Ω-gliadin Genes Research Articles

Molecular characterization of the IgE-binding epitopes in the fast ω-gliadins of Triticeae in relation to wheat-dependent, exercise-induced anaphylaxis

Fast ω-gliadins were minor components of wheat storage proteins but a major antigen triggering allergy to wheat. Sixty-six novel full-length fast ω-gliadin genes with unique characteristics were cloned and sequenced from wheat and its relative species using a PCR-based strategy. Their coding regions ranged from 177bp to 987bp in length and encoded 4.28kDa to 37.56kDa proteins. On the base of first three deduced amino acids at the N-terminal, these genes could be classified into the six subclasses of SRL-, TRQ-, GRL-, NRL-, SRP- and SRM-type ω-gliadin genes. Compared by multiple alignments, these genes were significantly different from each other, due to the insertion or deletion at the repetitive domain. An analysis of the IgE-binding epitopes of the 66 deduced fast ω-gliadins demonstrated that they contained 0–24 IgE-binding epitopes. The phylogenetic tree demonstrated that the fast ω-gliadins and slow ω-gliadins were separated into two groups and their divergence time was 21.64millionyears ago. Sequence data of the fast ω-gliadin genes assist in the study of the origins and evolutions of the different types of ω-gliadins while also providing a basis for the synthesis of monoclonal antibodies to detect wheat antigen content.

Molecular Cloning and Identification of Novel ω-gliadin Genes fromTriticumSpecies

Gliadin is a main component of gluten proteins that affect functional properties of bread making and contributes to the viscous nature of doughs. In this study, thirteen novel ω-gliadin genes were identified in several Triticum species, which encode the ARH-, ATDand ATN-type proteins. Two novel types of ω-gliadins: ATD- and ATN- have not yet been reported. The lengths of 13 sequences were ranged from 927 to 1269 bp and the deduced mature proteins were varied from 309 to 414 residues. All 13 genes were pseudogenes because of the presence of internal stop codons. The primary structure of these ω-gliadin genes included a signal peptide, a conserved N-terminal domain, a repetitive domain and a conserved C-terminus. In this paper, we first characterize ω-gliadin genes from T. timopheevi ssp. timopheevi and T. timopheevi ssp. araraticum. The ω-gliadin gene variation and the evolutionary relationship of ω-gliadin family genes were also discussed.

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain.

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7% reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.

Genome-, Transcriptome- and Proteome-Wide Analyses of the Gliadin Gene Families in Triticum urartu.

Gliadins are the major components of storage proteins in wheat grains, and they play an essential role in the dough extensibility and nutritional quality of flour. Because of the large number of the gliadin family members, the high level of sequence identity, and the lack of abundant genomic data for Triticum species, identifying the full complement of gliadin family genes in hexaploid wheat remains challenging. Triticum urartu is a wild diploid wheat species and considered the A-genome donor of polyploid wheat species. The accession PI428198 (G1812) was chosen to determine the complete composition of the gliadin gene families in the wheat A-genome using the available draft genome. Using a PCR-based cloning strategy for genomic DNA and mRNA as well as a bioinformatics analysis of genomic sequence data, 28 gliadin genes were characterized. Of these genes, 23 were α-gliadin genes, three were γ-gliadin genes and two were ω-gliadin genes. An RNA sequencing (RNA-Seq) survey of the dynamic expression patterns of gliadin genes revealed that their synthesis in immature grains began prior to 10 days post-anthesis (DPA), peaked at 15 DPA and gradually decreased at 20 DPA. The accumulation of proteins encoded by 16 of the expressed gliadin genes was further verified and quantified using proteomic methods. The phylogenetic analysis demonstrated that the homologs of these α-gliadin genes were present in tetraploid and hexaploid wheat, which was consistent with T. urartu being the A-genome progenitor species. This study presents a systematic investigation of the gliadin gene families in T. urartu that spans the genome, transcriptome and proteome, and it provides new information to better understand the molecular structure, expression profiles and evolution of the gliadin genes in T. urartu and common wheat.

Cloning and characterization of novel fast ω-gliadin genes in Triticum monococcum.

