Acid Polyacrylamide Gel Electrophoresis Research Articles

Molecular cloning of novel α-gliadin genes from Crithopsis delileana and the evolution analysis with those from Triticeae

The α-gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) were investigated by Acid polyacrylamide gel electrophoresis (A-PAGE) analysis. It was indicated that the electrophoresis mobility of gliadins from C.delileana had obvious difference with those from common wheat in α, γ and ω region. Using primers designed from published sequences of α-gliadin genes, three α-gliadin genes were isolated from C. delileana, which were designated as gli-ka1, gli-ka2 and gli-ka3, respectively. Two in-frame stop codons were found in the coding sequences of gli-ka3, indicating that gli-ka3 could be a pseudogene. The gli-ka2 was a gliadin with an odd number of cysteines, resulting from a non-synonymous mutation. This change might lead to the interactive behavior of gli-ka2. Three α-gliadin genes of C. delileana had the similar but not identical primary structures to the corresponding gene sequences from other wheat related species. By the alignment of α-gliadin genes from Triticeae, phylogenetic analysis indicated that three α-gliadin genes of C. delileana clustered together with all α-gliadin genes from Ee genome of Lophopyrum elongatum by an interior paralleled branch.

Diversity of seed storage protein patterns of Slovak accessions in jointed goatgrass (Aegilops cylindrica Host.)

Variations in seed storage protein patterns were investigated for six accessions of jointed goatgrass (Aegilops cylindrica) populations collected from Slovakia within the framework of the bilateral Co-operation in Science and Technology between the Slovak Republic and Hungary. The study covered populations collected from the southwestern (localities: Sered and Dunajská Streda), southern (localities: Chlaba and Kamenica nad Hronom) and southeastern (localities: Cierna nad Tisou and Dobra) parts of Slovakia. Analysis of profiles of seed storage proteins – glutenins and gliadins – was carried out using acid polyacrylamide gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis. All accessions have a uniform three-band high molecular weight glutenin pattern with CxCyDy subunit composition. The highest variations in gliadin bands among the populations were observed from Cierna nad Tisou. There were small differences among the populations from Chlaba and Dobra. The lowest variations were in populations from Sered, Dunajska Streda and Kamenica nad Hronom. The present investigation showed that these jointed goatgrass populations are valuable genetic resources for wheat crop improvement programmes.

Analyses of monomeric storage proteins “gliadins” in Iranian bread wheats

A collection of new and obsolete Iranian bread wheat cultivars were characterized for gliadins using acid polyacrylamide gel electrophoresis (A-PAGE). Extensive polymorphism (H) = 0.734 in gliadin patterns was found. A total of 26 band patterns including 13, 8 and 5 different mobility bands were identified, in the zones of ω-, β + γ- and α-gliadins, respectively. There were a few patterns specific to each region and some were common among all the regions. Patterns of α-gliadin C, β + γ-gliadins A, and ω-gliadins H, C and E patterns were significantly higher in temperate and tropical zones. β + γ-gliadin C and ω-gliadin Q were significantly higher in Caspian-cold regions. Variation was observed in gliadins patterns of cultivars grown in different regions in Iran. Individual cultivars showed unique gliadin fingerprints. There were larger variation in ω - and γ + β-gliadins than in α-gliadins. These results may provide complementary information for relating genetic diversity, and quality characterization of Iranian wheat cultivars.

Similarity of cultivars of wheat (Triticum durum) on the basis of composition of Gliadin alleles

Twenty one durum wheat cultivars originating from different world countries were investigated. Composition of gliadins was analyzed by acid polyacrylamide gel electrophoresis. Allele composition of gliadins was determined on the basis of identified gliadin blocks. Polymorphisms of Gli- loci was established and 27 different gliadin alleles were identified, namely, 5 at Gli-A1, 4 at Gli-B1, 9 at Gli-A2 and 9 alleles at Gli-B2 locus. The catalogue of determined alleles was presented. Frequency of alleles ranged from 4.76% to 42.86%. Heterozygous Gli-loci were identified at two durum cultivars. Similarity among cultivars was studied on composition of Gli-alleles and presented by UPGMA dendogram. On the base of Gli-allele composition, similarity varied from 0% to 100%.

