Inheritance of alleles for glutelin alpha-2 subunit genes in rice and identification of their corresponding cDNA clone

Rice glutelins consist of acidic (alpha) and basic (beta) subunits which are further separated into three polypeptide components assigned as alpha-1, alpha-2, and alpha-3 subunit components and beta-1, beta-2 and beta-3 subunit components. Nine rice mutant lines with a decreased amount of the glutelin alpha-2 subunit component (alpha-2L) were obtained by screening about 6,800 potential mutant lines derived from the fertilized egg treatment with N-methyl-N-nitrosourea (MNU) using the SDS-PAGE method. The mutants were classified into three types of the increased alpha-1 subunit (alpha-1H/alpha-2L), the decreased beta-2 subunit (beta-2L/alpha-2L) and the increased alpha-3 subunit (alpha-3H/alpha-2L) represented by EM278, CM1707 and EM659, respectively. Iso-electric focus (IEF) analysis revealed that all of the mutants had an extremely low amount of a polypeptide with a 6.71 pI value, whereas a polypeptide with either a 6.50 pI value or with a 6.90 pI value increased significantly in alpha-1H/alpha-2L mutants or in alpha-3H/alpha-2L mutants, respectively. The beta-2L/alpha-2L mutants had a decreased amount of a basic polypeptide with a 8.74 pI value. Genetic analysis revealed that the three types of mutants were controlled by a single incomplete dominant gene respectively, and the three are alleles. The gene was temporarily named glu4, which was found to be located on chromosome 1 linked with the eg and spl6 genes. Two-dimensional electrophoresis analysis revealed that the glu4 encoded polypeptides of pI 6.71/alpha-2 and pI 8.74/beta-2. Amino acid sequence analysis suggested that the mutated acidic polypeptide was the product of a GluA subfamily gene. Northern and RT-PCR analyses revealed that glu4 corresponded to the GluA-1 gene.