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.

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.

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 ℃.

RHEOLOGICAL PROPERTIES OF WHEAT GLIADINS IN AQUEOUS PROPANOL

Rheological properties of wheat gliadins in 50% （V/F） aqueous propanol were carried out as a function of gliadin concentration c and temperature. The solutions at 20 g L-1 to 200 g L-1 behave as Newtonian fluids with an flow activation energy of 23.5-27.3 kJ mol-1. Intrinsic viscosity [η] and Huggins constant kH are determined according to Huggins plot at c 〈 120 g L-1. The results reveal that gliadins are not spherical shaped and the molecular size tends to increase with temperature due to improved solvation.

Production, function, modification and application of zein

Gliadins are stored in plant seeds,such as gliadins in wheat and corn.Zein has good film forming,oil retention,water retention,stability,thermal plasticity,oxidation resistance and biodegradability,and zein can be dissolved in ethanol solution.These functional properties make zein have a wide application prospect in food,medicine,textile,environmental protection and other fields.This paper briefly introduces the source,structure and amino acid composition of zein,focuses on the extraction technology of zein,summarizes the functional properties of zein,and analyzes the modification methods of zein.In the long run,summarizing the functional characteristics of zein has a certain reference value for its application in the future and large-scale production.

Genetic Diversity of Gli-1, Gli-2 and Glu-1 Alleles in Sichuan Wheat Landraces

Genetic diversity at Gli_1, Gli_2 and Glu_1 loci was investigated in 89 Sichuan wheat (Triticum aestivum L.) landraces by using acid polyacrylamide gel electrophoresis (APAGE) and SDS_PAGE. In these landraces, a total of 32 gliadin and 3 high_molecular_weight (HMW) glutenin patterns were observed. In total, 14, 15 and 5 alleles were identified at Gli_1, Gli_2 and Glu_1, respectively. At each locus, the alleles in higher frequency were Gli_A1a (89%), Gli_B1h (46%), Gli_D1a (65%), Gli_A2a (64%), Gli_B2j (45%), Gli_D2a (48%), Glu_A1c (99%), Glu_B1b (99%) and Glu_D1a (100%). The Nei's genetic variation index (H) of Sichuan wheat landraces was 0.370?6, varying from 0 to 0.708?7. The highest genetic diversity was found at Gli_B2 locus, while the lowest was found at Glu_D1. The genetic diversity at Gli loci was higher than that of Glu_1 loci among these landraces, but it was much lower than that of modern wheat cultivars. These results indicated a narrow genetic base of Sichuan wheat landraces. In this study, “Chengdu_guangtou” had the identical gliadin and HMW_glutenin patterns with “Chinese Spring”, further supporting the proposal that “Chinese Spring” is a strain of “Chengdu_guangtou”.

Genetic Diversity of Gli-1,Gli-2 and Glu-1 Alleles Among Chinese Endemic Wheats

Genetic diversity at Gli_1, Gli_2 and Glu_1 loci was investigated in 32 accessions of Chinese endemic wheat by using acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulfate (SDS)_PAGE. There were 8 gliadin and 3 high_molecular_weight (HMW)_glutenin patterns in 14 Yunnan hulled wheat ( Triticum aestivum ssp. yunnanese King) accessions, 9 gliadin and 4 HMW_glutenin patterns in 9 Tibetan weedrace ( T. aestivum ssp. tibetanum Shao ) accessions, and 9 gliadin and 5 HMW_glutenin patterns in 9 Xinjiang rice wheat ( T. petropavlovskyi Udacz. et Migusch.) accessions. One accession (i.e. Daomai 2) carried new subunits 2.1+10.1 encoded by Glu_D1. Among the three Chinese endemic wheat groups, a total of 10, 14 and 11 alleles at Gli_1 locus; 11, 14 and 12 alleles at Gli_2 locus; and 5, 6 and 8 alleles at Glu_1 locus were identified, respectively. Among Yunnan hulled wheat, Tibetan weedrace and Xinjiang rice wheat, the Nei's genetic variation indexes were 0.3798, 0.5625 and 0.5693, respectively. These results suggested that Tibetan weedrace and Xinjiang rice wheat had higher genetic diversity than Yunnan hulled wheat.

