麦谷蛋白亚基对小麦品质特性的影响及其遗传转化

麦谷蛋白是小麦籽粒贮藏蛋白的重要组成部分,其亚基类型及组成与小麦品质密切相关,为了更进一步了解其与小麦蛋白质品质特性的关系,综合论述了小麦谷蛋白亚基的分类、结构及其不同亚基与小麦品质的关系,并对利用基因工程遗传转化方法改变小麦谷蛋白亚基组成从而提高小麦加工品质的现状及应用前景进行了讨论。     

Effects of post-anthesis fertilizer on the protein composition of the gluten polymer in a US bread wheat

Both genetic and environmental factors influence the types and amounts of wheat proteins that link together to form polymers essential for flour quality. To understand how plant growth conditions might influence gluten polymer formation, protein fractions containing small and large polymers were separated from flour from the US wheat Butte 86 grown in the absence or presence of post-anthesis fertilizer. Proteins in the polymer fractions were analyzed by quantitative two-dimensional gel electrophoresis (2-DE). The ratio of high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS) increased in both fractions in response to fertilizer, due in part to small increases in the proportions of individual HMW-GS. There were also changes within the LMW-GS. In particular, omega and alpha chain terminators increased in proportion in both polymer fractions, but changes were more pronounced in the large polymer fractions. Serpins also increased in both polymer fractions. Additionally, the study revealed differences in the proportions of traditional LMW-GS in small and large polymer fractions. LMW-s type proteins were more abundant in the large polymers while LMW-i type proteins were more prevalent in the small polymers, suggesting that these proteins may play different roles in the gluten polymer.     

Correspondence between two minor Glu-A3 genes of durum wheat and their encoded polypeptides by using a proteomic approach

The low molecular weight glutenin subunits (LMW-GS) are wheat storage proteins participating to the formation of glutenin polymers that, along with the other gluten proteins, allow the accumulation of a large quantity of protein in the endosperm tissue. The size and composition of the glutenin polymers are directly related to gluten visco-elastic properties. In particular, LMW-GS composition is the factor most influencing durum wheat quality.     

Characterization of seed proteome in Brachypodium distachyon

Brachypodium distachyon, an emerging model plant system for some economically important temperate grasses such as wheat, barley and switchgrass, has recently caught wide attention in modern biological research. In the current study, the glutenin, albumin and globulin components of 13 B. distachyon accessions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) followed by peptide mass finger printing (PMF) and MS/MS protein identification. Abundant wheat low molecular weight glutenin subunit (LMW-GS) like proteins and a few high molecular weight glutenin subunits (HMW-GS) with low expression level were detected in B. distachyon. A total of 18 storage proteins and 15 albumin proteins were identified through PMF and MS/MS. The results demonstrated that the major seed storage proteins in B. distachyon are wheat LMW-GS like proteins and globulins. The identified albumins and globulins were mostly various enzymes that were classified into five groups according to their functions. The 2-DE spot distribution and MS results suggested that post-translational modifications (PTMs) such as phosphorylations and glycosylations are common phenomena in B. distachyon seed proteome.     

Characterisation of an s-type low molecular weight glutenin subunit of wheat and its proline and glutamine-rich repetitive domain

The low molecular weight glutenin subunits (LMW-GS) are major components of the glutenin polymers which determine the elastomeric properties of wheat (Triticum aestivum L.) gluten and dough. They comprise a complex mixture of components and have proved to be difficult to purify for detailed characterisation. The mature LMW subunit proteins comprise two structural domains, with one domain consisting of repeated sequences based on short peptide motifs. DNA sequences encoding this domain and a whole subunit were expressed in Escherichia coli and the recombinant proteins purified. Detailed comparisons by spectroscopy (CD, FT-IR) and dynamic light scattering indicated that the repetitive and non-repetitive domains of the proteins formed different structures with the former having an extended conformation with an equilibrium between poly-L-proline II-like structure and type II' β-turns, and the latter a more compact globular structure rich in α-helix. Although the structures of these two domains appear to form independently, dynamic light scattering of the whole subunit dissolved in trifluoroethanol (TFE) suggested that they interact, leading to a more compact conformation. These observations may have relevance to the role of the LMW-GS in gluten structure and functionality.     