Molecular cytogenetic identification of a new wheat-Thinopyrum substitution line with stripe rust resistance

A new wheat-Thinopyrum substitution line AS1677, developed from a cross between wheat line ML-13 and wheat-Thinopyrum intermedium ssp. trichophorum partial amphiploid TE-3, was characterized by fluorescence in situ hybridization (FISH), sequential Giemsa-C banding, genomic in situ hybridization (GISH), seed storage protein electrophoresis, molecular marker analysis and disease resistance screening. Sequential Giemsa-C banding and GISH using Pseudoroegneria spicata genomic DNA as probe indicated that a pair of St-chromosomes with strong terminal bands were introduced into AS1677. FISH using pTa71 as a probe gave strong hybridization signals at the nuclear organization region and in the distal region of the short arms of the St chromosome. Moreover, FISH using the repetitive sequence pAs1 revealed that a pair of wheat 1D chromosomes was absent in accession AS1677. Seed storage proteins separated by acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that AS1677 lacked the gliadin and glutenin bands encoded by Gli-D1 and Glu-D1, further confirming the absence of chromosome 1D. The introduced St chromosome pair belonging to homoeologous group 1 was identified by newly produced genome specific markers. AS1677 is a new 1St (1D) substitution line. When inoculated with stripe rust and powdery mildew isolates, AS1677 expressed stripe rust resistance possibly derived from its Thinopyrum parent. AS1677 can be used as a donor source for introducing novel disease resistance genes to wheat in breeding programs aided by molecular and cytogenetic markers.

Characterization of two novel γ-gliadin genes encoded by K genome of Crithopsis delileana and evolution analysis with those from Triticeae

By acid polyacrylamide gel electrophoresis (A-PAGE) analysis, it was indicated that the electrophoresis mobility of gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) had obvious difference with those from common wheat in α, γ and ω region. Using homologous primers, two γ-gliadin genes (gli-Kr1 and gli-Kr2) were isolated from C. delileana, which had been deposited in the GenBank under accession numbers EU283818 and EU283821, respectively. Two γ -gliadin genes of C. delileana had the similar primary structures to the corresponding gene sequences from other wheat related species. The differences were mainly resulted from substitutions, insertions and deletions involving single amino acid residues or motifs of γ-gliadins. The repetitive domains of gli-Kr1 and gli-Kr2 from C. delileana are shorter than most of other sequences. By the alignment of γ-gliadin genes from A, B, D, Am, Au, S, Sl, Ssh, Ss and Sb genomes of Triticum and Aegilops, R genome of Secale (γ-secalin), Ee genome of Lophopyrum and K genome of Crithopsis in Triticeae, phylogenetic analysis indicated that two γ-gliadin genes of C. delileana could be clustered together with a γ-gliadin genefrom Ssh genome of Aegilops by an interior paralleled branch. It was the first time that the γ-gliadin genes encoded by K genome of C. delileana were characterized. These could offer precious information for better understanding the qualities associated with gliadins, the response in coeliac disease and studying the evolutionary relationship of gliadins in Triticeae.

Transfer to wheat (Triticum aestivum) of small chromosome segments from rye (Secale cereale) carrying disease resistance genes

One hundred wheat lines, derived from monosomic additions of chromosome 1R of rye inbred line R12 (Chinese rye), were detected by PCR amplification using rye-specific primer pairs. Only 5 wheat lines, 1R296, 1R330, 1R314, 1R725, and 1R734, were determined to contain rye chromatin. While 1R296 and 1R330 were highly susceptible to stripe rust and powdery mildew, 1R314, 1R725 and 1R734 were highly resistant to both diseases. Acid-polyacrylamide gel electrophoresis showed that the omega-secalin bands were absent in 1R314, but present in the other 4 wheat lines. Genomic in situ hybridization indicated that 1R296, 1R330, and 1R725 contained translocations involving the whole short arm of chromosome 1R. However, 1R314 and 1R734 contained a pair of wheat chromosomes with small, terminal, rye-derived chromosome segments. The results suggest that the translocation breakpoint of 1RS in 1R314 was located between the Sec-1 locus and the disease-resistance loci, while in line 1R734, the breakpoint was located between the Sec-1 locus and the centromere. Taking account of the improved disease resistance of 1R725, 1R314 and 1R734, the chromosome arm 1RS of R12 may represent new and valuable disease resistance resources for wheat improvement.

