Γ-gliadin Gene Research Articles

The γ-gliadin multigene family in common wheat (Triticum aestivum) and its closely related species

BackgroundThe unique properties of wheat flour primarily depend on gluten, which is the most important source of protein for human being. γ-Gliadins have been considered to be the most ancient of the wheat gluten family. The complex family structure of γ-gliadins complicates the determination of their function. Moreover, γ-gliadins contain several sets of celiac disease epitopes. However, no systematic research has been conducted yet.ResultsA total of 170 γ-gliadin genes were isolated from common wheat and its closely related species, among which 138 sequences are putatively functional. The ORF lengths of these sequences range from 678 to 1089 bp, and the repetitive region is mainly responsible for the size heterogeneity of γ-gliadins. The repeat motif P(Q/L/S/T/I/V/R/A)F(S/Y/V/Q/I/C/L)P(R/L/S/T/H/C/Y)Q1–2(P(S/L/T/A/F/H)QQ)1–2is repeated from 7 to 22 times. Sequence polymorphism and linkage disequilibrium analyses show that γ-gliadins are highly diverse. Phylogenic analyses indicate that there is no obvious discrimination between Sitopsis and Ae. tauschii at the Gli-1 loci, compared with diploid wheat. According to the number and placement of cysteine residues, we defined nine cysteine patterns and 17 subgroups. Alternatively, we classified γ-gliadins into two types based on the length of repetitive domain. Amino acid composition analyses indicate that there is a wide range of essential amino acids in γ-gliadins, and those γ-gliadins from subgroup SG-10 and SG-12 and γ-gliadins with a short repetitive domain are more nutritional. A screening of toxic epitopes shows that γ-gliadins with a pattern of C9 and γ-gliadins with a short repetitive domain almost lack any epitopes.Conclusionγ-Gliadin sequences in wheat and closely related Aegilops species are diverse. Each group/subgroup contributes differently to nutritional quality and epitope content. It is suggested that the genes with a short repetitive domain are more nutritional and valuable. Therefore, it is possible to breed wheat varieties, the γ-gliadins of which are less, even non-toxic and more nutritional.

The γ-gliadin gene content of a derivative from a somatic hybrid between bread wheat and tall wheatgrass

A PCR-based strategy was applied to obtain the DNA sequence of γ-gliadin open reading frames present in line II-12, a derivative from a somatic hybrid between bread wheat (Triticum aestivum L.) cv. Jinan177 and tall wheatgrass (Lophopyrum ponticum, 10×). A total 50 analysable sequences were obtained, 18 from II-12 and 16 each from the parents. Amplicon length ranged from 720 to 936 bp, corresponding to a putative mature protein of 239–309 residues. The primary structure of these putative proteins comprised five domains, of which only two varied in length. Phylogenetic analyses showed that the mature γ-gliadin sequences fell into four major clades. Group 1 contained sequences shared between II-12 and L. ponticum, suggesting that some L. ponticum γ-gliadin genes are present in the introgression line. Group 3 has five Jinan177 and five II-12 sequences, indicating that II-12 also carries wheat versions of Gli-1. Group 2 and 4 comprised four and two II-12, three and one Jinan177 as well as one and four L. ponticum sequences, respectively. Fewer genes encoding coeliac disease epitopes were present in II-12 than in the wheat donor parent. Three II-12 γ-gliadins and one from the wheat parent contained an odd number of cysteine residues, and two of them had an additional cysteine residue at the amino end of domain V. The possible use of II-2 for improving quality of bread wheat is discussed.

Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat

RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms. The elucidation of the mechanism of RNA silencing has stimulated its use as a reverse genetics tool, because RNA silencing strongly down-regulates the expression of the target gene in a sequence-specific manner. The major protein fraction of wheat grain is gluten which is largely responsible for the functional properties of dough. Gliadins contribute mainly to the extensibility and viscosity of gluten and dough, with the polymeric glutenins being responsible for elasticity. The aim of this work was therefore to silence the expression of specific γ-gliadins by RNA interference, to demonstrate the feasibility of systematically silencing specific groups of gluten proteins. The sequence of a γ-gliadin gene was used to construct the pghp8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. Two lines of bread wheat were transformed by particle bombardment. Gliadins were extracted from 30 mg of flour, separated by acid-PAGE and determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Seven transgenic lines were obtained and all of them showed reduced levels of γ-gliadins. All seven transgenic plants were fully fertile and their grain morphology and seed weight were comparable to the control lines. MALDI-TOF MS showed that six peaks, present in the untransformed line, were missing in transgenic lines of the BW208 genotype whereas three peaks were missing in the BW2003 genotypes. The proportion of γ-gliadins was reduced, by about 55–80% in the BW208 lines and by about 33–43% in the BW2003 lines. The ELISA assay based on the R5 antibody showed reductions in total gliadins (μg/mg flour) in three of the BW208 lines and in one BW2003 line, but an increase in one BW208 line (C613).

Cloning of nine γ-gliadin mRNAs (cDNAs) from wheat and the molecular characterization of comparative transcript levels of γ-gliadin subclasses

Nine new full-length cDNA sequences for γ-gliadins were generated from total mRNA from bread and durum wheat. The length of the open reading frames (ORFs) ranged from 720 to 936 bp, which corresponds to putative proteins of 239–311 amino acid residues, respectively. All nine sequences, reconstructed from phylogenetic data showed sequences similar to the γ-gliadins registered at GenBank. All known γ-gliadin sequences fall into four major groups in the tree constructed by the neighbour-joining method. Primer pairs were designed to discriminate between these four γ-gliadin groups during grain development in bread wheat using quantitative real time polymerase chain reaction (QRT-PCR). Expression profiles of two lines of bread wheat showed higher levels of gene transcription for γ-gliadins from group II, whereas γ-gliadins from group I were expressed at the lowest level. Thus, the γ-gliadins from group II are expressed four- and 10-fold higher in ‘Perico’ and ‘Anza’, respectively, than the other γ-gliadins. The results reported may help to understand the order of synthesis and deposition of γ-gliadins in the grain of developing bread wheat. In addition, the differences found in the expression levels among the four groups could be used to select genotypes with specific profiles of γ-gliadins. These findings will also facilitate the up- and down-regulation of γ-gliadin gene expression in transgenic wheat.

The wheat γ-gliadin genes: characterization of ten new sequences and further understanding of γ-gliadin gene family structure

Ten new wheat γ-gliadin gene sequences are reported and an analysis of γ-gliadin gene family structure is carried out using all known γ-gliadin sequences. The new sequences comprise four genomic clones with significantly more flanking DNA than previously reported, and six cDNA clones from a wheat endosperm EST project. Analysis of extended flanking DNA from the genomic clones indicates the limits of conservation of γ-gliadin DNA sequence that are similar to those previously found with other gliadin and glutenin genes and that are theorized to define the DNA sequence necessary for gene control. Most of the flanking DNA is not homologous to any reported DNA sequence, and one flanking region contains the first MITE-like (miniature inverted transposable element) DNA sequence associated with gliadin genes. About a quarter of the encoded polypeptides would contain a free cysteine residue – an observation that may relate to reports that at least some gliadins can participate in wheat endosperm glutenin polymer formation. The new sequences represent both genes closely related to those previously reported and a new sub-class of γ-gliadins.

