Small-angle X-ray scattering study by synchrotron orbital radiation reveals that high molecular weight subunit of glutenin is a very anisotropic molecule.

Small-angle X-ray scattering of one high molecular weight (HMW) subunit of wheat glutenin was measured at protein concentration ranges from 1.0 to 10.0 mg/ml. The radius of gyration of whole particles, RO, in aq. 50% (v/v) 1-propanol and 0.1M acetic acid was 16.6 +/- 0.1nm and 22.8nm, respectively, and the corresponding radius of gyration of the cross-section, RC, was 2.82 +/- 0.02 nm and 2.23 +/- 0.01 nm, which indicate that the glutenin HMW subunit exists as very anisotropic particles in both solutions. The RO and RC values of the subunit, and the drastic decrease in scattered intensity at small angles that occurs in the acetic acid solution with relatively low protein concentration are completely explained in terms of rod-like molecules of the glutenin HMW subunit.     

Mapping of glutenin and gliadin genes located on chromosome 1B of common wheat

Summary The inheritance of the high molecular weight (HMW) glutenins and of several gliadins controlled, respectively, by the long and short arms of chromosome 1B of common wheat was studied. Analysis was carried out on the progeny of two inter-varietal crosses in which the parental lines possessed differentially migrating subunits as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No recombination event was detected either within the fraction of the HMW glutenins or among most of the gliadin subunits studied indicating that they are controlled by tightly linked gene clusters. One gliadin subunit (B30) showed 25.5% recombination frequency with the rest of the gliadin subunits and 23.5% recombination frequency with the fraction of the HMW glutenin subunits. It has been concluded that this subunit is controlled by a separate locus (Gld-B6), proximal to the major gliadin gene cluster on the short arm of chromosome 1B. Consequently, the recombination percentage between the glutenin loci and most of the gliadin loci was calculated as 49.0 and the distance in centi-Morgans (cM) as 53.6. The estimated distance in cM is very close to the observed recombination percentage. A genetic map of these storage protein genes is presented.     

小麦种子贮藏蛋白质研究进展

小麦醇溶蛋白组成可以作为小麦品种鉴定的指纹图谱,其分离方法有酸性电泳、反相高压液相色谱（RP－HPLC）和毛细管电泳（CE）等手段,3种方法相互补充,而CE分辨率最高。对醇溶蛋白酸性电泳条件的改良和完善仍在进行中,利用最新的分离技术对小麦醇溶蛋白基因进行染色体定位和遗传行为分析是近年来醇溶蛋白研究的另一领域。小麦高分子量麦谷蛋白亚基（HMW－GS）与小麦面包烘烤质量密切相关,关于它的研究目前主要集中在3个方面;对各个迁3移率较近的亚基进行快速,准确分离方法的研究,HMW－GS与小麦面包烘烤质量关系的研究和通过基因工程来改良小麦的品质、提高面粉的加工特性等。低分子量麦保蛋白（LMW－Glutenin)影响小麦面粉的特性,截止目前已经获得了17个该基因的克隆,并对其基因结构进行了描述,有些低分子量麦谷蛋白亚基（LMW－GS）加入碱性面粉后改变了面筋的性质,报道了小麦醇溶蛋白,高分子量麦谷蛋白亚（HMW－GS）、低分子量麦谷蛋白亚基（LMW－GS）3个方面的最新研究进展。     

Suppression of gliadins results in altered protein body morphology in wheat

Wheat gluten proteins, gliadins and glutenins, are of great importance in determining the unique biomechanical properties of wheat. Studies have therefore been carried out to determine their pathways and mechanisms of synthesis, folding, and deposition in protein bodies. In the present work, a set of transgenic wheat lines has been studied with strongly suppressed levels of γ-gliadins and/or all groups of gliadins, using light and fluorescence microscopy combined with immunodetection using specific antibodies for γ-gliadins and HMW glutenin subunits. These lines represent a unique material to study the formation and fusion of protein bodies in developing seeds of wheat. Higher amounts of HMW subunits were present in most of the transgenic lines but only the lines with suppression of all gliadins showed differences in the formation and fusion of the protein bodies. Large rounded protein bodies were found in the wild-type lines and the transgenic lines with reduced levels of γ-gliadins, while the lines with all gliadins down-regulated had protein bodies of irregular shape and irregular formation. The size and number of inclusions, which have been reported to contain triticins, were also higher in the protein bodies in the lines with all the gliadins down-regulated. Changes in the protein composition and PB morphology reported in the transgenic lines with all gliadins down-regulated did not result in marked changes in the total protein content or instability of the different fractions.     

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species

Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.     

Characterization of HMW prolamines and their coding sequences from Crithopsis delileana

The high-molecular-weight (HMW) prolamines subunits and their coding sequences from wheat-related diploid species Crithopsis delileana were investigated. Only one HMW prolamine subunit with the similar electrophoresis mobility to the y-type HMW glutenin subunit of hexaploid wheat was observed in two accessions of C. delileana by SDS-PAGE analyses of the total storage protein fractions. It was confirmed by sequencing and expression analysis that this prolamine subunit was an x-type subunit. The amino acid sequence of this subunit had the similar typical structure to those of x-type HMW glutenin genes previously described in wheat. An in-frame stop codon was found in the coding sequences of y-type prolamine subunits. It was found by specifically extraction of HMW prolamines and sequence analysis that the coding regions of Ky prolamine subunit gene is very likely to be not expressed as a full-length protein. Phylogenetic analysis indicated that the Kx subunit could be clustered together with 1Ax1 subunit by an interior paralleled branch, and Ky subunit (inactive) was most closely related to the 1Ay subunit. The coding sequences of Kx subunit could successfully be expressed in bacterial expression system, and the expressed protein had the same electrophoresis mobility as the Kx subunit from the seed of C. delileana. It was the first time that the HMW prolamines subunits encoded by K genome of C. delileana were characterized.     

