Analysis of LMW-GS,α-and γ-Gliadin Gene Coding Sequences from Triticum macha

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.

澳冰草中一个新型α-gliadin基因的克隆与序列分析

以澳冰草(Australopyrum retrofractum)基因组DNA为模板,用小麦种子醇溶蛋白的保守引物进行PCR扩增,对扩增产物进行克隆测序。结果表明,获得的扩增片段总长度为936 bp,包含一个完整的262个氨基酸的编码区,基因库登录号为EF536330,序列比对表明该序列为α-gliadin基因家系成员。利用α-gliadin基因的编码氨基酸序列建立系统树分析表明,序列EF536330不能与源于普通小麦的A、B和D染色体组的α-gliadin基因序列聚在一起,而单独聚为一类,推测所获得的来自澳冰草W染色体组的序列EF536330为麦类α-liadin基因家系的新类型。

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

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.

Cytoskeleton reorganization and ultrastructural damage induced by gliadin in a three-dimensional in vitro model

AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure （by means of light microscopy and scanning electron microscopy）, and the effect of gliadin on actin （phalloidin fluorescence） and the tight-junction protein occludin and zonula occluden-1.RESULTS： The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gUadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system.CONCLUSION： Our data obtained from an ＂in vivolike＂ polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.

Effect of high-nitrogen fertilizer on gliadin and glutenin subproteomes during kernel development in wheat(Triticum aestivum L.)

Nitrogen(N),a macronutrient essential for plant growth and development,is needed for biosynthesis of protein and starch,which affect grain yield and quality.Application of high-N fertilizer increases plant growth,grain yield,and flour quality.In this study,we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and twodimensional difference gel electrophoresis(2D-DIGE).Application of high-N fertilizer led to significant increases in gluten macropolymer content,total gliadin and glutenin content,and the accumulation of individual storage protein components.Of 126 differentially accumulated proteins(DAPs)induced by high-N conditions,24 gliadins,12 high-molecularweight glutenins,and 27 low-molecular-weight glutenins were significantly upregulated.DAPs during five kernel developmental stages displayed multiple patterns of accumulation.In particular,gliadins and glutenins showed respectively five and six accumulation patterns.The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.

Genetic Diversity of Recurrent Selection Populations with Ms2 Gene Assessed by Gliadins in Common Wheat (Triticum aestivum L.)

The male-sterile lines with Ms2 gene were highly evaluated in recurrent selection in wheat （Triticum aestivum L.）. Three populations C6 （population after six cycles of selection）, C7 （population after seven cycles of selection）, and C8 （population after eight cycles of selection） were constructed through recurrent selection with 12 parental materials （P）. Acid polyacrymide gel electrophoresis （A-PAGE） analysis was used to identify gliadin patterns and evaluate the genetic diversity in 12 parents and three populations. A total of 63 bands were identified, of which 17 polymorphic bands and 7 unique bands were present in populations and seven polymorphic bands and four unique bands were present in parents. The number of polymorphic and unique bands decreased gradually from C6 to C8, especially for to- and y-gliadins. The genetic distances in C6, C7, and C8 were calculated. The distributions of genetic distance were different in three recurrent selection populations. From C6 to C8, the genetic distance was 0.2687, 0.2652 and 0.1987, respectively. Statistically significant differences were detected between C7 and C8 with the T value of 37.9718. The result of cluster analysis based on genetic similarity matrix of three populations fitted well to those of principle coordinates analysis （PCoA）. Compared with 12 parents, almost all individuals of three populations are new genotypes. Most of the individuals from C6 and C7 could be divided into two groups, while most individuals of C8 were in one cluster. In conclusion, the results indicated that the genetic diversity was decreased severely according to the information revealed by A-PAGE, although some variations could be created in the recurrent selection. It was necessary to introduce diverse germplasm based on the genetic database of recurrent population to maintain and improve the breeding efficiency in the further program.