Detection of wheat adulteration of spelt flour and products by PCR

To date, no general method to detect wheat [Triticum aestivum vulgare (Vill.) Mackey] adulteration of spelt [T. aestivum spelta (L.) Thell.] exists. Exploiting a published allelic difference in γ-gliadin gene GAG56D, we developed and evaluated three PCR-based methods to determine the proportion of wheat in spelt flour and products. A sensitive wheat-specific PCR system was designed. Moreover, a heterologous internal standard was constructed for quantitative competitive (QC-)PCR. The standard was arbitrarily calibrated to 5% wheat DNA in spelt DNA. A PCR-RFLP system consisting of a GAG56D-specific PCR system with subsequent restriction was used to estimate the relative proportions of wheat and spelt in DNA mixtures. The three methods were successfully applied to seven commercial spelt samples and one self-produced bread. Five and three of the seven samples were found to contain more than 5% and 10% wheat, respectively. One sample appeared to contain no spelt at all. Analyzing the bread crumb with PCR-RFLP, we were able to estimate the wheat content of the flour which was used to produce the bread and thus demonstrated the applicability of PCR-RFLP to quantify processed products.

Polymorphisms in two homeologous γ-gliadin genes and the evolution of cultivated wheat

The polymorphisms in two γ-gliadin genes GAG56D and GAG56B on the D- and B-genomes of polyploid wheat, respectively, were investigated by sequencing PCR products and by PCR-RFLP. Of GAG56D, two alleles fo and ok were previously known to occur in hexaploid wheat. Here, we found that 16 sequenced fragments of GAG56D from six recognized subspecies of Triticum aestivum, including 13 contributed by this study, were identical to either the fo or the ok allele. Considering published evidence, it was concluded that the investigated alleles of GAG56D stemmed from two different Aegilops tauschii plants and thus two independent origins of hexaploid wheat. Compared to GAG56D-sequences obtained from 10 accessions of Ae. tauschii, the fo and ok alleles clustered with fragments from three accessions collected in the Caspian region. By sequencing fragments of GAG56B, four distinct allelic groups were found among cultivated wheats, typical of bread wheat (p-aes), durum wheat of gliadin 45-type (a), durum wheat of gliadin 42-type (p-dur) and Timopheev's wheat (p-tim), respectively. Interestingly, the a allele found in gliadin 45-type durum wheat was shared by European spelt cultivars, which strongly supported the hypothesis that European spelt originated from a hybridization event between a tetra- and hexaploid wheat. The data also suggested that emmer might have been domesticated more than once. Phylogenetic analysis of GAG56-fragments obtained from putative B/G-genome donors excluded all candidate species as immediate donors of the B/G-genome, but instead indicated a monophyletic origin of all GAG56B alleles found in polyploid wheat, i.e. including T. timopheevii.

A spelt-specific γ-gliadin gene: discovery and detection

Polymerase chain reaction (PCR) primers GAG5 and GAG6 were designed based on published γ-gliadin gene sequences and applied to 35 cultivars of closely related spelt (Triticum spelta L.) and hexaploid wheat (T. aestivum L.). Eight tetraploid durum wheat (T. durum Desf.) cultivars were included in the analysis. The obtained PCR products originated from two γ-gliadin genes which were mapped to homeologous chromosomes 1B and 1D and termed GAG56B and GAG56D, respectively. Two alleles of GAG56D differing in a 9-bp deletion/duplication and single nucleotide polymorphism were found. The 18 spelts tested and wheat cultivar ’Chinese Spring’ were discovered to carry a previously unknown γ-gliadin gene, while 16 wheat cultivars possessed its longer, already published allele. Two PCR-based detection systems for the diagnostic alleles were developed and applied. The occurrence of two alleles of GAG56B among the investigated durum wheats correlated with their expression of gluten quality markers γ-gliadins 42 or 45.

Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease

BACKGROUND/AIMSCoeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin. The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for...

Endosperm specific expression of a gliadin-actin hybrid promoter in transgenic rice (Oryza sativa L.)