Wheat seed storage proteins: Advances in molecular genetics,diversity and breeding applications

Wheat seed storage proteins, especially glutenins and gliadins, have unique functional properties giving rise to a wide array of food products for human consumption. The wheat seed storage proteins, however, are also the most common cause of food-related allergies and intolerances, and it has become crucially important to understand their composition, variation and functional properties and interface this knowledge with the grain handling industry as well as the breeders. This review focuses on advances in understanding the genetics and function of storage proteins and their application in wheat breeding programs. These include: (1) The development and validation of high-throughput molecular marker systems for defining the composition and variation of low molecular weight glutenin subunits (LMW-GS) genes and a summary of the more than 30 gene-specific markers for rapid screening in wheat breeding programs; (2) The identification of more than 100 alleles of storage proteins in wild species provide candidate genes for future quality improvement; (3) The documentation of quality effects of individual LMW-GS and HMW-GS for improving end-use quality; and (4) The analysis of α-gliadin genes on chromosomes 6A and 6D with non-toxic epitopes as potential targets to develop less toxic cultivars for people with celiac disease. Genomic and proteomic technologies that will continue to provide new tools for understanding variation and function of seed storage proteins in wheat are discussed.     

The gluten protein and interactions between components determine mixograph properties in an F6 recombinant inbred linepopulation in bread wheat

One hundred and sixty-eight F₆ recombinant inbred lines (RILs) derived from Chinese wheat cultivars, PH82-2 and Neixiang188, were used to determine the cumulative effects of HMW-GS and LMW-GS composition and quantity of gluten protein fractions on dough mixograph properties. A wide range of variation for all parameters in the RILs was detected. Major gene loci of HMW-GS were associated with variation in mixograph characters, but accounted for no more than 25.3% of the phenotypic variations. Glu-D1, together with Glu-B3, played the most important role in determining the properties. Additive effects of HMW-GS and LMW-GS showed major contributions to most of the variation of mixograph parameters, and epistatic effects were also important and could be counter to additive effects of individual loci. The quantity of gluten protein fractions, especially the quantity of glutenin, LMW-GS, and Glu-B3, showed highly significant correlations with most of the quality parameters, but the correlation coefficients were influenced by grain hardness, protein content, or both. Protein quality could be greatly improved through increasing the quantity of glutenin, while holding desirable composition of HMW-GS and LMW-GS alleles, with an appropriate ratio of quantity of glutenin to gliadin.     

High throughput microchip-based separation and quantitation of high-molecular-weight glutenin subunits

Knowledge of glutenin subunit composition is important for the prediction of the genetic potential of breeding lines as these proteins are known to be responsible for the main differences in bread-making quality. In this study, a commercial high throughput microchip capillary electrophoresis-sodium dodecyl sulfate (microchip CE) platform, LabChip 90, was evaluated for qualitative and quantitative analyses of HMW-GS. 130 French common wheat varieties of known composition were analyzed for rapid identification and the allocation of individual HMW-GS. In addition, the HMW-GS were individually quantified and the ratio of HMW-GS to LMW-GS was determined for genotype comparison. The microchip CE analysis provides comparable resolution and sensitivity to conventional RP-HPLC for identification of the HMW-GS but at a time scale of approximately 100 times faster (45 s per sample analysis versus 80 min for RP-HPLC). The results show that the high throughput microchip CE method can be used for routine identification and quantitation of glutenin subunits, in particular for screening wheat quality and wheat cultivar development activities where large numbers of samples are to be evaluated.     