Fast ω-gliadin is the major allergen to wheat-dependent exercise-induced anaphylaxis (WDEIA). It is generally accepted that the proteins were expressed inGli-B1 loci located on the short arm of chromosome 1B in wheat. The knowledge of the orthologous genes in A and D genomes is still limited. In this study, the novel fastω-gliadin genes were cloned from genome A of diploid wheat Triticum monococcum. The deduced amino acid sequences of N-terminal domain showed that they referred to TRQ-type ω-gliadins. This type of ωgliadins have been identified in genome A by using mass spectroscopy and amino acid sequencing in previous studies (DuPont et al. 2004), but their coding sequences were lacking. Further analysis of IgE-binding epitopes suggested that the TRQ-typeω-gliadins have potential to trigger WDEIA. Wheat is one of the most important sources of food in the human diet. In the kernels of wheat, gliadins are the main components of the gluten. Traditionally, gliadins are further classified into α-gliadins, γ-gliadins and ω-gliadins on the basis of their mobility in electrophoresis at low pH (Wan et al. 2014). The ω-gliadins are minor components among wheat prolamins and they have not been studied well due to difficulties in cloning their genes caused by highly repetitive sequences (Anderson et al. 2009). In common wheat, most of the ω-gliadins were encoded by the Gli-A1, Gli-B1 and Gli-D1 on the short arm of chromosomes 1A, 1B and 1D, respectively (Pogna et al. 1990; Shewry et al. 1995; DuPont et al. 2000, 2004). Theω-gliadins have been categorized into ω1,ω2 andω5 types (DuPont et al. 2004). Theω5-gliadin, also referred as fastω-gliadin, are encoded byGli-B1 and the proteins can be distinguished by the amino acid sequences at the N-terminal domain, which are Ser-Arg-Leu (SRL) in ω5-gliadin, Ala-Arg-Gln (ARQ) or Lys-Glu-Leu (KEL)

Comparative and evolutionary analysis of new variants of ω-gliadin genes from three A-genome diploid wheats

A genomic polymerase chain reaction (PCR) cloning strategy was applied to isolate ω-gliadin sequences from three A-genome diploid wheats (Triticum monococcum, T. boeoticum and T. urartu). Amplicon lengths varied from 744 and 1,044bp, and those of the corresponding deduced mature proteins from 248 to 348 residues. The primary structure of the deduced polypeptides comprised a short N- and C-terminal conserved domain, and a long, variable repetitive domain. A phylogenetic analysis recognised several clades: the first consisted of three T. aestivum sequences; the second and the third two T. boeoticum and six T. monococcum sequences; and the rest four T. urartu and three T. aestivum sequences. Among the functional (non-pseudogene) ARQ/E-type ω-gliadin sequences, two were derived from T. boeoticum and three from T. monococcum; one of the latter sequences appeared to be a chimera originating via illegitimate recombination between the other two T. monococcum sequences. None of the 12 intact ω-gliadin sequences contained any cysteine or methionine residues. We discussed the variation and evolution of A-genome ω-gliadin genes.

Molecular cloning and variation of ω-gliadin genes from a somatic hybrid introgression line II-12 and parents (Triticum aestivum cv. Jinan 177 and Agropyron elongatum)

Gluten proteins, including gliadins and glutenins, together are responsible in large part for the end-use quality of the flour, have been extensively studied (Shewry and Tatham 1997; Shewry et al. 2003). The gliadins are monomeric polypeptides, classified into α-, β-, γandωtypes (Shewry et al. 2003). The ω-gliadin genes are encoded by the Gli-1 loci on the short arm of chromosome 1 which is tightly linked to Glu-3 loci, at which the LMW-GS are encoded (Tatham and Shewry 1995). There are also a few ω-gliadins encoded by ‘selfish genes’ at the Gli-A3, Gli-A5, Gli-B5 and Gli-A6 loci (Shewry et al. 2003). The number of ω-gliadins present in the hexaploid wheat endosperm ranges from 15 to 18 (Sabelli and Shewry 1991), and have been classified, according to the first three peptide residues present in the mature protein into ARQ/E-, KEL-, SRLor TRQ-types. ARQ/ E-type shares a 19-residue putative signal peptide, followed by a 10–11-residue N-terminal domain, a large repetitive region (encompassing 90–96% of the protein), and a 10– 11-residue C-terminal domain (Hsia and Anderson 2001). ω-Gliadins are deficient in both cysteine and methionine, and are therefore ‘sulphur poor’ polypeptides (Tatham and Shewry 1995). However, rather few ω-gliadin genes are represented (Hsia and Anderson 2001; Masoudi-Nejad et al. 2002; Matsuo et al. 2005; Hassania et al. 2008), possibly because their coding sequence includes a large repetitive domain, which hampers cloning. Further analysis ofω-gliadins at the DNA level would provide more information to define the evolution and function of this gene family (Hassania et al. 2008).