Molecular characterization of α-gliadin genes from common wheat cultivar Zhengmai 004 and their role in quality and celiac disease

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.

Construction of Gliadin Fingerprints Database for the Main Wheat Cultivars Grown in North China

The standard gliadin fingerprints and their database of 68 major cultivars and a part of backbone parents, which have ever been extensively grown in North China since the 1950' s, were constructed by using CAWGES software and an improved method of pH 3.2 A-PAGE. In the meantime, investigations were made on the utilization of the database in the area of gliadin fingerprints analysis, variety identification and genetic relationship study. The results showed that it provided an effective method for building core collections and variety identification.

Accumulation Pattern of Gliadin Fractions α, β, γ, ω and Regulation of Nitrogen

Gliadin, the major storage protein in endosperm, affects grain quality in spring wheat by its content and composition. Eighteen cultivars differing in HMW-GS were used in the study to approach the accumulation pattern of gliadin fractions α, β, γ, ω and regulation of three kinds of nitrogen source. The results showed that the content of gliadin in grains increased gradually along with the process of grain-filling, but the accumulation intensity and final content differed evidently among cultivars with different HMW-GS composition. Of all the subunit types used here, cultivars with subunits 7＋9 and 2＋12 had smaller accumulation intensity and lower final content. During grain-filling, 4 gliadin fractions had the same increase trend, but differed in accumulation course. The dynamic trends of gliadin accumulation were similar in different nitrogen treatments whose effects on initial amount, accumulation intensity and final level of accumulation varied with cultivars. Of three nitrogen fertilizer types, the amide-form nitrogen source was better to the formation and accumulation of gliadin as well as its four fractions.

New transcriptomic insights in two RNAi wheat lines with the gliadins strongly down-regulated by two endosperm specific promoters

The gluten proteins of wheat grain are responsible for visco-elastic properties of flour,but they also trigger the immune-response of celiac disease.In this work,two low-gliadin RNA interference(RNAi)wheat lines that differ for the promoter driving the silencing(D-hordein andγ-gliadin promoters for D783 and D793 lines,respectively),were characterized at transcriptomic,and protein fraction levels in the grain.Silencing of gliadins provides a readjustment in the grain protein fractions that also affects to the nongluten proteins(NGP),which were increased in both RNAi lines.Determination of wheat gluten by means of the R5 monoclonal antibody also showed a strong reduction in the content of gluten in both RNAi lines.Moreover,fructans,an oligosaccharide linked with the development of non-celiac wheat sensitivity(NCWS)were also significantly decreased in RNAi lines.The down-regulation of gliadins fractions also impacts to other metabolic processes,particularly on carbohydrate metabolism,enzyme regulator activity and response to stress.Genes and transcription factors regulated by ABA were up-regulated,which could suggest the implication of this phytohormone on the stress response observed in the RNAi lines.

A catalog of gliadin alleles:Polymorphism of 20th-century common wheat germplasm

A new, improved version of the catalog of 182 alleles at the six Gli loci of common wheat(T.aestivum L.) shown in electrophoregrams of 128 standard genotypes was used for analysis of1060 cultivars and lines bred in the 20 th century. The most frequent alleles in the studied germplasm occurred with frequencies of 18%–40%, with 30 unique alleles, one in each cultivar. Extremely high genetic diversity was found(average H for the six main Gli loci was0.870 ± 0.046), nearly identical in winter(H = 0.831) and spring(H = 0.856) wheats but differing among 28 groups of cultivars released in 22 countries. Each country or region was characterized by its own specific set of the most frequent Gli alleles, and the 28 cultivar groups formed five main relationship clusters if polymorphism at the six Gli loci was considered. However, different levels of similarity between groups of cultivars were found if polymorphism of the A, B, or D genomes of common wheat was tested separately. In general, the 20 th century germplasm of common wheat was differentiated and structured by country or region and cultivar type(spring or winter). Each elemental genome(in particular, A and D) contributed to the structure of the polymorphism studied. We propose that the structure of the wheat germplasm was a result of natural selection under the ecoclimatic conditions of cultivation specific to each country or region. As many as 27.4% of cultivars studied violated the requirement of the DUS rules for uniformity, being represented by mixtures of two or more closely related genotypes. However, the composition of a cultivar as a set of related but different genotypes may contribute to its adaptivity, and thereby to the known high plasticity of common wheat.