Molecular characterization of storage proteins for selected durum wheat varieties grown in different environments

The variations of the amounts of individual high molecular weight glutenin subunits (HMW-GS), of the ratios HMW-GSy to HMW-GSx and HMW-GS to low molecular weight glutenin subunits (LMW-GS) and of protein content were evaluated for eight durum wheat cultivars in two regions using four fertilizer combinations during two successive years. All measured parameters showed significant variation with genotypes (G), environments (E) and fertilizers (F). The interaction E × G × F was highly significant for glutenin amount variation. Amongst cultivars possessing HMW-GS 20, landraces seem to better value the N-fertilizer use for the accumulation of HMW-GSy than high yielding cultivars. Both HMW-GSy to HMW-GSx and HMW-GS to LMW-GS ratios were found to be positively correlated (p < 0.05) with total protein content.     

Characterisation of a specific class of typical low molecular weight glutenin subunits of durum wheat by a proteomic approach

Glutenin polymers are formed by high (HMW-GS) and low molecular weight glutenin subunits (LMW-GS). The latter group of subunits has been less characterised compared to the former due to their great number and heterogeneity.     

Simultaneous Isolation of Wheat High Molecular Weight and Low Molecular Weight Glutenin Subunits

A procedure was established for isolating simultaneously high molecular weight (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) from chloroform-defatted wheat flour. Temperature conditions were optimised to obtain maximum yield and purity. A pre-extraction of gliadin was performed with 50% (v/v) propan-1-ol at room temperature. Glutenin subunits were solubilised from the residue at 60 °C with 50% (v/v) propan-1-ol containing 1% (w/v) dithiothreitol. The HMW-GS in the glutenin mixture were then precipitated selectively by increasing the propan-1-ol concentration to 60%. Upon addition of propan-1-ol to a final concentration of 85%, LMW-GS were precipitated. It was shown by densitometric scanning of SDS–PAGE gels that the isolated glutenin subunits were very pure. Furthermore, a partial separation of LMW-GS into B and C type subunits was observed when the propan-1-ol concentration was increased gradually.     

Significance of LMW-GS and HMW-GS for Dough Extensibility: «Addition» versus «Incorporation» Protocols

Low molecular weight (LMW-GS) and high molecular weight glutenin subunits (HMW-GS) were added to a base flour using both «addition» and «incorporation» protocols. «Incorporation» of glutenin subunits into the glutenin network of the base flour was performed by partial (reversible) reduction and subsequent reoxidation of the glutenin network in the presence of the added glutenin subunits whereas, in the «addition» protocol, glutenin subunits were added without reduction/oxidation. The effects of both «addition» and «incorporation» of alkylated and unalkylated LMW-GS and HMW-GS on dough extension parameters maximum resistance (MR) and extensibility (EX) were compared and thoroughly discussed. HMW-GS and LMW-GS had totally different effects on dough extensibility. «Addition» of LMW-GS significantly decreased both MR and EX whereas HMW-GS caused a significant increase in MR. «Incorporation» of LMW-GS caused a decrease in MR whereas HMW-GS clearly increased MR. The similarity in effects obtained with «addition» and «incorporation» of glutenin subunits indicated that, even with «addition», glutenin subunits can be partially incorporated into the glutenin network in the presence of oxygen. Alkylated and unalkylated glutenin subunits had different effects. This was probably caused by the effect of free sulphydryl groups in unalkylated subunits (possibility of SS/SH exchanges and/or incorporation) and/or the effect induced by introduction of alkylated derived substituents. A protocol for «incorporation with excess KIO₃» was developed to exclude the possible effect of a lowering of the available oxidant concentration by oxidation of free sulphydryl groups in glutenin subunits. However, the use of high levels of oxidant in the «incorporation with excess KIO₃» protocol seems to overrule the effects of added glutenin subunits or may force glutenin subunits to incorporate differently from what can be observed under gentle oxidation conditions. Therefore, «incorporation with excess KIO₃» is not suitable for studying the effects of incorporation of glutenin subunits on dough extensibility.     

