Use of artificial neuronal nets in automation of analysis and genetic identification of electrophoretic spectra of gliadin from durum wheat]

Each wheat cultivar has a characteristic spectrum of gliadins. This makes it possible to use blocks of the components of reserve proteins as genetic markers when estimating seed quality. However, identification of the blocks that constitute the electrophoretic spectrum is a complicated task. For this purpose artificial neural network (ANN) technology is proposed. Based on experimental data, a teaching database and testing databases have been created. ANN was shown to be highly efficient (efficiency up to 100%) expert system for deciphering the electrophoretic spectra of gliadins of durum wheat cultivars.     

The Use of Artificial Neural Networks for Automatic Analysis and Genetic Identification of Gliadin Electrophoretic Spectra in Durum Wheat

Each wheat cultivar has a characteristic spectrum of gliadins. This makes it possible to use blocks of the components of reserve proteins as genetic markers when estimating seed purity and identity. However, identification of the blocks that constitute the electrophoretic spectrum is a complicated task. For this purpose artificial neural network (ANN) technology is proposed. Using experimental data, a teaching database and testing databases have been created. ANN was shown to be highly efficient (efficiency up to 100%) expert system for deciphering the electrophoretic spectra of gliadins of durum wheat cultivars.     

Aneuploid analysis of low molecular weight gliadins from wheat

Summary The chromosomal location of genes affecting five components from the low molecular weight gliadin (LMWG) fraction from wheat endosperm has been investigated by aneuploid analysis. Genes controlling these proteins were assigned to chromosomes 4B, 7A and 7D. Chromosomes from homoeologous groups 1, 2 and 6, where genes controlling classical gliadins are located, are not involved in the control of LMWG.     

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten‐free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α‐, γ‐, ω‐gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP‐HPLC and mass spectrometry (LC‐MS/MS), whereas gluten immunogenicity was assayed by an anti‐gliadin 33‐mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ‐ and α‐gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω‐gliadins and γ‐gliadins, and three of these also silenced α‐gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC‐MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α‐ and ω‐gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.     

不同提取剂对麦醇溶蛋白提取效果的电泳比较

以大麦、小麦品种为材料,分别采用醇溶蛋白提取剂乙醇、乙二醇、2-氯乙醇、2-巯基乙醇、尿素等配成不同浓度的单一组分提取剂和复合提取剂,在同一条件下提取大麦和小麦去胚种子的醇溶蛋白,通过A-PAGE分离样品液发现,单一组分提取剂中25％的2-氯乙醇效果较好,5％的2-巯基乙醇效果较差;复合提取剂中由2-氯乙醇和2-巯基乙醇组成的提取剂效果最好。这种复合提取剂制备的样品液经电泳后,在图谱中条带丰富,带型清晰,无论对大麦还是小麦种子,都是最佳的醇溶蛋白提取剂。     

A proteomic approach to verify in vivo expression of a novel gamma-gliadin containing an extra cysteine residue

Gliadins and glutenins are the main protein fractions present in wheat gluten. They are responsible for technological and nutritional quality of wheat based products. In particular, glutenins are mainly responsible for dough visco-elastic properties, whereas gliadins confer extensibility to dough and are the most important factor triggering celiac disease, the major human intolerance to gluten. Gliadins are monomeric proteins, whereas glutenins are polymers stabilized by disulfide bonds. Although they have distinctive structural characteristics, it is possible that some gliadins become part of the glutenin fraction because of mutations that affect cysteine number and distribution. Here, we provide evidence that a naturally mutated gamma-gliadin with an extra cysteine residue is incorporated into the polymeric fraction. This goal was achieved using an integrated approach involving heterologous expression, 2-DE, RP-HPLC and MS.     

Gliadins polymerized with cysteine: effects on the physical and water barrier properties of derived films

To study the effects of disulfide bonds on certain functional properties of films made from the wheat gluten proteins gliadin and glutenin, cysteine was used to promote the formation of interchain disulfide bridges between gliadins in 70% ethanolic solution. Disulfide-mediated polymerization of gliadins was confirmed by means of SDS-PAGE analysis. After chemical treatment of gliadins, films were solution cast and the effects of both glycerol (used as a plasticizer) and relative humidity were studied on water vapor permeability, moisture sorption isotherms at 23 degrees C, and the optical properties of the films. The results were compared with those obtained from analogous films made from untreated glutenin macromolecules. Cysteine-mediated polymerization of gliadins improved the water vapor resistance of films achieving values close to those obtained for glutenin films. Development of intra- and interchain disulfide bonds did not change the moisture sorption capacity of the films but transparency was slightly diminished.     