Large enhancements in gelation behavior of wheat gliadins by incorporation of low concentrations of methylcellulose

Influence of non-gelling methylcellulose （MC） on gelation behavior of wheat gliadins in 13 wt% alkaline propanol/water （50：50, v/v） solution was investigated using dynamic theological time sweep test. Increasing MC concentration （CMC） up to CMC = 1 wt% caused a significant reduction in gelation time （tget） of the solution and an increase in loss tangent （tan δ） value of the resultant gel at T 〈 30 ℃.

Correlation analysis of celiac sprue tissue transglutaminase and deamidated gliadin IgG/IgA

AIM:To indirectly determine if tissue transglutaminase(tTG)-specific T cells play a crucial role in the propagation of celiac disease.METHODS:Anti-deamidated gliadin peptide(DGP) and anti-tTG IgA and IgG were measured in the sera of celiac patients(both untreated and treated).The correlations were determined by Spearman's rank correlation test.RESULTS:In celiac patients,we found a very significant correlation between the production of DGP IgA and IgG(r = 0.75),indicating a simultaneous and ongoing production of these two isotypes reminiscent of oral vaccination studies.However,there was far less association between the production of tTG IgA and tTG IgG in celiac patients(r = 0.52).While tTG IgA was significantly correlated with DGP IgA(r = 0.80) and DGP IgG(r = 0.67),there was a weak correlation between production of anti-tTG IgG and the production of anti-DGP IgA(r = 0.38) and anti-DGP IgG(r = 0.43).CONCLUSION:These data demonstrate that the production of anti-tTG IgA is directly correlated to the production of anti-DGP IgG and IgA,whereas anti-tTG IgG is only weakly correlated.This result therefore supports the hapten-carrier theory that in well-established celiac patients anti-tTG IgA is produced by a set of B cells that are reacting against the complex of tTG-DGP in the absence of a tTG-specific T cell.

Damaging effects of gliadin on three-dimensional cell culture model

AIM： To evaluate the effects of gliadin on the oxidative environment in the “in vivo-like”model of a three-dimensional cell culture system. METHODS： LoVo cell line （intestinal adenocarcinoma） multicellular spheroids were treated with digested gliadin （with albumin used as a control）. Spheroid volumes, cell viability and morphology, lactate dehydrogenase （LDH） release, content of reduced glutathione （GSH） and activity of GSH-related enzymes were examined. The data were statistically analyzed using the Student＇s t-test （P〈0.05）. was considered statistically significant. RESULTS： Gliadin reduced cell viability （from 20% to 60%） and led to morphological alterations characterized by apoptotic findings and cytoskeletal injuries. LDH activity increased. The content of GSH reduced （-20% vscontrols）, and activity of GSH-related enzymes was significantly inhibited. CONCLUSION： Gliadin treatment induces an imbalance in the antioxidative mechanism of cells cultured by the three-dimensional technique. This alteration may explain cell damage directly caused by gliadin and the subsequentmorphological abnormalities.

