Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding t...Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.展开更多
A total of 43 unique clones(Z4A-1 to Z4A-43 with GenBank accession numbers of HM120221, HM120222, JX828270, JN831402 to JN831406, and KC715889 to KC715923, respectively) were amplified and cloned from common wheat cul...A total of 43 unique clones(Z4A-1 to Z4A-43 with GenBank accession numbers of HM120221, HM120222, JX828270, JN831402 to JN831406, and KC715889 to KC715923, respectively) were amplified and cloned from common wheat cultivar Zhengmai 004 using a PCR-based strategy. They included 22 full-ORF α-gliadin genes and 21 pseudogenes containing at least one in-frame stop codon. Comparative analysis of the deduced amino acid sequences showed that all the isolated genes displayed the typical structural features of α-gliadin genes and that the putative proteins of Z4A-7, Z4A-14, Z4A-17 and Z4A-20 had an extra cysteine residue in the unique domain II, while Z4A-15 lacked the second conserved cysteine residue in the unique domain I. The two fusion proteins of Z4A-15 and Z4A-20 were successfully detected by SDS-PAGE and Western blotting, although the protein level was relatively low. Based on the occurrence of the four major epitopes, as well as the lengths of the two glutamine repeats, 8, 6, and 8 genes were assigned to the Gli-2 loci on the respective chromosomes 6A, 6B, and 6D and a total of respectively 16, 0 and 23 immunogenic peptides were identified. In addition, 4 of the 5 genes with odd numbers of cysteine residues were assigned to chromosome 6D, suggesting that common wheat cultivar Zhengmai 004 has the potential to induce celiac disease(CD) and that the D genome exerts the most influence on gluten quality, but is the most deleterious for CD patients. By phylogenetic analysis, 11 exceptional α-gliadins with few or no immunogenic peptides from Triticum monococcum and Aegilops tauschii were detected, a finding that further supports the prospect of CD prevention. Finally, secondary structure prediction showed that most(98.48%) of the α-gliadins invariably contained five conserved α-helices(H1 to H5) in the two glutamine repeats and unique domains and 67.68% of the α-gliadins also contained a β-strand(S) in the C-terminal unique domains. An absent α-helix H2, 1–2 extra α-helices, or an additional β-strand(SE) also probably occurred in some cases. Of the 22 cloned genes in this work, 10 putative proteins contained 1–2 extra α-helices in addition to the five conserved α-helices or the additional β-strand. The observation that most of the α-helices and β-strands were present in the two unique domains and that an extra α-helix also probably occurred in the two glutamine repeats in some desirable genes strongly suggested that these two uniquedomains are the most important regions for the function of α-gliadins, although the glutamine repeats would also contribute in some cases.展开更多
Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions...Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.展开更多
基金supported by the National High Technology Research and Development Program of China(863 Program,2006AA10Z179,2006AA10ZEF87)National Natural Science Foundation of China(30300219 and 30571163)+2 种基金the Foundation for the Authors of National Excellent Doctoral Dissertation of China(200357 and 200458)supported by the Program for New Century Excellent Talents in Universifies of Chinasupported by the Program for Changjiang Scholars and Innovative Research Teams in Universities of China(IRT0453).
文摘Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.
基金supported by the National Natural Science Foundation of China (31271713)the "Twelfth Five-Year-Plan" in National Science and Technology for Rural Development in China (2011BAD07B01 and 2012AA101105)
文摘A total of 43 unique clones(Z4A-1 to Z4A-43 with GenBank accession numbers of HM120221, HM120222, JX828270, JN831402 to JN831406, and KC715889 to KC715923, respectively) were amplified and cloned from common wheat cultivar Zhengmai 004 using a PCR-based strategy. They included 22 full-ORF α-gliadin genes and 21 pseudogenes containing at least one in-frame stop codon. Comparative analysis of the deduced amino acid sequences showed that all the isolated genes displayed the typical structural features of α-gliadin genes and that the putative proteins of Z4A-7, Z4A-14, Z4A-17 and Z4A-20 had an extra cysteine residue in the unique domain II, while Z4A-15 lacked the second conserved cysteine residue in the unique domain I. The two fusion proteins of Z4A-15 and Z4A-20 were successfully detected by SDS-PAGE and Western blotting, although the protein level was relatively low. Based on the occurrence of the four major epitopes, as well as the lengths of the two glutamine repeats, 8, 6, and 8 genes were assigned to the Gli-2 loci on the respective chromosomes 6A, 6B, and 6D and a total of respectively 16, 0 and 23 immunogenic peptides were identified. In addition, 4 of the 5 genes with odd numbers of cysteine residues were assigned to chromosome 6D, suggesting that common wheat cultivar Zhengmai 004 has the potential to induce celiac disease(CD) and that the D genome exerts the most influence on gluten quality, but is the most deleterious for CD patients. By phylogenetic analysis, 11 exceptional α-gliadins with few or no immunogenic peptides from Triticum monococcum and Aegilops tauschii were detected, a finding that further supports the prospect of CD prevention. Finally, secondary structure prediction showed that most(98.48%) of the α-gliadins invariably contained five conserved α-helices(H1 to H5) in the two glutamine repeats and unique domains and 67.68% of the α-gliadins also contained a β-strand(S) in the C-terminal unique domains. An absent α-helix H2, 1–2 extra α-helices, or an additional β-strand(SE) also probably occurred in some cases. Of the 22 cloned genes in this work, 10 putative proteins contained 1–2 extra α-helices in addition to the five conserved α-helices or the additional β-strand. The observation that most of the α-helices and β-strands were present in the two unique domains and that an extra α-helix also probably occurred in the two glutamine repeats in some desirable genes strongly suggested that these two uniquedomains are the most important regions for the function of α-gliadins, although the glutamine repeats would also contribute in some cases.
基金supported by the National High Technology Research and Development Program of China(863 Program, 2006AA10Z179, 2006AAl0Z1F8)the Key Technologies R&D Program (2006BAD01A02,2006 BAD13B02)
文摘Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.