Effects of low temperature on wheat leaf proteins

Finnish wheat cultivars, winter wheat ‘Vakka’ and spring wheat ‘Apu’, grown for 9 days at 25° and then for 52 days at 2–4°, were called ‘hardened’. Proteins and esterases of young leaves were resolved by polyacrylamide gel electrophoresis (PAGE), porosity gradient PAGE (poroPAGE), isoelectric focusing (PAGIF) between pH 3 and 9, and by Na-dodecylsulfate (SDS)-PAGE. Hardened and unhardened leaves have different protein patterns after PAGE, PAGIF, poroPAGE and SDS-PAGE, respectively, as well as for esterases after PAGE and PAGIF. The PAGIF patterns of esterases show distinct changes of two bands especially for hardened leaves of winter wheat, one appearing, the other one disappearing.     

Salivary proteins of aphids, a pilot study on identification, separation and immunolocalisation

Salivary proteins (SPs) of Schizaphis graminum, Acyrthosiphon pisum and Myzus persicae were studied after probing and feeding on different artificial diets. Salivary sheaths as well as apical lumps of saliva were found, presumably representing subsequently excreted saliva of different types. Phenoloxidase, pectinase and peroxidase activities were detected by staining the enzyme-converted products, thus confirming these enzyme activities found earlier by others. Proteinase and cellulase were not found. SPs in three major SDS-PAGE bands, at 154 and 66/69 kDa, were collected in fluid diets (soluble fraction) and as sheath material (solid fraction) attached to the membranes covering these diets. Proteins of both fractions presumably represented the enzymatic activities found, although this could not be proven. The lack of electrophoretic mobility of the undenaturated (isoelectrofocusing and PAGE) active proteins meant that they could not be separated, whereas the mobile denaturated (SDS-PAGE) proteins had lost their enzyme activity. Polyclonal antibodies, anti-SP154 and anti-SP66/69, both cross-reacted to most salivary proteins in Western blots. They also reacted to sheath material and to the principal salivary glands. For further studies of saliva some monoclonal antibodies were developed. The complexity of salivation and the relation of the results obtained to the behaviourally known secretion periods is discussed.     

Null alleles for gliadin blocks in bread and durum wheat cultivars

Summary Wheat gliadin proteins are coded by clusters of genes (complex loci) located on the short arms of chromosomes of homoeologous groups 1 and 6 in bread (6x) and durum (4x) wheats. The proteins expressed by the various complex loci have been designated gliadin blocks. In a survey of accessions from the Germplasm Institute (C.N.R., Bari, Italy) collection, several different accessions have been found that lack particular blocks of proteins (null alleles). In some bread wheat accessions, seeds do not express gliadins that are coded by chromosomes 1D and 6A in normal cultivars. Similarly, some durum wheat accessions lack -gliadin components coded for by genes on chromosomes 1A and 1B. The missing proteins do not result from the absence of whole chromosomes, but may be the consequence of partial deletion of these genes at a complex locus or result from their silencing.     

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

Background  

The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.     

Polymorphism of omega-gliadins in durum wheat as revealed by the two-step APAGE/SDS-PAGE technique

Polymorphism of omega-gliadins was studied in 243 durum wheats from 27 countries using the two-step one-dimensional APAGE/SDS-PAGE technique. A total of 12 bands of different mobility were observed, and four of them were found to be different from those previously detected by Khelifi et al. (1992) in bread wheat. Fifteen alleles, six coded by the Gli-A1 locus and nine coded by the Gli-B1 locus, were identified, accounting for 19 different electrophoretic patterns. Seven new alleles were detected: two at the Gli-A1 locus and five at the Gli-B1 locus. The polymorphism found at the Gli-A1 and Gli-B1 loci was slightly greater than that found in bread wheat. Allelic differences between both species were higher at the Gli-B1 locus. A comparison of the frequencies of alleles in both species was carried out. The null allele, Gli-A1e, was more common in durum wheat than in bread wheat. The Gli-B1b allele, present in 60% of the bread wheats, was found in only 2% of the durum wheats and Gli-B1e, very common in durum wheat (45%), was rare in bread wheat (4%). The Gli-B1IV allele, common in durum wheat (28%), was not detected in bread wheat.     

