Identification of mutations in OASA1 gene from a gamma‐irradiated rice mutant population

Abstract

One thousand three hundred and fifty homologous rice M 8 mutant lines resistant to 5-methyltryptophan, a tryptophan analogue, were selected from embryogenic calli irradiated with 30-120 Gy of gamma rays. The mutant rice would be compromised in tryptophan homoeostasis in the cell if a negative feedback by anthranilate synthase (AS) in tryptophan biosynthesis was altered by gamma irradiation, consequently causing tryptophan over-accumulation. Therefore, the correlation between traits of over-accumulated tryptophan and the alteration of AS by mutagenesis including single-nucleotide polymorphisms (SNPs) would enable the visualization and selection of overaccumulated mutant lines from the rice mutant population. Targeting Induced Local Lesions IN Genome analysis with five fluorescentlabelled primer sets covering the OASA1 locus resulted in the selection of three rice mutant lines, MRVII-9, MRVII-33 and MRVII-37. The tryptophan contents in the seed storage protein of the three mutant lines were elevated when compared with the wild-type cv. ÔDonganÕ. Genomic DNA of OASA1 was re-sequenced and validated at the SNP and other polymorphism sites. Three mutant lines (MRVII-9, MRVII-33 and MRVII-37) not only contained more than five SNP sites in the total regions of OASA1 gene, but also contained SNPs in coding regions that resulted in amino acid changes such as E241G, A560G and V495F/S496F, respectively. The SNPs, which resulted in amino acid changes, may affect the enhanced tryptophan content in the seed storage protein. These different features could utilize the selection of mutant lines with over-accumulated tryptophan from large rice mutant populations.

Current Technologies in Plant Molecular Breeding

“…In humans, tryptophan defi ciency can induce pellagra and cause low levels of serotonin, which have been related to depression, anxiety, aggression, and overeating (Lucki 1998 ;Hegyi et al 2004 ). In this study, to identify the mutations that resulted in high levels of tryptophan in rice, a total of 1,350 mutant lines among the M8 generation were used in a TILLING analysis Chun et al 2012 ). In this study, to identify the mutations that resulted in high levels of tryptophan in rice, a total of 1,350 mutant lines among the M8 generation were used in a TILLING analysis Chun et al 2012 ).…”

“…The three mutant lines (MRVII-9, MRVII-33, and MRVII-37) with changed translated amino acid sequences have shown good agronomical traits and alterations (Chun et al 2012 ) in tryptophan biosynthesis compared with the cv. Dongan line.…”

“…(Chun et al 2012 ) Genetic engineering that encompasses transformation is an advanced breeding method that can overcome reproductive barriers and extend genetic variations. In traditional breeding programs, a lot of resources are usually required, including time, land, and money before the selected traits can be introduced successfully into existing commercial cultivars or elite lines.…”

“…11 Variation of tryptophan content between the wild-type cv. Dongan and three high-tryptophan-containing mutant lines that were genetically fi xed by self-pollination(Chun et al 2012 ) …”

Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site-Directed Mutagenesis of the Anthranilate Synthase Gene in Rice

“…Missense mutations in the OASA1 gene were discovered in some of these mutants, suggesting that these mutations might affect Trp accumulation [8]. Trp fortification in rice has also been achieved by the overexpression of mutated enzyme genes.…”