宁夏引黄灌区冬小麦谷蛋白亚基组成和1BL/1RS易位分析及品质性状研究

为全面了解宁夏引黄灌区冬小麦品质概况,为冬小麦品质改良和粮食生产提供理论依据。选用冬小麦主栽品种、亲本材料和高代品系30份。用SDS-PAGE分析了其中18份材料的HMW—GS、LMW—GS组成和1BL／1RS易位系分布状况。并对23个冬小麦品种的营养与加工品质进行了研究。结果表明,2#、7＋9、2＋12、Glu-A3a、Glu-A3c、Glu-B3h和Glu—B3j在宁夏引黄灌区冬小麦中分布较广,1BL／1RS易位系分布相当普遍。分布频率为27．8％。参试品种的籽粒硬度、面粉PPO活性、SDS沉淀值、形成时间、稳定时间和评价值的变异范围较大,变异系数分别为25．37％、33．68％、21．42％、26．58％、43．95％和29．31％。多数冬小麦品种（系）的千粒重、出粉率和湿面筋含量低于对照宁春4号。而蛋白质含量、SDS沉淀值、吸水率和稳定时间优于宁春4号。供试品种（系）中。HMW-GS品质评分、籽粒硬度、蛋白质含量、SDS沉淀值均较高,并且不含1BL／1RS易位系的有烟优361、济麦20、鲁875067和923—9等,可用于宁夏引黄灌区冬麦品质改良。     

Characterization of HMW glutenin subunits in common wheat and related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The sample preparation method of high molecular weight glutenin subunits (HMW-GS) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, without a separation step, by high-performance liquid chromatography (HPLC) was established in this study. Three major factors influencing mass spectra—the ratio of components of the solvent, the resolving time, and the sample volume—were optimized using HMW-GS mixtures extracted from Chinese cultivar Jing 411. The results showed that the optimized method for sample preparation was to resolve HMW-GS from 20 mg in an hour with 50 μl solvent of 0.4% TFA, 30.0% ACN and 69.6% H₂O. The stable mass spectra and accurate molecular weights of 16 major HMW glutenin subunits from common wheat and related species were obtained using the optimized MALDI-TOF-MS method. Seven subunits, where each was from 2–5 cultivars, showed very similar molecular weights. The determined molecular weights of 11 subunits were close to those calculated from their coding sequences. In addition, no positive reaction between HMW-GS and GelCode^® Glycoprotein Staining Reagent was observed. These results suggested that HMW-GS lack extensive post-translational modifications (PTMs), but low levels of glycosylation or phosphorylation present in some subunits cannot be ruled out. Because of its ability to obtain a rapid, complete and precise profile of HMW glutenin subunits without purifying procedures, MALDI-TOF-MS is expected to be a powerful technique for structural and functional studies of HMW glutenin subunits as well as other cereal proteins.     

中国小麦品种高分子量谷蛋白亚基和低分子量谷蛋白亚基组成分析

为给我国优质小麦品种的选育和改良提供科学依据,应用SDS-PAGE方法对我国235份推广品种和高代品系的HMW-GS和LMW-GS组成与分布进行了分析。结果表明,HMW-GS和LMW-GS分别具有39和40种带型组合;5个位点共发现34个等位基因。Glu-A1、Glu-B1和Glu-D1分别有3、8和6个变异位点;亚基1、Null、7＋9、5＋10和2＋12是主要的HMW-GS类型,频率分别为46．2％、46．2％、44．5％、47．0％和48．3％。Glu-A3和Glu-B3位点（本文不涉及Glu-D3）分别具有6和11个变异位点。Glu-A3c、Glu-A3d、Glu-B3e和Glu-B3j是主要的LMW-GS类型,频率分别为55．1％、21．6％、28％和28．8％。本研究还发现,在品种Z75和绵阳96-319的Glu-D1位点上,亚基组合形式为5＋12;在品种周92034的Glu-D1位点,亚基组合形式为2＋10。虽然这两种组合形式在本研究中出现的频率很低,但这两种亚基组合在以前的研究中很少出现。     

