| The high-molecular-weight glutenin subunits (HMW-GS) are main seed storage proteins encoded by multi-alleles at Glu-1 loci in wheat. There are two subfamilies called x-type and y-type subunits according to their molecular weight. It is well known that bread-making quality is highly associated with the composition of HMW glutenin subunits. Studies showed that the Gli-D1 loci play an important role in bread-making quality. The allelic variations at the Gli-Dl loci in bread wheat, however, are rather limited. Therefore, it is important to widen the genetic basis of bread wheat by using useful genes of related Triticum species. The D genome donor of bread wheat - diploid Asian goatgrass, Aegilops tauschii Coss (syn. Triticum tauschii, 2n = 2x = 14, DD) was found to possess extensive variations at the Gli-Dl loci, and therefore it is expected to serves as an important gene resource for bread wheat quality improvement. In this study, some specific HMW glutenin subunits from Aegilops tauschii were separated and identified by SDS-PAGE, A-PAGE., 2-DE (A-PAGE x SDS-PAGE) and High Performance Capillary Electrophoresis (HPCE): Allele specific PCR primers were designed to amplify and clone some specific HMW-GS genes. The main results were as the followings:Separation and identification of specific HMW-GS in Aegilops tauschii by PAGE and HPCESome novel HMW-GS in Aegilops tauschii were detected by one- and two-dimensional gel electrophoresis and they were named as 1Dx1.5t, 1Dx5.2t, IDy10.1t, lDy12.1t, 1Dy12.2t and lDy12.5t, respectively. They were further characterized by high performance capillary electrophoresis (HPCE). The high resolution CE separations of HMW-GS could be obtained by 0.1 M phosphate-glycine (pH2.50) buffer, containing 20% acetonitrile (ACN) and 0.05% Hydroxypropyl-methylcellulose (HPMC) with a 25.5 cm length capillary (50um i.d.). In addition, the multiple peaks of single HMW-GS under above acidic CE conditions were found, which may relate to post-translational modification of glutenin proteins. Our results showed that HPCE is a powerful tool for HMW-GS separation and characterization with low cost, minor sample requirement, good resolution, and rapidly automatic separation.Cloning, characterization and molecular evolutional analysis of HMW-GS genes at Glu-Dt 1 locusAS-PCR primers were designed to amplify the upstream and coding region of HMW-GS genes from TD159 with IDy12.1t gene and TD81 with 1Dy10.1t gene. Single strongly amplified band with about 1850bp from both accessions were obtained, and then the amplified products were cloned and sequenced. The complete nucleotide sequence of 1Dy12.1t gene was 2807bp, containing an open reading frame of 1950bp and 857bp of upstream sequence. A perfectly conserved enhancer sequence and the -300 element were present at positions of 209-246bp and 424-447bp upstream of the ATG start codon, respectively. The deduced mature protein of 1Dy12.1t subunit comprised 648 amino acid residues and had great similarity to 1Dy10 subunit from bread wheat with only seven amino acid substitutions, suggesting that 1Dy12.1t gene could have positive effect on bread-making quality. The complete nucleotide sequence of 1Dy10.1t gene was 1980bp. Sequence analysis showed that it is the coding sequence of typical y-type HMW-GS genes. Its deduced mature protein had 638 amino acid residues and was highly homologous with 1Dy12 subunit of bread wheat. The phenetic trees produced by nucleotide and protein sequences showed that the x-and y-type subunit genes were clustered together, respectively, and the 1Dy12.1t and 1Dy10.1t genes are closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat. Expression and identification of 1Dy12.1t gene in E. coliBecause of high similarity to 1Dy10 good-quality subunit, it could be concluded that 1Dy12.1t gene have positive effect on bread-making quality. Thus, in order to further understand its function, the expression characters of the 1Dy 12.1t gene were primarily investigated. Cloned 1Dy12.1t gene was connected... |
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