Establishment of 6VS Telocentric Lines of Haynaldia villosa Resistant to Powdery Mildew Induced by Immature Embryo Culture

The line of T240-6 was selected from 32 SC 2 lines of immature embryo culture of T240 (common wheat (Triticum aestivum L.)× Wheat-Haynaldia villosa (L.) Schur. 6D/6V substitution line) through powdery mildew characterization, glutamate oxaloacetate transaminase (GOT) enzyme and gliadin (Gli) analyses and in situ hybridization. All of the individual plants resistant to powdery mildew lacked the locus of GOT at 6VL arm (GOT-V 2) and had gliadin locus at 6VS arm (Gli-V 2) of Haynaldia villosa. All the plants resistant to powdery mildew had one or two telocentric chromosomes that did not pair with wheat chromosomes but paired between themselves. T240-6 was identified as a telocentric line through in situ hybridization.

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.

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.

Celiac disease serology in patients with different pretest probabilities: Is biopsy avoidable?

AIM: To establish the diagnostic performance of sev-eral serological tests, individually and in combination, for diagnosing celiac disease (CD) in patients with different pretest probabilities, and to explore potential se- rological algorithms to reduce the necessity for biopsy. METHODS: We prospectively performed duodenal biopsy and serology in 679 adults who had either high risk (n = 161) or low risk (n = 518) for CD. Blood samples were tested using six assays (enzyme-linked immunosorbent assay) that detected antibodies to tissue transglutaminase (tTG) and deamidated gliadin peptide (DGP). RESULTS: CD prevalence was 39.1% in the high-risk population and 3.3% in the low-risk group. In high-risk patients, all individual assays had a high diagnostic efficacy [area under receiving operator characteristic curves (AU ROC): 0.968 to 0.999]. In contrast, assays had a lower diagnostic efficacy (AU ROC: 0.835 to 0.972) in the low-risk group. Using assay combinations, it would be possible to reach or rule out diagnosis of CD without biopsy in 92% of cases in both pretest populations. We observed that the new DGP/tTG Screen assay resulted in a surplus compared to more conventional assays in any clinical situation. CONCLUSION: The DGP/tTG Screen assay could be considered as the best initial test for CD. Combinations of two tests, including a DGP/tTG Screen, might be able to diagnose CD accurately in different clinical scenarios making biopsy avoidable in a high proportion of subjects.

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.

Seroreactivity against Saccharomyces cerevisiae in patients with Crohn's disease and celiac disease

AIM:To explore whether there was anti-Saccharomyces cerevisiae antibodies (ASCA) positivity in our patients with biopsy-confirmed celiac disease. METHODS:A cohort of patients with inflammatory bowel diseases (42 patients with Crohn's disease and 10 patients with ulcerative colitis) and gluten sensitive enteropathy (16 patients) from Debrecen,Hungary were enrolled in the study. The diagnosis was made using the formally accepted criteria. Perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA), antiendomysium antibodies (EMA),antigliadin antibodies (AGA) and anti human tissue transglutaminase antibodies (tTGA) were investigated. RESULTS:The results showed that ASCA positivity occurred not only in Crohn's disease but also in Celiac disease and in these cases both the IgG and IgA type antibodies were proved. CONCLUSION:It is conceivable that ASCA positivity correlates with the (auto-) immune inflammation of small intestines and it is a specific marker of Crohn's disease.

Prevalence of celiac disease in Iranian children with idiopathic short stature

AIM： To determine the prevalence of celiac disease （CD） in children with idiopathic short stature （ISS） and the diagnostic value of immunoglobulin （Ig） A G antigliadin antibodies （AGA） and transglutaminase （TTG） antibodies for CD.METHODS： A total of 104 children （49 male, 55 female） with ISS without a specific etiology were studied. Extensive endocrine investigations had shown no abnormalities in any subject. Anthropometric parameters and IgA AGA and IgA TTG antibodies were evaluated in this study group. These antibodies were measured by enzyme-linked immunosorbent assay. All patients were referred for an endoscopic intestinal biopsy. The biopsy samples were classified according to revised Marsh criteria （UEGW 2001）.RESULTS： We detected positive IgA TTG antibodies in 36 and IgA AGA in 35 of these patients. Thirty one IgA TTG antibody positive and 28 IgA AGA positive subjects showed histological abnormalities compatible with celiac disease （33.6%）. Sensitivity, specificity, positive predictive value （PPV） and negative predictive value for IgA AGA were found to be 80%, 88.4%, 77.8% and 89.7%, respectively. Sensitivity, specificity and PPV for IgA TTG antibodies were 88.6%, 94.2% and 88.6%, espectively.CONCLUSION： We conclude that the prevalence of celiac disease is high in patients with ISS and it is important to test all children with ISS for celiac disease by measuring serologic markers and performing an intestinal biopsy.

