| The high molecular weight glutenin subunits (HMW-GS) are a group of 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 firmly associated with the composition of HMW glutenin subunits. Studies showed that the extensive allelic variations of HMW-GS were found in the related Triticum species, which are expected to be useful gene resources for quality improvement of bread wheat.To acquire new HMW glutenin subunits from closely related species of bread wheat, we chose T. dicoccum, T. durum, T. spelta and T. compactum as materials, and used SDS-PAGE, 2-D A-PAGE SDS-PAGE and capillary electrophoresis (CE) to detect the allelic variations of HMW-GS. Some novel subunits e.g. Bx6.1, By22.1, Bxl3* and Byl9* were found in different tetraploid and hexaploid species. The high resolution and repeatable CE separations of HMW-GS could be obtained by 0.1M phosphate-glycine (pH2.50) buffer, containing 20% acetonitrile (ACN) and 0.05 % Hydroxypropylmethyl-cellulose (HPMC) with a 25.5 cm length capillary (50 m i.d.). The electroelution times and orders of several novel subunits were established, namely Bx6.1 at about 10min, By22.1 at 7-8min, Bxl3* at 8-9min and Byl9* at 7-8min. The order of all kinds of HMW glutenin subunits according to migration time is: By, Dy- Ax- Bx- Dx. In addition, the multiple peaks of single HMW-GS under CE conditions used were found, which may relate to post-translational modification of protein. Our results showed that CE is a powerful tool for HMW-GS characterization with low cost, minor sample requirement, good resolution and reproducibility, and rapidly automatic separation.According to conservation sequences of 5' and 3' regions of HMW glutenin genes, we designed 4 pairs of primers to amplify the coding sequences of x- and y-type genes and upstream sequence of Ay gene, respectively. Single bands of strong amplifications were obtained through allelic-specific PCR of gDNA from closely related species of bread wheat. After cloning and sequencing, we got complete coding sequences of Ayld and Ay1s genes,upstream sequence of Ayld, and partial coding sequences of Bx6.1 and two y-type genes.The upstream and coding sequence of Ayld gene have 2609bp in length with 97% homology with the reported Ay gene. There is a 'TATA' box of sequence TATAAA 85-91bp upstream from start codon. Three sequences similar to the 'CCAAT' box are present between -233bp---119bp. The 5' flanking region also contains 3 direct repeats with homology to '-300 element' of prolamin genes. There are no introns in the coding sequence of Ayld gene, but four stop codons occur at 442bp, 451bp, 856bp and 1063bp, respectively. Ay 1s gene possesses 1827bp in length with 96% homology with Ay gene. Besides 5 basis substitutions, there is another stop codon at 988bp compared with Ayld gene. Ayld and Ay1s genes have been registered in GenBank with accession No. of AY260548 and AY303766, respectively.The amino acid sequences deduced from Ayld and Ay 1s genes show that the first 21 residues work as signal peptide. The mature protein has 3 domain in structure: N-terminal domain of 104 amino acids, repetitive domain of 441 amino acids and C-terminal domain of 42 amino acids. The hexapeptide and nonapeptide repeats make up 75% of the final protein. The sequence of Ayld and Ayls predict 6 cysteine residues, five within N-terminal domain and 1 within C-terminal domain. Ayld gene has another cysteine residue located in the repetitive domain near C-terminal domain.Partial coding sequence of Bx6.1 gene has 94% homology with Bx7. Subunit Bx6.1 also has a signal peptide of 21 amino acids. There are tripeptide (DQQ), hexapeptide and nonapeptide in the central repetitive domain. Partial coding sequences of the other two y-type genes show considerable homology with Dyl2.The 5 new genes are compared and analysed with the HMW glutenin genes deposited in GenBank. Their relationship d...
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