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。     

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。     

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.     

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.     

Absence of subunits in high-molecular weight proteins (spectrin) of red cell membranes

It is shown that the high-molecular weight proteins (spectrin), which make up a large part of the total protein of the red cell membrane, are single polypeptide chains. They do not generate smaller subunits on treatment with dilute acid: it is shown that recent observations of the appearance of electrophoretic components of low molecular weight, following incubation in acid solution, and interpreted in terms of a subunit structure for the spectrin chains, can be attributed to degradation by endogenous proteases. It is shown that the experimental conditions used favour such degradation.     

Characterization of the genes coding for the high molecular weight glutenin subunits in Lophopyrum elongatum

In order to reveal the evolution of Glu-E1 loci of Lophopyrum elongatum (Host) A. L?ve and find novel alleles for wheat quality breeding, four Glu-1 alleles were isolated and characterized via genomic PCR, from this wheat-related species. Of them, 1Ex2 and 1Ey2 were novel alleles, which differ from all the previously known HMW-GS alleles of L. elongatum. Two alleles 1Ex1 and 1Ey2, which contain intact open reading frames, have been successfully expressed in E. coli. The expressed proteins showed similar electrophoresis mobility with the candidate high molecular weight glutenin subunit bands found in seeds. Sequence alignment indicated that proteins encoded by the novel alleles showed similar primary structure with those of wheat and other wheat-related grasses, however, they possess some unique modifications in their own structure. For example, the number of residues in the N-terminal domain is different from those of wheat, an irregular tripeptide present between two nonapeptide motifs and a unique cysteine position in the repetitive region. Phylogenetic analyses using N-terminal conserved sequences showed that 1Ex2 was homologous to those from the D genome; but 1Ey2 was homologous to a y-type allele 1Ky from the K genome. The evolution relationship of Glu-E1 alleles and the possible utilization of the alleles in wheat breeding are discussed.     

Gluten characteristics imparting bread quality in wheats differing for high molecular weight glutenin subunits at Glu D1 locus

Physiology and Molecular Biology of Plants - High yielding genotypes differing for high molecular weight glutenin subunits at Glu D1 locus in national wheat programme of India were examined for...     

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。     

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     

Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats

Synthetic hexaploid wheats (2n=6x=42, AABBDD) involving genomes from Triticum turgidum (2n= 4x=28, AABB) and Aegilops tauschii (2n=2x=14, DD) have been produced as a means for introducing desirable characteristics into bread wheat. In the present work we describe the genetic variability present at the Glu-D ^t 1 and Glu-D ^t 3 loci, encoding high- (HMW) and low-molecular-weight (LMW) glutenin subunits respectively, derived from Ae. tauschii, using electrophoretic and chromatographic methods, in a collection of synthetic hexaploid wheats. A wide variation both in mobility and surface hydrophobicity of HMW glutenin subunits was observed between different accessions of Ae. tauschii used in the production of the synthetic hexaploids. A combination of electrophoretic and chromatographic methods improves the identification of HMW glutenin subunits; in fact subunits with identical apparent mobility were revealed to have a different surface hydrophobicity by reversed-phase high performance liquid chromatography. None of the Dx5^t subunits present in Ae. tauschii showed the presence of the extra cysteine residue found in the HMW glutenin subunit Dx5 of Triticum aestivum, as revealed by selective amplification with polymerase chain reaction (PCR). The wide variability and the high number of subunits encoded by the Glu-D ^t 3 locus suggests that Ae. tauschii may be a rich source for enhancing the genetic variability of glutenin subunits in bread wheat and improving bread-making properties. Received: 3 March 2001 / Accepted: 23 March 2001     

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.     

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.     

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.     

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.     

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.     

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.     

Homocysteinylation score of high-molecular weight plasma proteins

Ultrafiltration rates (filterability) of protein-bound homocysteine (Hcy), unlike glutathione (Glt), are significantly decreased in patients with cardiovascular disorders, end-stage renal disease, and prothrombotic conditions. Reduced filterability of Hcy through 300,000 MWCO PES membranes in these groups of patients was observed, regardless of hyperhomocysteinemia (HHcy) degree. Filterability of Hcy, but not of glutathione, was impaired in plasma preparations. It is possible that the patients with impaired filterability of Hcy in mixed disulfide state are characterized by increased ability of partial Hcy retention by proteins and complexes with higher than albumin molecular weight. These findings led us to conclude that evaluation of protein-bound Hcy filterability may provide more complete diagnostic data, to interpret clinical significance of HHcy. The proposed Hcy filterability test may be performed as a simple laboratory procedure, in addition to conventional tests for total Hcy.     

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.     

Development of haplotype-specific molecular markers for the low-molecular-weight glutenin subunits

Low-molecular-weight glutenin subunits (LMW-GSs) are one of the major components of gluten, and their allelic variation has been widely associated with different wheat end-use quality parameters. These proteins are encoded by multigene families located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3); the genes at each locus are divided by large intergenic and highly recombinogenic regions. Among the methods used for the LMW-GS allele identification, polymerase chain reaction (PCR)-based molecular markers have the advantages of being simple, accurate, and independent from the plant stage of development. However, the available LMW-GS molecular markers are either incapable of capturing the complexity of the LMW-GS gene family or difficult to interpret. In the present study, we report the development of a set of PCR-based molecular markers specific for the LMW-GS haplotypes present at each Glu-3 locus. Based on the LMW-GS gene sequences available in GenBank, single nucleotide polymorphisms (SNPs) specific for each Glu-3 haplotype were identified and the relevant PCR primers were designed. In total, we developed three molecular markers for the Glu-A3 and Glu-B3 loci, respectively, and five molecular markers for the Glu-D3 locus. The markers were tested on 44 bread wheat varieties previously characterized for their LMW-GS genic profile and found to be equally or more efficient than previously developed LMW-GS PCR-based markers. This set of markers allows an easier and less ambiguous identification of specific LMW-GS haplotypes associated with gluten strength and can facilitate marker-assisted breeding for wheat quality.