Mixing properties and dough functionality of transgenic lines of a commercial wheat cultivar expressing the 1Ax1, 1Dx5 and 1Dy10 HMW glutenin subunit genes

In this work we report the effects of the HMW-GS 1Ax1, 1Dx5 and 1Dy10 on the breadmaking quality of the bread wheat cultivar Anza that contains the HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8, and is null for the Glu-A1 locus. This allows the characterization of individual subunits 1Dx5 and 1Dy10 in the absence of subunit 1Dx5, and the interactions between these subunits and subunits 1Dx2 and 1Dy12 to be determined. Three transgenic lines termed T580, T581 and T590, containing, respectively, the HMW-GS 1Ax1, 1Dx5 and 1Dy10 were characterized over 3 years using a range of widely-used grain and dough testing methods. The transgenic subunits 1Ax1, 1Dx5 and 1Dy10 accounted for 25.2%, 20.3% and 17.9%, respectively, of the total HMW-GS in the three transgenic lines. Although lines T581 and T590 expressed similar levels of subunits 1Dx5 and 1Dy10 they had different effects on other aspects of protein composition, including changes in the ratios of glutenin/gliadin, of HMW/LMW-GS, the 1Dx2/1Dy12, the x-type/y-type HMW-GS and the proportions of high molecular mass glutenin polymers. In contrast, lines transformed to express subunits 1Ax1 and 1Dx5 showed similar changes in protein composition, with higher protein contents and decreased ratios of glutenin/gliadin and 1Dx2/1Dy12. In addition, both transgenic lines showed similar increases in the ratio of x-type/y-type subunits compared to the control line. The transgenic lines were analysed using Farinograph, Mixograph and Alveograph. This confirmed that the expression of all three subunits resulted in increased dough strength (and hence breadmaking quality) of the cultivar Anza. A beneficial effect of subunit 1Dx5 has not been reported previously, transgenic wheat lines expressing this subunit giving overstrong dough unsuitable for breadmaking. However, the expression of subunit 1Dy10 had a greater effect on breadmaking quality than subunits 1Ax1 and 1Dx5. The Farinograph parameters such as dough stability and peak time were increased by 9.2-fold and 2.4-fold, respectively, in line T590 (expressing 1Dy10) with respect to the control line. Similarly, the Mixograph mixing time was increased by four-fold and the resistance breakdown decreased by two-fold in line T590 compared with the control line. The Alveograph W value was also increased by 2.7-fold in line T590 compared to the control line. These transgenic lines are of value for studying the contribution of specific HMW-GS to wheat flour functional properties.     

HMW-GS affect the properties of glutenin particles in GMP and thus flour quality

Using a unique set of deletion lines, (Olympic×Gabo, varying in high molecular weight glutenin subunit (HMW-GS) composition, but with the same genetic background) it was shown that the presence of glutenin particles in glutenin macropolymer (GMP) is directly related to the presence of certain HMW-GS. In the absence of HMW-GS only a small amount of insoluble glutenin protein (GMP) could be recovered from the flour that contained only LMW-GS. No particles were observed in this fraction. When one subunit (HMW-GS Glu-Ax1) was present some particles could be observed, but when two or more HMW-GS were present particles could be clearly identified. The amount of GMP increased with the increasing number of HMW-GS. All particles had the same LMW-GS composition irrespective of HMW-GS-composition. Since the relative proportion of LMW-GS in GMP was dependent on the number of HMW-GS, we postulate that LMW-GS become part of GMP through disulfide cross-linking with HMW-GS. GMP wet weight is correlated with the average HMW density of the glutenin particle. These data were combined with previously published technological data from the same set of wheats. Significant statistical relationships were observed between optimal mixing time and glutenin particle size and between thimble-loaf height and GMP content. Taken together, these studies suggest that glutenin HMW-GS composition affects flour technological properties through glutenin particle size.     

