Large-scale expression and purification of high-molecular-weight glutenin subunits

The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough. The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours. Consequently, it is difficult to purify these proteins separately in appreciable amounts. Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins. However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized. Previous studies have been analytical or small scale (5-100 ml) and in most cases only partial purity was obtained. In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter. Our results include optimization of expression conditions to increase yield and stability of proteins. Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression. We also developed a large-scale purification procedure. The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour. Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour. These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality.     

High molecular weight glutenin subunit in durum wheat (T. durum)

Summary The diversity of high molecular weight (HMW) glutenin subunits of 502 varieties of durum wheat (Triticum durum) from 23 countries was studied using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-nine types of patterns were observed with 18 mobility bands. A total of 18 alleles were identified by comparing the mobilities of their subunits to those previously found in hexaploid wheat (T. aestivum) and in Triticum turgidum var. dicoccum. Five new alleles were detected: two on the Glu A1 and three on the Glu B1 locus. Comparison of the frequency of alleles in the three species T. aestivum, T. dicoccum and T. durum was investigated. Significant differences exist between each of these species on the basis of the frequency distributions of their three and four common alleles at the Glu A1 and Glu B1 locus, respectively. The Glu B1c allele occuring very frequently in hexaploid wheats was not found in the two tetraploid species. More than 83% of the T. durum analysed were found to have the Glu A1c (null) allele.     

In vitro phosphorylation of high molecular weight glutenin subunits from wheat endosperm

We have investigated the in vitro phosphorylation of high molecular weight glutenin subunits (HMW-GS), a group of non-soluble proteins present in wheat endosperm. Computer aided searches of potential biological sites in the known sequences of these proteins have evidenced the presence of sequence motifs specific for protein kinase C (PKC), calcium-dependent protein kinase from wheat, casein kinase II, tyrosine protein kinase and glycosylation. We have demonstrated that subunit 1Bx7 is a substrate of a partially purified PKC from rat brain. Further experiments have shown that this subunit is phosphorylated by an endogenous protein kinase activity found in wheat flour. These preliminary results are important for the possible implications on the structure-function relationships of these proteins and could probably suggest, for the first time, a potential physiological role in particular situations for some HMW-GS.     

Conformational studies of wheat flour high relative molecular mass glutenin subunits by circular dichroism spectroscopy

Conformational studies of 1Dx2, 1Bx7, and 1Dy12 high relative molecular mass glutenin subunits, extracted from Alisei 1 flour, are reported. Circular dichroism (CD) spectroscopy is employed to study their conformational polymorphism induced by urea and by urea in the presence of 1% sodium dodecyl sulfate (SDS). The CD spectra indicate that SDS promotes ordered structures. The addition of urea to the SDS-acetate solution of 1Dx2, 1Bx7, and 1Dy12 subunits eliminates the effect of SDS. Its addition to the acetate solution of proteins induces conformational transitions to form a poly-L-proline II-like structure. All the changes induced by urea follow a multistep transition process that is typical of proteins consisting of different domains.     

部分小麦高分子量谷蛋白亚基组成分析

利用十二烷基硫酸钠聚丙烯胺凝胶电泳（SDS－PAGE）分析了85个小麦材料的高分子量谷蛋白亚基的构成,其结果表明：（1）目前生产中应用的优质小麦品种,大部分具有1A上的优质亚基1,1B上的14＋15／17＋18或1D上的5＋10,个别品种还同时聚合有1A,1B,1D上的优质亚基;（2）在所分析的28个八倍体小偃麦中,多数材料含有1,2^*和5＋10等优质亚基;（3）在本实验室创造的材料中,来源于中间偃麦草和普通小麦杂交的后代材料中大部分具有14＋15亚基。此外,个别种质材料还含有Payne亚基命名系统中未命名的一些稀有的高分子量谷蛋白亚基。     

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.     

Inheritance of glutenin protein subunits of wheat

Summary The inheritance of the high-molecular-weight (HMW) glutenin protein subunits in hexaploid wheat has been investigated by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis to examine the segregation of these subunits in 496 test-cross seeds. The parents of the f₁ hybrid were chosen so that the test-cross seeds segregated for all the HMW glutenin bands. Two glutenin subunits from one parent, believed to be controlled by genes on chromosome 1D, segregated as alternatives to two glutenin subunits from the other parent, a result that supports the assumption that these subunits are controlled by allelic genes at each of two loci that are very closely linked. Similar results were obtained for glutenin subunits believed to be controlled by chromosome IB, which suggests that these subunits are controlled also by allelic genes at each of two loci that are very closely linked. A single glutenin subunit band, believed to be controlled by chromosome 1A, segregated as an alternative to a single glutenin band from the other parent, except that one seed did not possess either band. It was concluded that these bands are controlled either by allelic genes or by nonallelic genes that are very closely linked.     