Cross-Reaction between Gliadin and Different Food and Tissue Antigens

A subgroup of coeliac disease patients continues to experience symptoms even on a gluten-free diet (GFD). We attempted to determine whether these symptoms could be due to either cross-contamination with gluten-containing foods or cross-reactivity between α-gliadin and non-gluten foods consumed on a GFD. We measured the reactivity of affinity-purified polyclonal and monoclonal α-gliadin 33-mer peptide antibodies against gliadin and additional food antigens commonly consumed by patients on a GFD using ELISA and dot-blot. We also examined the immune reactivity of these antibodies with various tissue antigens. We observed significant immune reactivity when these antibodies were applied to cow’s milk, milk chocolate, milk butyrophilin, whey protein, casein, yeast, oats, corn, millet, instant coffee and rice. To investigate whether there was cross-reactivity between α-gliadin antibody and different tissue antigens, we measured the degree to which this antibody bound to these antigens. The most significant binding occurred with asialoganglioside, hepatocyte, glutamic acid decarboxylase 65, adrenal 21-hydroxylase, and various neural antigens. The specificity of anti-α-gliadin binding to different food and tissue antigens was demonstrated by absorption and inhibition studies. We also observed significant cross-reactivity between α-gliadin 33-mer and various food antigens, but some of these reactions were associated with the contamination of non-gluten foods with traces of gluten. The consumption of cross-reactive foods as well as gluten-contaminated foods may be responsible for the continuing symptoms presented by a subgroup of patients with coeliac disease. The lack of response of some CD patients may also be due to antibody cross-reactivity with non-gliadin foods. These should then be treated as gluten-like peptides and should also be excluded from the diet when the GFD seems to fail.

Cloning and characterization of novel γ-gliadin genes from Aegilops markgrafii in relation to evolution and wheat breeding

Gliadins are the major components of storage proteins in wheat and play an important role in determining the extensibility properties of dough.In the present work,six novel full-length γ-gliadin genes were cloned from the C genome of Aegilops markgrafii using a PCR-based strategy.Analysis of the deduced amino acid sequences showed that the cloned genes had primary structures that were similar,but not identical,to published γ-gliadins from other wheat-related species.The lengths of the open reading frames(ORFs)ranged from 909 to 963 bp,and the repetitive and glutamine-rich domains were mainly responsible for the size of the proteins.An extra cysteine residue was present in the repetitive domain of sequence JX566513.All amino acid sequences of γ-gliadin genes from Ae.markgrafii were searched for the five peptides identified as T cell stimulatory epitopes in celiac disease(CD)patients.Peptide Gliγ-3 was present in sequences JX566513 and JX566514.Peptide Gliγ-5 was present only in JX566513.The otherγ-gliadins contained no toxic epitopes.These results provide information to better understand the use of Ae.markgrafii in wheat breeding and the evolutionary relationship of theγ-gliadin genes in Ae.markgrafii and other Triticeae species.

Identification of a Group of Novel γ-Gliadin Genes

γ-Gliadins are an important component of wheat seed storage proteins. Four novel γ-gliadin genes （Gli-ngl to Gli-ng4） were cloned from wheat （Triticum aestivum） and Aegilops species. The novel γ-gliadins were much smaller in molecular size when compared to the typical γ-gliadins, which was caused by deletion of the non-repetitive domain, glutamine-rich region, 3＂ part of the repetitive domain, and 5＇ part of the C-terminal, possibly due to illegitimate recombination between the repetitive domain and the C-terminal. As a result, Gli-ngl and Gli-ng4 only contained two and three cysteine residues, respectively. Gli-ngl, as the representative of novel γ-gliadin genes, has been sub-cloned into an Escherichia coli expression system. SDS- PAGE indicated that the both cysteine residues of Gli-ngl could participate in the formation of intermolecular disulphide bonds in vitro. Successful cloning of Gli-ngl from seed cDNA of T. aestivum cv. Chinese Spring suggested that these novel γ-gliadin genes were normally transcribed during the development of seeds. Phylogenic analysis indicated that the four novel γ-gliadin genes had a closer relationship with those from the B （S） genome of wheat.

