Antisense Waxy Genes with Highly Active Promoters Effectively Suppress Waxy Gene Expression in Transgenic Rice

Terada, Rie; Nakajima, Mitsutoshi; Isshiki, Minoru; Okagaki, Ron J.; Wessler, Susan R.; Shimamoto, Ko

doi:10.1093/pcp/pcd008

Cited by 59 publications

(25 citation statements)

References 36 publications

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“…The first is to suppress GBSS activity. Expression of antisense GBSSI has been reported to yield a rice starch with reduced AC Itoh et al 1997;Terada et al 2000). The second mechanism is through modulation of GBSSI promoter activity.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of a novel Waxy allele from a Yunnan rice landrace

Liu

et al. 2009

Plant Mol Biol

102

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Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. To gain a better understanding of the molecular mechanism controlling AC formation, we screened 83 Yunnan rice landraces in China and identified a rice variety, Haopi, with low AC. Genetic analyses and transgenic experiments revealed that low AC in Haopi was controlled by a novel allele of the Wx locus, Wx(hp), encoding a granule-bound starch synthase (GBSSI). Sequence comparisons of Wx(hp) and Wx(b) alleles (from Nipponbare) showed several nucleotide changes in the upstream regulatory regions (including the promoter, 5'-untranslated region, and first intron 5' splicing junction site). Interestingly, these changes had no obvious effect on the expression level and splicing efficiency of Wx transcripts. In addition, an examination of the coding region revealed that the Wx(hp) allele carries an A-to-G change at nucleotide position +497 from the start codon, resulting in an Asp(165)/Gly(165) substitution. The amino acid substitution had no detectable effects on GBSSI activity in vitro; however, it notably reduced the binding of GBSSI to starch granules, resulting in a reduction of AC in rice seeds. Moreover, three other Yunnan landraces with low AC also carry a nucleotide substitution identical to Haopi at the +497 position of the Wx gene, suggesting common ancestry. Based on the single-nucleotide polymorphism, we have developed a new derived cleaved amplified polymorphic sequence marker for use in breeding practice to manipulate AC in rice endosperm.

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Section: Discussionmentioning

confidence: 99%

Identification and characterization of a novel Waxy allele from a Yunnan rice landrace

Liu

et al. 2009

Plant Mol Biol

102

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“…More recent studies have found that mutations at other loci, such as du-1 and du-2, also cause a reduced amylose content through their effects on the splicing of the waxy pre-mRNA , and that complete absence of amylose or its reduction was observed in transgenic rice whose wild-type wx gene expression was suppressed by antisense genes (Terada et al 2000). These studies indicate that a variety of mechanisms are responsible for the waxy expression, including transcriptional and post-transcriptional processing, and translational regulation.…”

Section: Discussionmentioning

confidence: 99%

Microsatellites in starch-synthesizing genes in relation to starch physicochemical properties in waxy rice (Oryza sativa L.)

2002

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Rice starch is composed of amylose and amylopectin. Amylose content, an important determinant of rice starch quality, is primarily controlled by the waxy gene, encoding granule-bound starch synthase (GBSS). The starch branching enzyme (SBE) and soluble starch synthase (SSS) play major roles in the synthesis of amylopectin. Microsatellite polymorphisms in the three genes, the wx gene encoding granule-bound starch synthase I, the SBE gene encoding starch branching enzyme I and the SSS gene encoding soluble starch synthase I, were studied for 56 accessions of waxy rice ( Oryza sativa L.). Four (CT)(n) microsatellite alleles, (CT)(16), (CT)(17), (CT)(18) and (CT)(19), at the wx locus were detected in this set of waxy rice, of which (CT)(17) was the most frequent. Three (CT)(n) microsatellite allele classes were found at the SBE locus, (CT)(8) or (CT)(10) together with an insertion sequence of CTCTCGGGCGA, and (CT)(8) alone without the insertion. There were multiple microsatellites clustered at the SSS locus. However, these alleles can also be grouped into three classes, i.e. the allele class SSS-A = (AC)(2) em leader TCC(TC)(11) em leader (TC)(5)C(ACC)(11), the allele class SSS-B = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(9), and the allele class SSS-C = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(8). The analyses of starch physicochemical properties among different microsatellite genotypes indicated that the waxy rice group with the (CT)(19) allele, the SBE-A allele and the SSS-B allele was quite different from other groups. Nine out of 15 accessions with a high gelatinization temperature (GT) belonged to the wx (CT)(19) group, all of them belonged to the SBE-A group and 13 of them belonged to the SSS-B group. These microsatellites might be useful in marker-assisted breeding for the improvement of rice grain quality.

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“…Previous studies demonstrated that the introduction of the antisense Wx gene into Japonica rice causes a reduction of amylose in the endosperm (Shimada et al 1993, Terada et al 2000. These studies showed that the differential regulation of the antisense gene expression by using low-or high-active promoters led to various levels of suppression of the endogenous Wx gene expression.…”

Section: Introductionmentioning

confidence: 99%

Introduction of Wx Transgene into Rice wx Mutants Leads to Both High- and Low-Amylose Rice

Itoh

Ozaki

Okada

et al. 2003

Plant and Cell Physiology

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;The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx a , which produces a large amount of amylose, and Wx b , which produces a smaller amount of amylose because of the mutation at the 5¢ splice site of intron 1. Wx b is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx a cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx a allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx a transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.

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Antisense Waxy Genes with Highly Active Promoters Effectively Suppress Waxy Gene Expression in Transgenic Rice

Cited by 59 publications

References 36 publications

Identification and characterization of a novel Waxy allele from a Yunnan rice landrace

Identification and characterization of a novel Waxy allele from a Yunnan rice landrace

Microsatellites in starch-synthesizing genes in relation to starch physicochemical properties in waxy rice (Oryza sativa L.)

Introduction of Wx Transgene into Rice wx Mutants Leads to Both High- and Low-Amylose Rice

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