English

  This study aimed at assessing the genetic diversity of 102 lines doubled haploid wheat (sent from CIMMYT) using acid-polyacrylamide gel electrophoresis (A-PAGE) method, morphological traits and baking quality. Cluster analysis according to morphological traits divided all genotypes into four groups, so that genotypes with high yield were placed in one group. However, by cluster analysis according to their qualitative traits, the genotypes were classified into three main groups, while genotypes with higher amount of protein was placed in a separate group. In the studied lines, 48 bands and 47 different patterns were detected and polymorphism was observed in most of the bands. In the ω area, 18 bands and 19 different patterns were observed and the most amount of band was observed in this area. In γ and β areas, 12 and 9 bands, 19 and 12 patterns were observed, respectively. The least pattern variety was seen in α area, presumably because the bands did not separate properly in the one-dimensional electrophoresis in this area. Seven patterns and nine bands were observed in this area. Using Nei formula and according to the patterns, the genetic diversity for all four areas (α, β, γ, ω) was calculated, according to which γ area with H = 0.872 had the most genetic diversity, then came ω and β areas with H = 0.767 and H = 0.714, respectively, and the least genetic diversity was observed in α area with H = 0.646. Cluster analysis according to protein bands has classified genotypes into 9 main groups. Although the lines studied in this research had the same parents, considerable diversity was observed among them. Therefore, the electrophoresis of polyacrylamide gel of gliadins can be used as a strong system for identifying similar varieties. While comparing the observed patterns, one pattern in ω area was proved to be relevant to the trait of the number of grain per spikelet, which can be used as a marker in order to increase yield.   Key words: Genetic diversity, gliadin patterns, morphological traits, backing quality, doubled haploid wheat.

Starch-bound 2S proteins and kernel texture in einkorn, Triticum monococcum ssp monococcum

The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE. All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B), as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured common wheat, "monococcum" accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules, respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele Eti-A(m) 1a and "valine-type" ETI encoded by allele Eti-A(m) 1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low as -2.05 +/- 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in "monococcum" wheat, as determined by visual assessments in counting chambers, was estimated at 764 mm(2)/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification of factors that cause the extra-soft texture of einkorn kernels.

Genetic variation of 1RS arm between sibling wheat lines containing 1BL.1RS translocation

3 sets of sibling 1BL.1RS translocation wheat lines (each set containing 2 sibling lines) derived from 3 different F4 single plant. Between 2 sibling wheat lines, different resistance to powdery mildew was observed. In addition, different bands in α-gliadin mobility zone between 2 sibling wheat lines were also significantly observed by acid polyacrylamide gel electrophoresis (A-PAGE). However, thenucleotide sequence of α/β-gliadin precursor gene cloned from 2 sibling wheat lines were 100% similarity. Polymerase chain reaction (PCR) analysis using 180 wheat microsatellite markers dispersed on 7 homeologous groups of wheatindicated that little genetic variation occurred in wheat A, B and D genomes of these sibling wheat lines. Structural variation of 1BL.1RS chromosome arm was detected by PCR analysis. Different rye-specific repetitive DNA exhibited different variation. The different structure of 1RS chromosome arms may be related to the genetic variation between the 2 sibling wheat lines. The results lead us to think that the variation of 1RS arm within a species should be utilized in wheat breeding program. To detect variation of repetitive DNA can provide better understanding of the genetic variation of 1BL.1RS translocation and will be useful for utilization of 1RS arm in wheat breeding program. Key words: 1BL.1RS translocation, repetitive DNA, sibling wheat lines, gentic variation.