Physicochemical and structural characteristics of the Venn components of wheat gliadin

The aim of this study was to analyze the physicochemical and structural characteristics of the Venn components of wheat gliadin to provide theoretical basis of gliadin for processing in dough and Chinese steamed bread. Eight Venn components, Gli-8, Gli-9, Gli-10, Gli-11, Gli-12, Gli-13, Gli-14, and Gli-15, were extracted from wheat gliadin based on their solubility. The results of physicochemical characteristics showed that the differences in the contents, TDS,electrical conductivity, particle size and zeta potential of Venn components were significant, respectively. The content of Gli-15 in gliadin was the highest, and the content of Gli-9 was the lowest. The TDS value of Gli-9 was the highest(336.0), and the TDS value of Gli-15 was the lowest(52.0). The electrical conductivity of Gli-9 was the highest,which was 7.54 times the lowest value of Gli-11. The zeta potential of Gli-9 was -25.2 mV, and the zeta potential of the Gli-15 was -7.64 mV. However, the difference in the p H values was not significant. The results of UV spectrum and FTIR analysis showed that the secondary structures of the Venn components had significant differences. The results of the XRD patterns indicated that the Venn components might not be a single substance. The results of CLSM images implied that the molecular interactions among the components were varied. Hence, the results could provide research materials and basic data for deep processing and utilization of gliadin.

Null Alleles in Gliadin Coding Loci and Wheat Allergenic Properties

Wheat gliadin proteins-an important, nutritional component of many food products may also act as allergenic proteins causing various, clinical symptoms of IgE-mediated food allergies. Gliadins are coded by six complex loci on the chromosomes 1A, 1B, 1D,6A, 6B and 6D of wheat genome. Each of the loci coding from a few to a dozen of polypeptides may spontaneously mutate to inactive gene variants called null alleles that do not code any proteins at all. The aim of the present work was to find out whether null alleles in some gliadin coding loci may decrease wheat allergenic properties. Six winter wheat genotypes: gliadin deletion lines (GDL) containing null alleles on 1D, 1B and 6B chromosomes and control lines (CL) containing active gene variants in all gliadin coding loci, were developed using plant breeding methods. Allergenic properties of the six analyzed hybrids were estimated by ELISA using polled sera of five patients allergic to gluten. Estimated immunoreactivity of GDLs was from 6% to 18% lower as compared with CLs. The obtained results evidenced that gliadin null alleles decrease wheat allergenic properties and may be used as parental forms for breeding of hypoallergenic wheat genotypes.

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies

Some wheat gliadin proteins are strong allergens that may cause various symptoms of food allergies and baker’s asthma. The most immunoreactive ω-5 gliadin fractions are the main allergens in wheat dependent exercise induced anaphylaxis (WDEIA). While the allergenicity of ω-5 is quite well understood, knowledge about α, β, γ and ω-1.2 gliadins is much more scanty. This study examines allergenic properties of other fractions as compared to ω-5. Gliadins were extracted from flour of winter wheat (Triticum aestivum L.) cultivar Ostka strzelecka. Purified samples representing proteins belonging to α, β, γ, ω-1.2 and ω-5 classes were isolated using preparative gel electrophoresis. Immuno-reactivity and allergenic properties of these proteins were analyzed by ELISA using sera from allergic patients with elevated sIgE (> 2KU/L), and by skin prick test (SPT). ELISA showed that ω-5 and ω-1.2 differed considerabely from α-, β- and γ-gliadins in respect of immunoreactivity. Responses of both ω-gliadins were almost twice as high as for other fractions. Significant differences were also observed among individual ω-gliadin fractions as evidenced by ANOVA. SPT showed that patient with symptoms of baker’s asthma and WDEIA had a positive results to all gliadins tested. Another patient with baker’s asthma (but not WDEIA) reacted positively only to ω-5 gliadins. In two patients with skin allergy SPT were negative with all analyzed proteins. Results show ω-1.2 gliadins to be almost as immunorective as ω-5. The α-, β- and γ-gliadins also recognize specific IgE antibodies, but their binding capacity is only about half that of ω-fractions. This kind of immunoreactivity could still be important since a cumulative effect of individual fractions may intensify disease symptoms in allergic patients.