A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[OPEN]</a>}

Abstract

Gene targeting (GT; that is, modification of a specific DNA sequence in an endogenous gene by replacement of the target gene with a GT vector through homologous recombination [HR]) is a useful tool in both basic and applied studies. In earlier studies in higher plants, most transferred DNA (T-DNA) containing sequences homologous to the endogenous target gene was found to have integrated randomly into the plant genome through the nonhomologous end joining (NHEJ) pathway, leading to decreased GT frequency (0.01%–0.1% compared with random integration; Paszkowski et al., 1988; Offringa et al., 1990; Puchta et al., 1996). More recently, GT has developed to become a reproducible and general approach, at least in rice (Oryza sativa), through the use of a positive-negative selection system with the hygromycin resistance gene as a positive selection marker and the diphtheria toxin A subunit (DT-A) gene as a negative selection marker (Terada et al., 2002, 2007; Yamauchi et al., 2009; Moritoh et al., 2012; Ono et al., 2012; Ozawa et al., 2012; Dang et al., 2013; Tamaki et al., 2015). Toxic to rice plants, expression of DT-A is also toxic in some dicots, such as Arabidopsis (Arabidopsis thaliana; Czakó et al., 1992; Thorsness et al., 1993; Day et al., 1995; Nilsson et al., 1998; Tsugeki and Fedoroff, 1999; Weijers et al., 2003), tobacco (Nicotiana tabacum; Czakó et al., 1992; Day et al., 1995; Twell, 1995; Uk Kim et al., 1998), and Brassica campestris (Lee et al., 2003). However, successful examples of targeted gene modification through GT with a positive-negative selection system using DT-A have not been reported in higher plants other than rice, even in model plants, such as Arabidopsis or tobacco. This raises the possibility that DT-A negatively affects the growth of GT cells or the nontransformed cells surrounding DT-A-transformed cells because of its strong toxicity to higher plant cells other than rice. Alternatively, transient expression of the DT-A gene from the GT vector before T-DNA integration into the plant genome might kill potential GT cells, leading to their loss.

Previous studies have shown that expression of an antisense transcript of the neomycin phosphotransferaseII (nptII) gene in transgenic tobacco-carrying sense nptII expression cassettes led to a significant reduction in sense nptII gene transcripts and reduced kanamycin resistance, suggesting that a combination of nptII and antisense neomycin phosphotransferaseII (antinptII) genes could be utilized as a positive-negative selection system for GT experiments (Xiang and Guerra, 1993). In our previous work, transgenic rice calli expressing nptII under the control of the Cauliflower mosaic virus (CaMV) 35S promoter but not the nopaline synthase promoter could be selected on medium containing 35 mg L ^{of geneticin (}G418), suggesting a narrow range of optimal conditions for G418 selection. Thus, we hypothesized that an effective positive-negative selection system could be established by the use of nptII under the control of the CaMV35S and antinptII under the control of the rice elongation factor 1a (Pef) or maize (Zea mays) polyubiquitin1 promoter (Pubi), which confer higher levels of gene expression than the CaMV35S promoter in rice calli as positive and negative selectable markers, respectively. With the goal of establishing a more generally applicable and more publicly acceptable GT approach in plants, this study investigated whether concomitant expression of the antinptII gene in nptII-expressing transgenic rice would function as a negative selection marker in rice. Furthermore, this approach was also applied successfully to knockout of the endogenous waxy gene, encoding a granule-bound starch synthase (Wang et al., 1995), and the 33-kD globulin (Glb33) gene, encoding glyoxalase I (defined as a major allergen of rice; Usui et al., 2001).

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