Two-dimensional Electrophoresis Of Glutenin Subunits And Molecular Cloning Of LMW-GS Coding Genes In T. Dicoccoides

Both high- and low-molecular-weight glutenin subunits are the important compnents of wheat seed storage proteins and play the major role in determining bread-making quality. The AABB genome donor of bread wheat - wild emmer wheat (Triticum dicoccoides, AABB,2n=4x=28 ) was found to possess high protein content and extensive glutenin variation, therefore it is expected to serve as an important gene resource for bread wheat quality improvement. In this study, HMW- and LMW-glutenin subunits from 28 wild emmer accessions were separated and identified by SDS-PAGE, A-PAGE and A-PAGE X SDS-PAGE. A pair of allele specific PCR primers was used to amplify and clone some LMW-GS genes. The main results were as the followings:Separation and identification of HMW-GS and LMW-GS in Triticum dicoccoides by PAGEHMW-glutenin subunits of 28 wild emmer accessions were analyzed using SDS-PAGE and extensive variations at Glu-3 loci were detected. Four allelic variants (Null, 1, 1*, 2*) at Glu-A1 locus and twelve allelic variants (6+8, 7+8, 13+19, 6+ 16, 6+19, 6*+8, 6. 1+22. 1, 13+22. 1,13+22*, 14+15, 13, 16) at Glu-Bl locus were found. Except for 6+8, 7+8 and 13+19, HMW-glutenin patterns controlled by Glu-Bl locus were new allelic variants. Nine wild emmer accessions with novel HMW glutenin subunits were further characterized and confirmed by A-PAGE and two-dimensional gel electrophoresis (A-PAGE x SDS-PAGE). Some HMW-GS separated by A-PAGE showed different mobilities comparing to SDS-PAGE.LMW-glutenin subunits in Triticum dicoccoides were analyzed by SDS-PAGE, A-PAGE X SDS-PAGE. The allelic variants of four wild emmer accessions were identified based on the nomenclature of alleles proposed by Jackson et al.(1996). The results showed that eleven wild emmer accessions possessed the LMW-2 detected in cultivated durum wheat, which was found to have positive effect on bread-making quality.Cloning, characterization and molecular evolutional analysis of LMW-GS genesA pair of LMW-GS gene AS-PCR primers X13306-F595 and X13306-R2103 was used to amplify the LMW-GS genes from Triticum dicoccoides accessions. Single strongly amplified band with about 1600bp from three accessions were obtained, and then the amplified products were cloned and sequenced. The complete nucleotide sequences of three LMW-GS genes have 1649bp, 1472bp and 1754bp, respectively, containing an open reading frame, 5'-upstream sequence and 3'-downstream sequence.First amino acid of the deduced mature proteins of three LMW-GS genes was isoleucine amino acid residues, so they were all classed to LMW-i type glutenins. Three LMW-GS gene sequences showed a clear structural organization of the polypeptide comparing with other LMW-glutenin DNA sequences previously published. Except the domain I, three deduced proteins are all composed of signal peptide, domain II, domain III, domain IV and domain V. The expected number of eight cysteine residues encoded by three genes was all located in the C-terminal region, which are different from LMW-m and LMW-s type subunits. The phenetic trees produced by nucleotide and protein sequences showed that the LMW-i type subunit genes and LMW-m, LMW-s type subunit genes were clustered together respectively. LMW-m and LMW-s type genes were also separated into two subclasses. This suggested that different LMW genes have its own evolutionary way after genetic differentiation. But the LMW-i type subunit genes have accumulated more variations than LMW-m and LMW-s type genes during the evolutional process.