Catalogue of alleles of gliadin-coding loci in durum wheat (Triticum durum Desf.)

Gliadins are seed storage proteins which are characterized by high intervarietal polymorphism and can be used as genetic markers. As a result of our work, a considerably extended catalogue of allelic variants of gliadin component blocks was compiled for durum wheat; 74 allelic variants for four gliadin-coding loci were identified for the first time. The extended catalogue includes a total of 131 allelic variants: 16 for locus Gli-A1(d), 19 for locus Gli-B1(d), 41 for locus Gli-A2(d), and 55 for locus Gli-B2(d). The electrophoretic pattern of the standard cultivar and a diagram are provided for every block identified. The number of alleles per family is quite small for loci Gli-A1(d) and Gli-B1(d) of durum wheat, as contrasted to loci Gli-A2(d) and Gli-B2(d) that are characterized by large families including many alleles. The presence of large block families determines a higher diversity of durum wheat for loci Gli-A2(d) and Gli-B2(d) as compared to Gli-A1(d) and Gli-B1(d). The catalogue of allelic variants of gliadin component blocks can be used by seed farmers to identify durum wheat cultivars and evaluate their purity; by breeders, to obtain homogenous cultivars and control the initial stages of selection; by gene bank experts, to preserve native varieties and the original biotypic composition of cultivars.     

The structure and genetic control of a new class of disulphide-linked proteins in wheat endosperm

Summary Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced total protein extracts from the endosperm of hexaploid wheat revealed three high molecular weight protein bands (triplet bands) in a zone of heavy background streaking. Electrophoretic examination of 135 hexaploid cultivars showed at least five different patterns of these triplet bands. Nine durum wheat cultivars showed a single band only. Analysis of nullisomic-tetrasomic and ditelocentric lines of Chinese Spring wheat revealed that the slowest moving band (Tri-1) of the triplet was controlled by gene(s) on chromosome arm 1DS and the fastest moving band (Tri-3) by 1AS. The band with intermediate mobility (Tri-2) was found to be a hybrid aggregate of the subunits controlled by 1DS and 1AS. Using a non-reducing/reducing form of 2-dimensional (2-D) electrophoresis, these triplet bands were shown to be heterotetramers of four subunits designated D (M.W. 58,000), (22,000), A (52,000) and (23,000) where Tri-1=DD, Tri-2 = DA and Tri-3 = AA. With very low concentrations of 2-mercaptoethanol (ME), the tetramers dissociated into dimeric subunit pairs (D, A), the monomers being observed with higher concentrations of ME. The structure of these subunit pairs resembles that of the subunit pairs in the globulin storage proteins of oats and some legumes. The 2-D method employed in this study was useful also for separating low molecular weight (LMW) subunits of glutenin from the monomeric gliadins which have similar electrophoretic mobility in 1-D separation. It was shown that at least four of these LMW glutenin subunits are controlled by genes on 1DS and 1AS and at least one subunit is controlled by gene(s) on 1BS. This electrophoretic separation method has proven useful in understanding the aggregation behaviour of the seed proteins of wheat.     

Demonstration of high opioid-like activity in isolated peptides from wheat gluten hydrolysates

Because of a possible relationship between schizophrenia and celiac disease, a condition in some individuals who are sensitive to wheat gluten proteins in the diet, there has been interest in observations that peptides derived from wheat gluten proteins exhibit opioid-like activity in in vitro tests. To determine the origin of the peptides exhibiting opioid activity, wheat proteins were fractionated by size (gel filtration), by charge differences (ion exchange chromatography) and by differences in hydrophobicity (reversed-phase HPLC). These fractions were hydrolyzed by pepsin or pepsin and trypsin and the resulting peptides separated by gel filtration chromatography. The separated peptides were tested for opioid-like activity by competitive binding to opioid receptor sites in rat brain tissue in the presence of tritium-labeled dihydromorphine. The peptides showed considerable differences in activity; while some peptides exhibited no activity, 0.5 mg of the most active peptides were equivalent to 1 nM of morphine in the binding assay. The most active peptides were derived from the gliadin fraction of the gluten complex.     