Identification of novelω-gliadin gene inAegilops tauschiiusing RFLP

A RFLP approach was used to investigate polymorphism of ω -gliadin genes in Ae. tauschii using a F 2 population from the cross of accessions AUS18913 and CPI110856. A set of 150 F 2 progenies was genotyped by acid polyacrylamide gel electrophoresis (A-PAGE) and only one recombinant line of Gli-D t 1/Gli-D t T1 was observed. Twelve restriction enzymes were initially tested on genomic DNA of the two parents of which four restriction enzymes revealed polymorphism. Of these four, only Dra I was associated with the novel ω -gliadin gene (T 1 ) using a 1,200 bp DNA fragment of a ω -gliadin gene as a gene-specific probe. The ω -gliadin gene (T 1 ) may be of interest for further studies relating storage proteins and wheat bread-making quality.

Impact of Peroxynitrite Modification on Structure and Immunogenicity of H2A Histone

Peroxynitrite is an extremely reactive entity and has in vivo existence. Its interaction with biomolecules may cause oxidation and nitration. In this study, commercially available H2A histone was exposed to peroxynitrite generated in vitro. The peroxynitrite-mediated structural changes in histone were studied by ultraviolet & fluorescence spectroscopy, high performance liquid chromatography, anilinonaphthalene-8-sulfonic acid binding and polyacrylamide gel electrophoresis. Analysis of results revealed that carbonyl, nitrotyrosine and dityrosine contents were significantly increased in peroxynitrite-modified H2A compared with native H2A. Rabbits challenged with peroxynitrite-modified H2A induced high titre antibodies. The immunogenicity of peroxynitrite-modified H2A was directly proportional to protein nitrotyrosine content and induced antibodies showed specificity for the immunogen and good cross-reaction with nitrated epitopes of other modified proteins. Formation of high molecular weight immune complex with retarded mobility further supports the specificity of induced anti-100 mum peroxynitrite-modified H2A antibodies for the immunogen. It may be concluded that induction of anti-H2A histone antibodies could be due to protection of peroxynitrite-modified histone from proteolytic breakdown and its subsequent recognition by immunoregulatory cells as foreign molecule.

Hordein gene dose effects in triploid endosperm of barley (Hordeum vulgare L.)

The presence of two maternal chromosome sets in triploid barley endosperm allows the distinction of maternal and paternal hordein bands in an electrophoregram: the maternal bands are stronger due to the higher gene dose. In the F1 generation there are differences between reciprocal crosses and in the F2 generation all 16 classes that are theoretically possible for a pair of polymorphic loci can be distinguished. This full classification is rarely possible in genetic studies, and allows more accurate estimates of recombination rates. Two hordein gene clusters (Hor1 and Hor2, corresponding to hordein C and hordein B respectively) were analyzed in hybrids obtained by crossing two winter barley cultivars Partizan and HWV-247. Hordein separation was performed by acid-polyacrylamide gel electrophoresis at pH 3.2 (A-PAGE). A set of most informative bands of B and C hordeins was selected in each cross by two criteria: (1) presence or absence of bands in the parents and (2) signal strength to allow doses scoring. The average genetic distance between Hor1 and Hor2 loci was 11 cM. Distances in male and female maps were not significantly different, suggesting a similar recombination rate in male and female meiosis.

Subunit stoichiometry determination by perfluorooctanoic acid polyacrylamide gel electrophoresis (PFO-PAGE)

Subunit stoichiometry determination by perfluorooctanoic acid polyacrylamide gel electrophoresis (PFO-PAGE)

Variation in hordein polypeptide pattern within Ethiopian barley, Hordeum vulgare L. (Poaceae)

From a large collection of Ethiopian barley (Hordeum vulgare L.) that was cultivated by the author in a sheltered bird-proof enclosure in Addis Abeba, 229 fully ripe spikes were sampled to represent the range of variation in morphology and distribution in regions and agroecological zones of the country. Hordeins were extracted from kernels and the polypeptide patterns obtained with the use of acid polyacrylamide gel electrophoresis. The major hordein bands were scored and the data subjected to the TWINSPAN classification procedure, giving 68 landrace groups. The findings document a considerable variation in hordeins in Ethiopian barley. Some hordein patterns were found to be widespread and abundant while others were restricted and less frequent. This variation in hordeins could be used as a supportive criterion for distinguishing groups within the Ethiopian landrace types.

