This study focused on optimizing phosphate-based buffers and other capillary electrophoresis (CE) parameters for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), emmer (Triticum dicoccum, AABB, 2n = 4x = 28) and Aegilops tauschii (DD, 2n = 2x = 14). The fast and high-resolution separation of HMW-GS was achieved using 0.1 M phosphate-glycine buffer (pH 2.5, containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose) at 12.5 kV and 40 degrees C with 25 microm inside diameter (ID)x27 cm uncoated fused-silica capillary. In general, one sample separation can be analyzed in 15 min. The good run-to-run repeatable separation of HMW-GS could be obtained with a relative standard deviation of less than 1% when capillaries were rinsed with 1 M phosphoric acid for 2 min, followed by separation buffer for 2 min after each separation. The HMW-GS from some bread wheat cultivars as well as tetraploid and diploid accessions was separated by the CE method described above, and all subunits detected were well characterized and readily identified. Some HMW-GS showed reversed mobilities and elution order compared to the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-CE. Particularly, most of the HMW-GS analyzed with the CE buffer used were separated into multiple peaks, generally a high peak plus a minor peak. CE appears to be capable of separating and characterizing HMW-GS with fast and high-resolution features, therefore it is expected to be useful for specific germplasm screening and desirable HMW-GS identification in wheat quality improvement.
A novel y-type high-molecular-weight glutenin subunit possessing a slightly faster mobility than that of subunit 1Dy12 in SDS-PAGE, designated 1Dy12.1(t) in Aegilops tauschi, was identified by one- and two-dimensional gel and capillary electrophoresis. Its coding gene at the Glu-D(t) 1 locus was amplified with allele-specific-PCR primers, and the amplified products were cloned and sequenced. The complete nucleotide sequence of 2,807 bp containing an open reading frame of 1,950 bp and 857 bp of upstream sequence was obtained. A perfectly conserved enhancer sequence and the -300 element were present at positions of 209-246 bp and 424-447 bp upstream of the ATG start codon, respectively. The deduced mature protein of 1 Dy12.1(t) subunit comprised 648 amino acid residues and had a Mr of 67,518 Da, which is slightly smaller than the 1Dy12 (68,695 Da) but larger than the 1Dy10 (67,495 Da) subunits of bread wheat, respectively, and corresponds well with their relative mobilities when separated by acid-PAGE. The deduced amino acid sequence indicated that the 1Dy12.1(t) subunit displayed a greater similarity to the 1Dy10 subunit, with only seven amino acid substitutions, suggesting that this novel gene could have positive effect on bread-making quality. A phenetic tree produced by nucleotide sequences showed that the x- and y-type subunit genes were respectively clustered together and that the Glu-D(t) 1y12.1 gene of Ae. tauschii is closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat.
Cereal Chem. 81(5):561-566High molecular weight glutenin subunits (HMW-GS) from three hexaploid wheat species (AABBDD, 2n=6x=42, Triticum aestivum L., T. spelta L., and T. compactum L.) were separated and identified by acidic capillary electrophoresis (A-CE) with phosphate-glycine buffer (pH 2.5) in uncoated fused-silica capillaries (50 µm, i.d. × 25.5 cm) at 12.5 kV and 40°C. The rapid separations (<15 min) of HMW-GS with good repeatability (RSD < 2%) were obtained using a fast capillary rising protocol. All 17 HMW-GS analyzed could be well separated and their relative migration orders were ranked. In particular, the good quality subunit pair 5+10 could be differentiated from poor quality subunit pair 2+12. In addition, the other three allelic pairs of 13+16, 17+18, and 7+8 subunits that were considered to have positive effects on dough properties, as well as three pairs of novel subunits 13+22
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