The far upstream regulatory region of the γ-gliadin gene was isolated from a wheat genomic library and its tissue specific effect on the expression of a reporter gene driven by a constitutive promoter was studied in transgenic rice (Oryza sativa L.). Since in earlier studies the wheat gliadin promoter containing −300 box has been previously shown to be active specifically in the endosperm of transgenic plants, therefore a hybrid promoter construct was created from γ-gliadin gene promoter far upstream region spanning from −421 to −71 bp and rice actin1 promoter from -95 to +12 bp. Downstream of the hybrid promoter a β-glucuronidase reporter gene (gusA) was fused. This construct was introduced into rice embryo by particle bombardment. Transgenic rice plants carrying the hybrid promoter linked to gusA were regenerated from bombarded embryos and grown up to T2 generation for further studies. RNA analysis and histochemical localization of GUS in transgenic rice plants revealed that the expression of the gene was highly specific in the endosperm, although the inserted transgene could be detected in every tissue (leaf, root and seed) investigated.

Molecular analysis of gliadin and glutenin genes in T. durum cv. Lira. A model system to analyse the molecular bases of quality differences in durum wheat cultivars

As a model system to study the molecular bases of differences between high- and lowquality durum wheat cultivars, designated as biotypes γ-45 and γ-42, respectively, the cultivar Lira, which contains both biotypes, was examined. Polymerase chain reaction analysis of biotypes 42 and 45 was carried out by using oligonucleotide primers from flanking nucleotide sequences coding for α-, β- and γ-gliadin and for high molecular weight (HMW) and low molecular weight (LMW) glutenins. A different amplified band for γ-gliadin sequences was observed between the two biotypes. Southern blot analysis of the same biotypes showed different hybridization patterns for both γ-gliadin and LMW glutenin genes. The hybridization patterns of LMW glutenin sequences revealed the presence of additional bands in biotype 45, suggesting that the quantitative differences in LMW glutenins between biotypes 42 and 45, considered responsible for their quality differences, are most probably due to a higher number of LMW genes in biotype 45. This conclusion seems also to be supported by the results obtained in other durum wheat cultivars belonging to types 42 and 45.

Molecular analysis of γ-gliadin gene families at the complex Gli-1 locus of bread wheat (T. aestivum L.)

A cDNA clone (pTag1436) carrying a complete coding sequence for a γ-gliadin polypeptide has been identified and sequenced. By hybridisation to size fractionated poly A(+) RNA from wheat nullisomic-tetrasomic lines, homologous transcripts from the Gli-A1, Gli-B1 and Gli-D1 loci were identified. These mRNAs differed from those complementary to a low molecular weight (LMW) glutenin cDNA clone. Hybridization of pTag1436 to digested wheat DNA produced a pattern of fragments unrelated to that obtained using a LMW glutenin cDNA probe. These results indicate that the γ-gliadin and LMW glutenin families, although both located at the Gli-1 loci, are distinct by hybridisation.

The nucleotide sequence of a wheat γ-gliadin genomic clone

γ-Gliadins are a subclass of the storage proteins from wheat. We have isolated from a wheat genomic library a clone encoding a γ-gliadin gene. The amino acid sequence derived from the gene sequence reveals a mature protein (308 amino acids, excluding a 19 amino acid signal sequence) with a characteristic repeated structure of the consensus heptapeptide PQQPFPQ corresponding to the high glutamine (35.4%) and proline (17.5%) content. The gene does not contain an intron, and possesses a typical eukaryotic promoter.

Structure of wheat gamma-gliadin genes

We have cloned and sequenced two linked members of the wheat ( Triticum aestivum) gliadin multigene family. One gene encodes a γ-gliadin which is 292 amino acids (aa) long. S1 mapping indicates that this gene is transcriptionally active. The second gene, which is only marginally active by S1 mapping, is closely related to the first gene. Moreover, it is probably incapable of encoding a full-length γ-gliadin due to the presence of two premature in-frame stop codons. Neither gene contains introns. Nucleotide sequences of the two genes show the presence of characteristic repeats which have a derived aa consensus sequence Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Pro-Gln. A comparison of the promoter regions of these γ-gliadin genes with those of the α-gliadin genes shows the presence of a highly conserved region that could be involved in tissue specific and developmental regulation.