Protocol: Precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli.

Abstract

Plant genome sequence data now provide opportunities to conduct molecular genetic studies at the level of the whole gene locus and above. Such studies will be greatly facilitated by adopting and developing further the new generation of genetic engineering tools, based on homologous recombination cloning in Escherichia coli, which are free from the constraints imposed by the availability of suitably positioned restriction sites. Here we describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs coordinately driven by locus-specific regulatory elements.