Homologous recombination-dependent gene targeting and an active DNA transposon nDart-promoted gene tagging for rice functional genomics

Abstract

Rice is an important staple food for more than half the world’s population, and it has become the first crop plant to have its 389-Mb genome sequenced (IRGSP 2005). Even though various functional genomic tools for elucidating the function of rice genes are available (Hirochika et al. 2004; Leung and An 2004; Sasaki et al. 2005; Upadhyaya 2007), developing new methods for characterizing genes of interest by both forward and reverse genetic approaches has become particularly important. This chapter contains a description of the current state of gene targeting mediated by homologous recombination (HR) and gene tagging promoted by a non-autonomous DNA-based active rice transposon, nDart, for rice functional genomics, both of which are being developed (Terada et al. 2002, 2007; Tsugane et al. 2006; Takagi et al. 2007). Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome and, often, to the conversion of the endogenous gene into a designed sequence. Gene targeting has been shown to lead to both gene replacements and base changes (Reiss 2003; Iida and Terada 2004, 2005); by the end of 2006, there was only one report describing reproducible true gene targeting (TGT) in rice, and it dealt with the inactivation or knockout of the endogenous Waxy gene by gene disruption (Figs. 1a and 2; Terada et al. 2002). Because independent gene targeting events by HR should generate an identical genomic structure with a designed sequence alteration(s), the experimental demonstration of the capability for the reproducible isolation of recombinants with the anticipated gene structure would be very important.