带芒草属物种新型高分子量谷蛋白亚基的鉴定

采用SDSPAGE方法对牧草带芒草属3个种8份材料的高分子量谷蛋白进行了检测和鉴定。结果显示,带芒草物种具有的高分子量谷蛋白亚基与普通小麦中发现的不一样,其迁移率存在较大差异。其中,x型亚基均比Dx2亚基迁移率小或接近,y型亚基均比Dx12亚基迁移率大。8份材料中共发现了4种x型亚基新类型(Tax1,Tax2,Tax3和Tax4),5种y型亚基新类型(Tay1,Tay2,Tay3,Tay4和Tay5)和6种亚基组合类型(Tax1+Tay3,Tax3+Tay2,Tax4+Tay1,Tax1+Tay1,Tax2+Tay5,Tax4+Tay2),该项研究结果揭示了带芒草属植物可能具有与普通小麦类似的高分子量谷蛋白亚基,这些亚基在小麦品质遗传改良中具有潜在的利用价值。     

小麦高分子量谷蛋白亚基对加工品质影响的效应分析

分析了 2 50份小麦材料的高分子量谷蛋白亚基 (HMW- GS)组成以及其中 66份材料的加工品质及面条制作品质。回归分析表明 :HMW- GS与 1 0种加工品质性状均有显著的线性关系。不同亚基对综合品质效应的得分大小依次为 :Glu- Al,1 >2 * >null;Glu- Bl,1 4 +1 5>7+8>1 7+1 8>>7+9;Glu- Dl,5+1 0 >>2 +1 2 >4+1 2。不同基因位点对品质的贡献大小顺序为 :Glu- Dl>Glu- Al>Glu- Bl。首次提出了 HMW- GS综合品质评分系统     

Characterization of B- and C-type low molecular weight glutenin subunits by electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry

Low molecular weight glutenin subunits (LMW-GS) are typically subdivided into three groups, according to their molecular weights and isoelectric points, namely the B-, C-, and D groups. Enriched B- and C-type LMW-GS fractions extracted from the bread wheat cultivar Chinese Spring were characterized using high performance liquid chromatography (HPLC) directly interfaced with electrospray ionization mass spectrometry and HPLC coupled off-line with matrix-assisted laser desorption/ionization mass spectrometry, in order to ascertain the number and relative molecular masses of the components present in each fraction and determine the number of cysteine residues. About 70 components were detected in each of the fractions examined by the combined use of these two techniques, with 18 components common to both fractions. Analysis of the fractions after alkylation with 4-vinylpyridine allowed determination of the number of the cysteines present in about 40 subunits. The proteins detected were tentatively classified based on the relative molecular masses and number of cysteine residues. Cross-contamination was found in both B- and C- fractions, along with the presence of D-type LMW-GS. The two fractions also contained unexpected components, probably lipid transfer proteins and omega-gliadins. The presence of extensive microheterogeneity was suggested by the detection of several co-eluting proteins with minor differences in their molecular masses.     

小麦高分子量麦谷蛋白亚基鉴定及其品质效应研究进展

高分子量谷蛋白亚基(HMW-GS,high molecular weight glutenin subunits)是小麦子粒贮藏蛋白的重要组成成分,其组成、搭配、表达水平及含量决定面团弹性和面包加工品质。本文主要介绍了小麦HMW-GS编码基因的克隆、分子特征、分子标记开发及其在小麦育种中的应用,并综述了不同HMW-GS与面粉加工品质之间的关系,以及HMW-GS基因遗传转化、微量配粉和突变体培育等方面的研究进展,分析了目前研究中存在的主要问题,认为通过分子标记辅助选择和转基因技术聚合优质亚基,培育优质面包小麦品种和明确各个HMW-GS基因的品质效应是今后的研究重点。     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.     

Comparison of repetitive sequences derived from high molecular weight subunits of wheat glutenin, an elastomeric plant protein

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.     

Variation and classification of B low-molecular-weight glutenin subunit alleles in durum wheat

 The B low-molecular-weight (LMW) glutenin subunit composition of a collection of 88 durum wheat cultivars was analyzed. Extensive variation has been found and 18 different patterns were detected. Each cultivar exhibited 4–8 subunits, and altogether 20 subunits of different mobility were identified. The genetic control of all these subunits was determined through the analysis of nine F₂ populations and one backcross. Five subunits were controlled at the Glu-A3 locus, 14 at Glu-B3 and 1 at Glu-B2. At the Glu-A3 locus each cultivar possessed from zero to three bands and eight alleles were identified. At the Glu-B3 locus each cultivar showed four or five bands and nine alleles were detected. Only one band was encoded by the Glu-B2 locus. A nomenclature for these alleles is proposed and the relationship between them and the commonly used LMW-model nomenclature is discussed. Received: 10 February 1997 / Accepted: 25 April 1997     