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2^t and T2, and was considered as part of the same allele at the Glu-D ^t 1 locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2^t and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an ω-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of ω-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F₂ grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii (Glu-D ^t 1 and Gli-D ^t 1) segregated as alleles of the Glu-D1 and Gli-D1 loci of bread wheat. A new locus encoding the ω-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively. Received: 13 October 2000 / Accepted: 18 April 2001     

Accumulation of Gliadin and Glutenin Polypeptides during Development of Normal and Sulphur-deficient Wheat Seed: Analysis using Specific Monoclonal Antibodies

A panel of monoclonal antibodies with various specificitiesfor wheat (Triticum aestivum L.) gluten polypeptides has beenused to analyse the accumulation of these polypeptides in theendosperm of developing wheat seeds grown under normal and sulphur-deficientconditions. Immunoblots of polypeptides fractionated by SDS-PAGEallowed a qualitative analysis of gliadin and HMW glutenin accumulationfor high- and low-sulphur seeds 8 d to 30 d after anthesis (d.a.a.).In addition, quantitative analysis of the deposition of variousgluten polypeptides was performed, with a solid-phase radioimmunoassayon extracts of seeds harvested 4–36 d.a.a. The initialaccumulation of HMW glutenin subunits was detectable at an earlierstage of development than that of gliadins for both normal andsulphur-deficient seeds. The initiation of detectable gliadinaccumulation was asynchronous with an order of alpha-gliadins,beta-, gamma- and some omega-gliadins and finally the remainingomega-gliadins. In sulphur deficiency, all gliadins reacheda constant proportion of the dry weight of the endosperm earlierthan in normal wheat, while a more marked increase in the proportionof HMW glutenin occurred late in grain development. The proteinblot studies also identified a putative omega-gliadin polypeptidewhich was detectable late in seed development and only in sulphur-deficientseeds. Key words: Wheat, seed maturation, immunoassay     

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     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS-PAGE检测了2份类大麦属(Crithopsis delileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2052bp．编码长度为661个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C．delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

Molecular cloning, heterologous expression, and phylogenetic analysis of a novel y-type HMW glutenin subunit gene from the G genome of Triticum timopheevi.

A novel y-type high molecular weight (HMW) glutenin subunit gene from the G genome of Triticum timopheevi (2n=4x=28, AAGG) was isolated and characterized. Genomic DNA from accession CWI17006 was amplified and a 2200 bp fragment was obtained. Sequence analysis revealed a complete open reading frame including N- and C-terminal ends and a central repetitive domain encoding 565 amino acid residues. The molecular weight of the deduced subunit was 77,031, close to that of the x-type glutenin subunits. Its mature protein structure, however, demonstrated that it was a typical y-type HMW subunit. To our knowledge, this is the largest y-type subunit gene among Triticum genomes. The molecular structure and phylogenetic analysis assigned it to the G genome and it is the first characterized y-type HMW glutenin subunit gene from T. timopheevi. Comparative analysis and secondary structure prediction showed that the subunit possessed some unique characters, especially 2 large insertions of 45 (6 hexapeptides and a nonapeptide) and 12 (2 hexapeptides) amino acid residues that mainly contributed to its higher molecular weight and allowed more coils to be formed in its tertiary structure. Additionally, more alpha-helixes in the repeat domain of the subunit were found when compared with 3 other y-type subunits. We speculate that these structural characteristics improve the formation of gluten polymer. The novel subunit, expressed as a fusion protein in E. coli, moved more slowly in SDS-PAGE than the subunit Bx7, so it was designated Gy7*. As indicated in previous studies, increased size and more numerous coils and alpha-helixes of the repetitive domain might enhance the functional properties of HMW glutenins. Consequently, the novel Gy7* gene could have greater potential for improving wheat quality.     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.     

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.     

DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat

Restriction enzyme digests of DNA from nullisomic-tetrasomic and intervarietal chromosome substitution lines of wheat were probed with a high molecular weight (HMW) glutenin cDNA. Three restriction endonucleases were used to investigate restriction-fragment differences among five wheat varieties. The results suggest that the hybridizing fragments contain single gene copies and permit the identification of the subunit encoded by each gene. Restriction-fragment variation associated with previously established allelic differences between varieties was observed. Also, there is a clear relationship between the electrophoretic mobility of a HMW subunit and the length of the central repetitive section of the gene encoding it. These results are discussed with reference to the evolution of the HMW glutenin gene family and the uses of restriction-fragment variation in plant breeding and genetics.N.P.H. was supported by a MRC Training Fellowship in Recombinant DNA Technology and a grant from the Perry Foundation. D.B. is supported by EEC Contract GBI-4-027-UK.     

小麦HMW-G12亚基基因启动子克隆及序列分析

为了研究高分子量谷蛋白基因启动子在种子中的特异性表达,以小麦品种“东农7742”的基因组DNA为模板,根据已发表序列设计并合成引物,用PCR的方法克隆了小麦贮藏蛋白中高分子量谷蛋白12亚基基因的上游调控序列。序列测定结果表明：所克隆的启动子片段大小为424bp与Thomspon报道的序列比较,同源性为97.9%,有9个核苷酸发生了改变。推测的TATA box位于-27— -30bp,Prolamin-box位于-175— -181bp,认为该元件可能与转录速率的调控有关。     

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F₂ populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.