| High-molecular-weight glutenin subunits (HMW-GS) are components of wheat storage proteins and are responsible in part for the viscoelasticity of the dough. Therefore, it is highly important to have fast methods for HMW-GS identification and to isolate their coding genes, which is useful for quality improvement of bread quality.In this research, HMW-GS from hexaploid, tetraploid and diploid wheat species were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Acid-PAGE (A-PAGE). The Two-dimentional PAGE (A-PAGE X SDS-PAGE) patterns of Triticum durum cultivar Simeto and Triticum dicoccum PI355463 indicated that each HMW glutenin subunit showed single component encoded by different genes at Glu-Al and Glu-Bl loci. The HMW-GS in Simeto were further characterized by High Performance Capillary Electrophoresis (HPCE) and the same results were obtained.The different HPCE conditions for HMW-GS separation were investigated. The results showed that 0.1M phosphate-glycine (pH2.50) buffer, containing 20% acetonitrile (ACN) and 0.05% Hydroxypropylmethyl-cellulose (HPMC) with a 25.5 cm length capillary (50u m i.d.) appeared to be optimal. By using this method, HMW-GS from different wheat species could be separated completely within about 20 minutes. Some HMW-GS, including good-quality subunits and novel subunits detected, e.g. 1B+17, 1By18, 18+14,. 1By, 18x13, 1 By 16 and lBy22,were identified and their migration orders were deduced judging by their electroelution time. It is clearly that HPCE has the advantages of fast and good resolution, small sample amounts and automation compared with regular gel electrophoresis. Thus, high performance capillary electrophoresis is a powerful tool for the identification of specific HMW-GS.The complete open reading frames of three HMW-GS genes including the upstream sequence of one gene were amplified directly from genome DNA by allele-specific PCR. The amplified products were cloned and sequenced. The nucleotide and deduced amino-acid sequence indicated that the 2.1kb PCR product obtained from durum cultivar Simeto was confirmed to be the coding sequence of IBy8 gene. This gene comprise 3020bp including 854bp upstream sequence. Some typical sequences of HMW-GS and eukaryotic genes, such as TATA box, the CCAAT-like sequence, -300 element and an enhancer sequence, were found. In comparison with By9 subunit, the By8 displayed 2 residue substitutions at the position 78 and 442 and one 15 residue insertion at the position 342. The nucleotide sequence of the same size PCR product obtained from Triticum dicoccum PI355463 showed highly homologous with y-type subunit genes, and was therefore identified as the partial coding sequence of 1By22gene. The PCR product about 1.9 kb from Triticum dicoccum TT268 had greater homology with the reported lAy silent gene, which possessed 5 stop codons in the repeated domain. Thus it was confirmed to be Ay silent gene and named 1 Ay2d.Compared with T. aestivum, tetraploid wheat species T. durum and T. dicoccum have the same A and B genome donor and possess less HMW-GS genes. This may be more convenient to isolate HMW-GS genes at Glu-Al and Glu-B1 loci by AS-PCR. This work, for the first time reported the HPCE patterns of some good-quality and novel subunits and complete By8 gene sequence in durum wheat as well as the coding sequence of Ay2d silent gene and partial coding sequence of By22* gene in cultivated emmer wheat. These genes have been deposited in GeneBank with the numbers of AY245797 (Glu-lBy8) and AY260549 (Glu-lAy2d). |
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