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.

Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

Diagnostic accuracy of a new point-of-care screening assay for celiac disease

AIM: To determine the diagnostic accuracy of a new point-of-care assay detecting anti-deamidated gliadin peptides in celiac disease(CD) patients.METHODS: One-hundred-and-twelve patients(age range: 1.8-79.2 years old) with clinical symptoms suggestive of CD and/or first-degree relatives(FDR) of CD patients(n = 66),and confirmed CD on a gluten-free diet(GFD)(n = 46),were prospectively enrolled in the study at Gastroenterology outpatient clinics for adult patients and from the Gastroenterology Consultation Ward at the Pediatric Department of the University Hospital of Geneva.Written informed consent was obtained from all subjects enrolled.The study received approval from the local ethics committee.The original CD diagnosis had been based on serum-positive IgA anti-tissue transglutaminase enzyme-linked immunosorbent assay(ELISA)(QuantaLite,Inova Diagnostics,San Diego,CA,United States) and on biopsy results.Serum samples from all study participants were tested by the new CD lateral flow immunochromatographic assay(CD-LFIA) device,Simtomax Blood Drop(Augurix SA,BioArk,Monthey,Switzerland) to detect immunoglobulin(Ig)A and IgG antibodies against deamidated gliadin peptides.The diagnostic performance was evaluated using receiver operating characteristic curves with 95%CIs.A cut-off of 2 on the Rann colorimetric scale was used to calculate the device's sensitivity and specificity.RESULTS: CD-LFIA was highly accurate in detecting untreated celiac patients.In the group of patients with CD symptoms and/or FDR,eight new cases of CD were detected by ELISA and biopsy.All of these new cases were also correctly identified by CD-LFIA.The test yielded four false positive and four false negative results.The false positive results were all within the groups with clinical symptoms suggestive of CD and/or FDR,whereas the false negative results were all within the GFD group.The test yeld a sensitivity of 78.9%(95%CI: 54.4-93.9) and specificity of 95.7%(95%CI: 89.4-98.8),and the area under the curve reached 0.893(95%CI: 0.798-0.988).The Kappa coefficient,calculated according to the values obtained by two readers from the same device,was of 0.96(SE: 0.06).When the GFD patients were excluded from the analysis,the area under the curve reached 0.989(95%CI: 0.971-1.000) and the Kappa coefficient,calculated according to the values obtained by two readers from the same device,became 0.96(SE: 0.07).Furthermore,using the Rann scale cut-off of 2 without the GFD patients,sensitivity was 100% and specificity was 93.1%(95%CI: 83.3-98.1).CONCLUSION: The new CD-LFIA rapid screening test shows good diagnostic accuracy,sensitivity and specificity,and may rule out CD in patients with CD-related symptoms.

Calcium glycerophosphate preserves transepithelialintegrity in the caco-2 model of intestinal transpo

AIM To assess the direct effects of ischemia onintestinal epithelial integrity. Furthermore, clinicalefforts at mitigating the effect of hypoperfusion on gutpermeability have focused on restoring gut vascularfunction.METHODS： we report that, in the Caco-2 cell modelof transepithelial transport, calcium glycerophosphate（CGP）, an inhibitor of intestinal alkaline phosphataseF3, has a significant effect to preserve transepithelialelectrical resistance （TEER） and to attenuate increasesin mannitol flux rates during hypoxia or cytokinestimulation.RESULTS： The effect was observable even at concentrationsas low as 1 μmol/L. As celiac disease is alsomarked by a loss of gut epithelial integrity, the effectof CGP to attenuate the effect of the α-gliadin peptide31-55 was also examined. In this instance, CGP exertedlittle effect of preservation of TEER, but significantlyattenuated peptide induced increase in mannitol flux.CONCLUSION： it appears that CGP treatment mightsynergize with other therapies to preserve gut epithelialintegrity.