A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice

Abstract

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.

A Novel Rice Cytochrome P450 Gene, CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

“…A BS-insensitive mutant ALS gene has been used widely as a positive marker to obtain transformed cells and plants under BS selection. For example, a combination of a mutated rice ALS gene and BS selection has been applied in rice (Osakabe et al, 2005;Okuzaki et al, 2007;Wakasa et al, 2007;Taniguchi et al, 2010), wheat (Ogawa et al, 2008), tall fescue (Festuca arundinacea; Sato et al, 2013), Arabidopsis (Kawai et al, 2010), and soybean (Glycine max; Tougou et al, 2009). Like the mutated ALS gene, the CYP81A6 gene has also been reported as a selection marker with BSM in Arabidopsis, cotton (Gossypium hirsutum), and tobacco plants (Ke et al, 2012;Liu et al, 2012).…”

Transgenic Tall Fescue and Maize with Resistance to ALS-Inhibiting Herbicides

“…Mutated ALS genes derived from Arabidopsis have been reported to be useful as selectable markers in various plants, such as tobacco (Gabard et al, 1989), rice (Li et al, 1992), potato (Anderson et al, 2003), oilseed mustard (Brassica juncea) (Ray et al, 2004) and maize (Zhang et al 2005). Mutated OsALS genes have been demonstrated to be useful as selectable markers in rice (Osakabe et al, 2005;Okuzaki et al, 2007). Using these mutated OsALS genes, transgenic plants have been produced in various plants such as rice (Osakabe et al, 2005;Okuzaki et al, 2007), soybeans (Tougou et al, 2009), tall fescue (in preparation) and wheat (Triticum aestivum L.) (Ogawa et al, 2008).…”

“…Mutated OsALS genes have been demonstrated to be useful as selectable markers in rice (Osakabe et al, 2005;Okuzaki et al, 2007). Using these mutated OsALS genes, transgenic plants have been produced in various plants such as rice (Osakabe et al, 2005;Okuzaki et al, 2007), soybeans (Tougou et al, 2009), tall fescue (in preparation) and wheat (Triticum aestivum L.) (Ogawa et al, 2008). Some studies have suggested that homology-dependent gene silencing is associated with the presence of either multiple copies of homologous transgenes and promoters (Matzke & Matzke, 1995) or a transgene and a homologous endogenous gene (Meyer, 1995).…”

“…The CaMV 35S promoter was used for two genes (OsALS (dm), hpt) in the same binary vector; therefore, the chances of gene silencing may have increased by overuse of the CaMV 35S promoter. Okuzaki et al (2007) reported that some transgenic rice calli with multiple copies of a mutated ALS gene driven by the maize ubiquitin promoter did not regenerate, whereas transgenic calli with only one or two transgenes did. On the other hand, no relationship between herbicide resistance and copy number was apparent in wheat transformation using the rice ALS promoter (Ogawa et al, 2008).…”

Application of the acetolactate synthase gene as a cisgenic selectable marker for Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa ssp. pekinensis)

“…An amino acid substitution at a particular position in ALS has been shown to confer resistance to herbicides such as sulfonylureas and imidazolinones and to pyrimidinyl carboxy herbicides. Mutant forms of the ALS gene have been reported to be useful as a selectable marker in many plant species, such as potato (Andersson et al 2003), oilseed mustard (Ray et al 2004), rice (Okuzaki et al 2007;Osakabe et al 2005), maize (Zhang et al 2005), cotton (Rawat et al 2008), wheat (Ogawa et al 2008), and soybean (Tougou et al 2009). The use of an ALS gene derived from the host crop itself would be expected to result in a more publicly acceptable genetically modified (GM) crop compared with the use of a conventional antibiotic selectable marker.…”