Modification of Endogenous Natural Genes by Gene Targeting in Rice and Other Higher Plants

Abstract

The capability to modify a genomic sequence into a designed sequence is a powerful tool for biologists and breeders to elucidate the function of an individual gene and its cis-acting elements of multigene families in the genome. Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome. In higher plants, however, the overwhelming occurrence of the random integration of transgenes by non-homologous end-joining is the main obstacle to develop efficient gene targeting. Two approaches have been undertaken to modify a genomic sequence in higher plants- chimeric RNA/DNA oligonucleotide-directed gene targeting to generate a site-specific base conversion, and homologous recombination-dependent gene targeting to produce either a base change or a gene replacement in a sequence-specific manner. The successful and reproducible targeting of an endogenous gene by homologous recombination, independently of gene-specific selection by employing a strong positive-negative selection, has been demonstrated for the first time in rice, an important staple food and a model plant for other cereal species. This review addresses the current status of targeting of an endogenous natural gene in rice and other higher plants and discusses possible models for Agrobacterium- mediated gene targeting by homologous recombination using a strong positive-negative selection.

Commentary: Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

“…Successful in vivo gene modification has been demonstrated notably in maize, rice, tobacco and wheat, e.g. to create plants insensitive to the action of a specific herbicide (Dong et al, 2006;Iida and Terada, 2005;Kochevenko and Willmitzer, 2003;Okuzaki and Toriyama, 2004;Zhu et al, 2000). Altered genes have been shown to be stably maintained during mitosis (Beetham et al, 1999;Kochevenko and Willmitzer, 2003), and transmitted in a Mendelian fashion to subsequent generations (Zhu et al, 1999(Zhu et al, , 2000.…”

Evaluation of Endogenous Gene Validation: An Important Contrivance for Differential Gene Study in Pearl Millet

“…Modification of endogenous gene is done by homologous recombination-dependent gene targeting and chimeric RNA/DNA oligonuclieotide -directed gene targeting to generate a site-specific base conversion, and homologous recombination-dependent gene targeting to produce either a base change or a gene replacement in a sequence-specific manner (Iida and Terada, 2005).…”

An efficient gene targeting system using homologous recombination in plants

“…식물에는 kanamycin, geneticin (G418)에 저항성을 보이는 NPTII 유전자와 hygromycin에 저항성을 보이는 HPT 유전자가 positive marker로 사용되며, 식물의 생존에 영향을 미치는 치사유전자인 cytosine deaminase를 번역하 는 codA유전자와 diptheia toxin A fragment를 번역하는 DT-A유전자가 negative marker로 사용된다 (Iida and Terada 2005). 벼에 이 시스템을 적용하여 Waxy (Terada et al 2002;Iida and Terada 2004;Iida and Terada 2005;Ozawa et al 2012), Alcohol dehydrogenase2 (ADH2) (Terada et al 2007), Methyltransferase1a (Met1a) (Yamauchi et al 2009), Repressor of silencing1 (ROS1) , Domains rearranged methylase1 (DRM2) (Moritoh et al 2012), 그리고 β1,2-xylosyltransferase (Ozawa et al 2012) 의 변이체가 제작되었다 ( Table 1). 그러나 상동재조합을 통한 진타겟팅의 성공율 은 positive selection된 개체 중 약 1.1%만 negative selection 에서 생존하였다 .…”