Heterologous expression of new antifungal chitinase from wheat

Chitinases (EC 3.2.1.14) have been grouped into seven classes (class I-VII) on the basis of their structural properties. Chitinases expressed during plant-microbe interaction are involved in defense responses of host plant against pathogens. In the present investigation, chitinase gene from wheat has been subcloned and overexpressed in Escherichia coli BL-21 (DE3). Molecular phylogeny analyses of wheat chitinase indicated that it belongs to an acidic form of class VII chitinase (glycosyl hydrolase family 19) and shows 77% identity with other wheat chitinase of class IV and low level identity to other plant chitinases. The three-dimensional structural model of wheat chitinase showed the presence of 10 alpha-helices, 3 beta-strands, 21 loop turns and the presence of 6 cysteine residues that are responsible for the formation of 3 disulphide bridges. The active site residues (Glu94 and Glu103) may be suggested for its antifungal activity. Expression of chitinase (33 kDa) was confirmed by SDS-PAGE and Western hybridization analyses. The yield of purified chitinase was 20 mg/L with chitinase activity of 1.9 U/mg. Purified chitinase exerted a broad-spectrum antifungal activity against Colletotrichum falcatum (red rot of sugarcane) Pestalotia theae (leaf spot of tea), Rhizoctonia solani (sheath blight of rice), Sarocladium oryzae (sheath rot of rice) Alternaria sp. (grain discoloration of rice) and Fusarium sp. (scab of rye). Due to its innate antifungal potential wheat chitinase can be used to enhance fungal-resistance in crop plants.     

Allelic variation of high molecular weight glutenin subunits of bread wheat in Hebei province of China

In common wheat (Triticum aestivum L.), allelic variations of Glu-1 loci have important influences on grain end-use quality. The allelic variations in high molecular weight glutenin subunits (HMW-GSs) were identified in 151 hexaploid wheat varieties representing a historical trend in the cultivars introduced or released in Hebei province of China from the years 1970s to 2010s. Thirteen distinct alleles were detected for Glu-1. At Glu-A1, Glu-B1 and Glu-D1, we found that the most frequent alleles were the 1 (43.0%), 7+8 (64.9%), 2+12 (74.8%) alleles, respectively, in wheat varieties. Twenty two different HMW-GS compositions were observed in wheat. Twenty-five (16.6%) genotypes possessed the combination of subunits 1, 7+8, 2+12, 25 (16.6%) genotypes had subunit composition of 2*, 7+8, 2+12; 20 (13.2%) genotypes had subunit composition of null, 7+8, 2+12. The frequency of other subunit composition was less than 10%. The Glu-1 quality score greater than or equal to 9 accounted for 20.6% of the wheat varieties. The percentage of superior subunits (1 or 2* subunit at Glu-A1 locus; 7+8, 14+15 or 17+18 at Glu-B1 locus; 5+10 or 5+12 at Glu-D1 locus) was an upward trend over the last 40 years. The more different superior alleles correlated with good bread-making quality should be introduced for their usage in wheat improvement efforts.     

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

Summary The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except ‘NapHal’ contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F₁ of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.     

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

Summary The genes controlling the synthesis of the high-molecular-weight subunits of glutenin on the long arms of chromosomes 1A and IB were mapped to the -gliadin genes on the short arms by analysing the progeny of three test crosses by sodium dodecyl sulphate, polyacrylamide-gel electrophoresis. Only very weak linkages were detected: the percentage recombination ranged from 39% to 47% and as the values did not significantly differ from each other, the data was pooled. A mean recombination of 43% was obtained and the map distance between glutenin and gliadin genes was calculated to be 66 cM. The analysis of three crosses involving telocentric lines revealed that the glutenin subunit genes on chromosomes 1A, IB and ID are tightly linked to the centromere, the mean map distance being 9.0 cM.     

Structural homology of endosperm high molecular weight glutenin subunits of common wheat (Triticum aestivum L.)

Summary Several high molecular weight endosperm glutenin subunits, coded by genes located on chromosomes 1A, 1B and 1D of common wheat, Triticum aestivum L. em. Thell., were isolated from excised gel segments and subjected to amino acid analysis and peptide mapping; the latter was carried out following a limited digestion with trypsin, chymotrypsin or Staphylococcus aureus — V8 protease. Generally, all high molecular weight glutenins had a similar amino acid composition but several significant differences were observed in some of them. Both analyses revealed that the structural similarity among the various subunits was related to the homology of the genes coding them: subunits coded by homoalleles, i.e., different alleles of the same gene, were most similar; those coded by homoeoalleles, i.e., alleles of homoeologous genes, were less similar; whereas subunits coded either by alleles of different genes of the same gene cluster, or by nonhomoeoalleles of homoeologous clusters, were the least similar. Several small peptides derived from protease digestion of various subunits had a higher than expected staining intensity indicating that small peptide repeats may be interspersed within the glutenin subunits. The evolutionary course of the high molecular weight glutenins is discussed.