Positive-negative-selection-mediated gene targeting in rice.

Zenpei Shimatani,Masaki Endo,Rie Terada,Seiichi Toki,Ayako Nishizawa-Yokoi

doi:10.3389/fpls.2014.00748

Abstract

Gene targeting (GT) refers to the designed modification of genomic sequence(s) through homologous recombination (HR). GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ) occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive–negative selection (PNS) is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology based on PNS are discussed.

Highlights

ADVANTAGES OF DEVELOPING A Positive–negative selection (PNS) SYSTEM IN RICE In higher plants, the establishment of Gene targeting (GT) of endogenous natural genes through homologous recombination (HR) has been hampered by the overwhelming occurrence of non-homologous end joining (NHEJ), i.e., random recombination, even when the transformed gene carries sequence(s) homologous to the target gene locus
Induction of a double strand break (DSBs) at the target site using an artificial endonuclease is progressing as a means of establishing GT in several plant species (Shukla et al, 2009; Zhang et al, 2013; Endo and Toki, 2014; Puchta and Fauser, 2014), most DSBs re-connected by NHEJ result in target gene disruption
Osros1a-GUS was detected in pollen and unfertilized ovules; concomitantly, an arrest of endosperm growth was observed in heterozygous knock-in rice (Ono et al, 2012). All these results show that the PNS-mediated GT procedure is able to generate novel mutant rice plants based on information gleaned from DNA sequence(s) and encoded protein motif(s), and that the functions of both endogenous promoters and genes can be studied effectively with plants segregated for the targeted locus

Summary

Introduction

ADVANTAGES OF DEVELOPING A PNS SYSTEM IN RICE In higher plants, the establishment of GT of endogenous natural genes through HR has been hampered by the overwhelming occurrence of NHEJ, i.e., random recombination, even when the transformed gene carries sequence(s) homologous to the target gene locus. Because almost all PNS-mediated GT in rice occurs in a heterozygous manner without any additional insertion of the GT vector (Terada et al, 2002, 2007), endogenous promoter activity in the original gene locus can be detected in the targeted heterozygote (Figure 1J).

Results

Conclusion