Wx-1基因变异和优质HMW-GS组合对中筋小麦品质的影响

高分子量谷蛋白亚基(HMW-GS)和Wx蛋白是两个影响小麦理化品质和制成品品质的重要因素。为研究HMW-GS和Wx基因变异对小麦理化品质、面条感官评分和质构特性的影响,以镇麦9号和扬糯麦1号为亲本的2个高代系为试验材料,研究了优质HMW-GS 5+10及Wx基因缺失对小麦理化品质及面条加工品质的效应。结果表明,品系1与品系2的湿面筋含量和SDS沉淀值与镇麦9号相近,但显著好于扬糯麦1号。相较于镇麦9号,品系1与品系2的直链淀粉含量分别降低了1.5%和2.5%,其峰值黏度和稀懈值均显著高于镇麦9号,膨胀势也高于镇麦9号。5+10亚基显著提高了面条质构参数中的硬度和咀嚼性,而Wx基因缺失显著提高了面条的软硬度评分和光滑性评分。综合来看,具有5+10亚基和 Wx-D1缺失型的品系2具有较高的SDS沉淀值、面团形成时间和稳定时间、较低的直链淀粉含量、较高的峰值黏度、稀懈值和膨胀势,蛋白质和淀粉综合品质表现较好。品系2面条总评分最高,显著高于镇麦9号和扬糯麦1号,其主要体现在适中的面条软硬度和较好的光滑性。推测Wx基因缺失和优质HMW-GS聚合可显著提高小麦淀粉品质和蛋白质品质,有效改善面条的蒸煮品质和感官评分。     

Ultra-fast separation of wheat glutenin subunits by reversed-phase HPLC using a superficially porous silica-based column

A relatively new, unique column packing material for reversed-phase high-performance liquid chromatography (RP-HPLC) was evaluated for rapid separation of wheat glutenin protein subunits. The product named “Poroshell” by the manufacturer consists of a solid core and a porous coat instead of solid silica spheres used in conventional RP-HPLC column packing. This architecture favours rapid mass transfer, facilitating faster reversed-phase separations of biomolecules compared to conventional silica columns. The main objective of this study was to evaluate the quality of separations of glutenin subunits (GS), as well as to optimize conditions to produce the fastest possible run times without sacrificing resolution using a Poroshell 300SB-C₈ 2.1×75 mm column. The stability of GS separations over time was also assessed. Two different bread wheat genotypes were used for optimization of separation conditions and six more common and durum wheat genotypes possessing different subunit combinations were used for further evaluation. Glutenin protein was extracted with 0.08 M Tris–HCl buffer (pH 7.5) containing 50% 1-propanol under reducing conditions after pre-extraction of soluble proteins with 50% 1-propanol. Optimization of GS resolution and sample throughput by RP-HPLC was assessed in response to variation in eluent flow rate, acetonitrile (ACN) gradient, and column temperature. The best resolution of both HMW- and LMW-GS was obtained in 13 min using a 23–44% ACN gradient with a flow rate of 0.7 mL/min at 65 °C. Subunit elution times and integrated areas were highly repeatable even after several hundred injections. Highly satisfactory separation of HMW-GS and quantification of ratio of HMW- to LMW-GS were achieved in less than 4 min per sample using a modified HPLC gradient. Ratio of HMW- to LMW-GS was unaffected by the speed of the separations. As well, the elution order of HMW- and LMW-GS was unaffected by the rapid analysis, compared to conventional RP-HPLC separations, so no new learning was required for interpreting chromatograms and classification of subunits. The rapid RP-HPLC method using the Poroshell column appears to be very well suited for routine quantification of HMW-GS and LMW-GS especially for purposes of wheat quality screening and wheat cultivar development activities where large numbers of samples are typically encountered.