1BL/1RS translocation in durum wheat and its effect on end use quality traits

The gluten proteins document the genotypic identity of a wheat variety, in addition to providing valuable clues about its ancestry and technological properties. In this study, an Indian durum wheat genotype B662 was identified to carry 1BL/1RS translocation and characterized further for its effect on end use quality traits. Comparison of the end use quality traits of B662 with five other durum cultivars without 1BL/1RS, showed decreased gluten content, lower swelling index of glutenins and low MSDS-SV indicating that, B662 with 1BL/1RS is not good for pasta making. In F_2:3 seeds from a durum wheat cross between the 1BL/1RS cultivar B662 and HI8498 without the translocation, the secalin Sec-1 loci segregated in theoretically expected 3:1 proportion and were inherited as a block of the rye chromosome arm. The analysis of F_2:3 harvests for the two most important durum wheat quality tests showed that the presence of 1BL/1RS translocation did not alter the grain protein content values, but was associated with significant reduction of micro SDS-sedimentation volume indicating inferior quality, thus limiting the commercial exploitation of durum wheat genotypes with 1BL/1RS translocation. The cautious use of rye translocation in Indian durum wheat breeding is suggested.     

Genetic control of endosperm proteins in wheat

Summary Total endosperm proteins extracted from both several common wheat cultivars and some intervarietal substitution lines derived from them were fractionated according to their molecular weight in a high resolution one-dimensional gel electrophoresis. The four donor cultivars and the recipient one — Chinese Spring, possessed differentially migrating protein bands in the fractions of high molecular weight (HMW) glutenins and gliadins. Several of these bands were identified for the first time in this study. By utilizing intervarietal substitution lines the control of the HMW glutenins and gliadins by chromosomes of homoeologous group 1 was either reaffirmed or, for the new bands, established. Several HMW gliadin subunits showed a considerable variation in their staining intensity in the intervarietal substitution lines indicating that their expression was dependent on the genetic background.This paper is based on a portion of a dissertation to be submitted by G. Galili in partial fulfilment of the Ph.D. requirements of the Feinberg Graduate School, The Weizmann Institute of Science, RehovotThe Marshall and Edith Korshak Professor of Plant Cytogenetics     

Use of selected enterococci and Rhizopus oryzae proteases to hydrolyse wheat proteins responsible for celiac disease

Aims:  This work aimed at using a pool of selected enterococci and fungal proteases to hydrolyse wheat gluten during long-time fermentation.
Methods and Results:  A liquid dough made with wheat flour (20% w/w) was fermented with three Enterococcus strains (dough A) or with the combination of enterococci and Rhizopus oryzae proteases (dough B). After 48 h of fermentation, dough A and B had a concentration of water-soluble peptides approximately threefold higher than the chemically acidified dough (CAD), used as the control. The same was found for the concentration of free amino acids, being higher in dough B with respect to dough A. SDS-PAGE analysis showed that albumin and glutenin fractions were partially hydrolysed, while gliadins almost disappeared in dough A and B, as confirmed by two-dimensional electrophoresis, RP-HPLC and R5-ELISA analyses.
Conclusions:  The combined use of enterococci and fungal proteases showed a decrease of the gluten concentration of more than 98% during long-time fermentation.
Significance and Impact of the Study:  The use of the mixture of selected enterococci and R. oryzae proteases should be considered as a potential tool to decrease gluten concentration in foods.     

单克隆抗体技术在小麦贮藏蛋白研究及其遗传改良中的应用进展

近20年来,人们制备了许多小麦种子贮藏蛋白的单克隆抗体(Monoclonal Antibody,McAb),一方面作为有效工具研究胚乳贮藏蛋白(主要是麦谷蛋白聚集体、特定的谷蛋白亚基及醇溶蛋白)的结构与功能关系;另一方面用作诊断试剂(diagnostic tools),为筛选具有合适加工品质的小麦品种提供依据。本文综述了国内外单克隆抗体技术在小麦贮藏蛋白研究及其遗传改良中的应用进展,并展望其应用前景。