Allelic variation of HMW Glutenin subunits and1BL.1RStranslocation in Slovak common wheats

High molecular weight glutenin and 1BL.1RS translocation employing the standard sodium dodecyl sulphate (SDS-PAGE) and acid (A-PAGE) polyacrylamide gel electrophoresis methods were classified in 43 Slovak wheat cultivars registered between 1976 and 2006. Total number of 9 alleles was detected at all Glu-1 loci. The most frequent HMW-GS alleles were “Null” for Glu-1A , 7+9 for Glu-1B and 5+10 for Glu-1D , respectively. At the same time these alleles also constituted the most frequent HMW-GS genotype and phenotype-0, 7+9, 5+10. Such HMW-GS combination was found in 48.8% of all genotypes analyzed in Triticum aestivum L. ssp. aestivum . Eleven different HMW-GS genotype-phenotype combinations were found, occurring at various frequencies.

Diversity in seed storage proteins in substituted hexaploid triticale cultivars (×TriticosecaleWittmack)

This study was undertaken to investigate the allelic diversity in seed storage proteins in 11 substituted hexaploid triticale cultivars (all spring). These cultivars were developed at the Cereal Institute of Thessaloniki, Hellas, in the 1970s and 1980s, after selection on segregating material originating from International Maize and Wheat Improvement Center (CIMMYT). Seeds from each line were used to determine alleles at the loci for high molecular weight glutenin subunits (HMW-GS) Glu-A1, Glu-B1, Glu-R1 (or Sec-3) and gliadins (the loci Gli-A1 and Gli-B1). For this to be done, acid polyacrylamide gel electrophoresis for gliadins and SDS-electrophoresis for high molecular weight glutenins (HMW-GS) were applied. Analysis of the electrophoretic patterns obtained from the above-mentioned material revealed that only 5 out of the 11 cultivars were biochemically uniform (cv. ‘Vryto’, ‘Thisvi’, ‘Dada’, ‘Leto’ and ‘Ekate’). On the contrary, the rest of the cultivars, despite they were under seed production process, exhibited heterogeneity. Cv. ‘Dada’, which was found to be uniform, is of special interest, due to its productivity, especially under drought stress conditions.

High-Molecular-Weight Glutenin Subunit Composition of Chinese Wheat Germplasm

Abstract The objective of the present study was to characterize the high molecular glutenin subunits (HMW-GS) composition and the presence of 1B/1R translocation in newly developed wheat ( Triticum aestivum L.) germplasm, which have one or more traits that are useful in wheat improvement. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and acid polyacrylamide-gel electrophoresis (A-PAGE) were used to detect HMW-GS composition and the presence of 1B/1R wheat-rye ( Secale cereale L.) chromosome translocation in the wheat germplasm. Bread-making quality scores of these lines were determined. A high level of variations in HMW-GS encoded by Glu-1 locus was observed. Sixteen major HMW-GS, with 30 combinations, were detected. The percentage of cultivars with more than two desirable subunits was 38.7%. Thirteen cultivars had bread-making quality scores of 10 in combination with one or two desirable agronomical traits, such as high-yield potential, dwarfing stem, resistance to diseases, and/or tolerance to abiotic stress. Sixty-eight (36.6%) cultivars possessed 1B/1R translocation. The newly developed germplasm with HMW-GS for good quality can be promising resources for improving bread-making quality of wheat.

Purification and three-dimensional electron microscopy structure of the Neisseria meningitidis type IV pilus biogenesis protein PilG.

Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 A in length and 80 A in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower "waist" region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

Polymorphism of Gli-D1 alleles of Kragujevac’s winter wheat cultivars (Triticum aestivum L.)

Composition of gliadins encoded by Gli-D1 allele as well polymorphisms of Gli-D1 allele investigated in 25 wheat cultivars by using acid polyacrylamide gel electrophoresis. Electrophoregrams obtained by polyacrylamide gel electrophoresis were used for estimation variability of gliadin components and identification of gliadin blocks. Five gliadin blocks encoded by different alleles at Gli-D1 locus were apparently expressed and identified. Gliadin blocks differed according to number of components and their molecular mass. Variability of determined block components indicates that existing polymorphisms of gliadins alleles. Frequency of identified 5 alleles at Gli-D1 locus was in ratio from 4% to 52%. The highest frequency of b allele and the of g allele was found.