1 candidate substrates, and that the GFP- and YFP-tagged kinase subunits co-immunoprecipitate with endogenous wild type AKIN10 and SNF4.
基因重排（recombineering）通过噬菌体编码的 Red 蛋白介导的同源重组，实现了对细菌人工染色体（BAC）中基因的精确修饰，这是一种在小鼠、秀丽隐杆线虫和果蝇遗传学中广泛应用的强大工具。由于农杆菌介导的将二元 BAC 和 TAC 中的大 DNA 插入片段转移到植物中的效率较低，因此迄今为止，基因重排在植物基因功能分析中很少被利用。我们构建了二元植物转化载体，这些载体适合使用以前为其他生物开发的基因重排方法，从 BAC 中进行基因的缺口修复克隆。在这里，我们展示了基因重排如何促进基于 PCR 的荧光报告基因和植物蛋白之间的精确翻译融合的产生，这种融合是通过基于 galK 的交换重组产生的。经过基因重排的修饰靶基因可以单独或以更大的基因簇的一部分，通过高频缺口修复进入植物转化载体，并在农杆菌中稳定维持，且不发生改变地转化到植物中。通过体内成像分析拟南芥 Snf1 相关（SnRK1）蛋白激酶的互作 AKIN10 催化亚基和 SNF4 激活亚基的发育调控和细胞定位，展示了植物 BAC 基因重排的广泛应用。为了验证标记的 SnRK1 亚基的完整功能和体内相互作用，证明了免疫沉淀的 SNF4-YFP 与能够磷酸化 SnRK1 候选底物的激酶结合，并且 GFP 和 YFP 标记的激酶亚基与内源性野生型 AKIN10 和 SNF4 共免疫沉淀。