The wheat ω-gliadin genes: structure and EST analysis

A survey and analysis is made of all available ω-gliadin DNA sequences including ω-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active ω-gliadin genes, two pseudogenes, and four fragments of the 3′ portion of ω-gliadin sequences. Comparison of ω-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of ω-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of ω-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some ω-gliadin genes and discussion of problems in studying the ω-gliadin gene family.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-009-0122-2) contains supplementary material, which is available to authorized users.

Characterisation of a ω-gliadin gene in Triticum tauschii

A ω-gliadin gene at the Gli-D t 1 locus of Triticum tauschii accession AUS18913 was isolated using PCR primers, designed from published sequences of ω-gliadin genes of bread wheat cv Cheyenne, and deduced sequences of the N-terminal amino acids of ω-gliadin proteins. Further, the derived protein was isolated from A-PAGE and was sequenced. The protein sequence contained a signal peptide of 19 amino acids followed by a short N-terminal sequence of 11 amino acids, a central repetitive domain that covers approximately 90% of the sequence and a short C-terminal domain of 12 amino acids. The sequence comparison with other ω-gliadins showed a high level of similarities between them. Further analysis of the ω-gliadins using A-PAGE revealed that there are three ω-gliadin proteins in AUS18913 accession. Comparison of N-terminal sequences of these proteins revealed that two of these proteins have very high homologies with ω-gliadins of Cheyenne while the third one was significantly different.

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5´ and 3´ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available.

Chromosome 1B-encoded gliadins and glutenin subunits in durum wheat: Genetics and relationship to gluten strength

The progenies of crosses between Berillo and four durum wheat cultivars were analysed for storage protein composition (by four different electrophoresis procedures), genetic segregation and gluten quality (by SDS sedimentation test and Viscoelastograph). The crosses enabled the segregation patterns of alleles at Gli-B1, Glu-B3 and Glu-B1 on chromosome 1B, and at Gli-A2 on chromosome 6A to be determined. The gene order on chromosome 1B was deduced to be Glu-B1-centromere-Glu-B3-Gli-B1, with 47% recombination between Glu-B1 and Glu-B3, and 2% between Glu-B3 and Gli-B1. Genes coding for γ-gliadins at Gli-B1 were distal to ω-gliadin genes with respect to the centromere. Analyses of the progeny (F4 grains) from single F2 plants, indicated that gliadins γ-42 and γ-45 are only genetic markers of quality, whereas allelic variation for low molecular weight (LMW) glutenin subunits encoded at the Glu-B3 locus is primarily responsible for differences in SDS sedimentation volume and gluten viscoelastic properties. High molecular weight (HMW) glutenin subunits 7+8 also gave larger SDS sedimentation volumes and higher gluten elastic recoveries than subunits 6+8 and 20. The positive effects of the so-called LMW-2 glutenin subunits and HMW subunits 7+8 were additive, with LMW-2 being the most important proteins for pasta-making quality as evaluated by SDS-sedimentation and gluten viscoelasticity (both parameters related to firmness of cooked pasta). Two alleles at Gli-A2 coding for α-gliadins were also found to have different effects on gluten firmness.

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

The genes controlling the synthesis of the high-molecular-weight subunits of glutenin on the long arms of chromosomes 1A and IB were mapped to the ω-gliadin genes on the short arms by analysing the progeny of three test crosses by sodium dodecyl sulphate, polyacrylamide-gel electrophoresis. Only very weak linkages were detected: the percentage recombination ranged from 39% to 47% and as the values did not significantly differ from each other, the data was pooled. A mean recombination of 43% was obtained and the map distance between glutenin and gliadin genes was calculated to be 66 cM. The analysis of three crosses involving telocentric lines revealed that the glutenin subunit genes on chromosomes 1A, IB and ID are tightly linked to the centromere, the mean map distance being 9.0 cM.