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.     

Introgression of transgenes into a commercial cultivar confirms differential effects of HMW subunits 1Ax1 and 1Dx5 on gluten properties

Transgenes encoding the HMW subunits 1Ax1 and 1Dx5 have been transferred from “model” wheat lines into the commercial French bread wheat cultivar Soissons, using three backcrosses. Five pairs of BC3 expressing and null lines were isolated from each cross and multiplied to provide grain for functionality studies. Analysis of white flour samples confirmed the expression of the transgenes. SE-HPLC and Reomixer studies showed that the two transgenes had differential effects on dough functional properties. Thus, subunit 1Dx5 resulted in detrimental effects on dough development which were associated with decreased extractability of large glutenin polymers. In contrast, lines expressing subunit 1Ax1 contained increased proportions of extractable large glutenin polymers with three lines showing higher torque at similar mixing times (i.e. increased dough strength). This confirms the results obtained with the model wheat lines and shows that the 1Ax1 transgene can be used to increase dough strength in commercial cultivars.     

Stacking HMW-GS transgenes in bread wheat: Combining subunit 1Dy10 gives improved mixing properties and dough functionality

We have determined the technological properties of four lines containing combinations of three HMW-GS transgenes, encoding HMW-GS 1Ax1, 1Dx5 and 1Dy10. These lines were produced by conventional crossing of three single transgenic lines of the bread wheat cultivar Anza that contains the endogenous HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8 and is null for the Glu-A1 locus. Consequently, the total number of HMW-GS ranged from 4 in the control line Anza to 7 in line T618 which contains all three HMW-GS transgenes. The lines were studied over two years using a range of widely used grain and dough testing methods. All lines with transgenic subunits showed higher levels of glutenin proteins than the Anza control, and these differences were highly significant for lines T616, T617 and T618, containing, respectively, the transgenes encoding HMW-GS 1Ax1 and 1Dy10, 1Dx5 and 1Dy10 and 1Ax1, 1Dx5 and 1Dy10. These increases in glutenin levels are compensated by lower levels of gliadins present in transgenic lines. These changes affected the ratio of polymeric to monomeric gluten proteins (poly:mono), the ratio of HMW-GS to LMW-GS (HMW:LMW) and the contents of individual 1Ax, 1Bx, 1By, 1Dx and 1Dy subunits. Transgenic lines expressing subunit 1Dy10 together with x-type subunits (T616, T617 and T618) were superior to line T606, which had only increases in x-type subunits. In particular, the combination of transgenic subunits 1Dx5 and 1Dy10 (line T617) gave better dough rheological properties than the other combinations of transgenic subunits. For example, dough development time and stability were increased by 3.5-fold and 8.5-fold, respectively, while the mixing tolerance index (MTI) was decreased by 3.3-fold in line T617 with respect to the control line. Alveograph analyses showed that all four transgenic combinations had increased P values compared to the Anza control but subunit 1Dx5 greatly reduced the extensibility (L). These results show that stacking HMW-GS transgenes by conventional crossing is a valid strategy for the improvement of wheat quality, with different effects being related to the different HMW-GS combinations.     

Milling and baking properties of field grown wheat expressing HMW subunit transgenes

Milling and baking tests were carried out on three transgenic wheat lines and their parental varieties grown in the field at two UK sites. The transgenic and control lines were essentially similar in their milling properties but the subunit 1Ax1 and 1Dx5 transgenes had different effects on breadmaking. The subunit 1Dx5 transgene resulted in a low loaf volume and poor crumb structure when expressed in lines with two or five endogenous HMW subunits, and this was accompanied by a greatly increased elastic modulus of the gel protein fraction. In contrast, the 1Ax1 transgene resulted in improved breadmaking quality and a more modest increase in the gel protein elastic modulus when expressed in the two subunit background. Blending of flour from a line expressing the 1Dx5 transgene with flour from a normal breadmaking wheat variety resulted in decreased breadmaking quality, even at a ratio of 1:9. The difference in the results obtained with the 1Ax1 and 1Dx5 transgenes may relate to the presence of an additional cysteine residue in the protein encoded by the latter, which promotes a more highly cross-linked glutenin network.     

Expression of an extended HMW subunit in transgenic wheat and the effect on dough mixing properties

Wheat line L88-31 was transformed with a gene encoding an extended form of subunit 1Dx5 to study the relationship between subunit size and the effect on dough mixing properties. Four transgenic lines were recovered, one of which expressed a truncated form of the protein with mobility between those of the wild type and extended subunits. Comparison of the Mixograph profiles and gluten protein compositions with those of the control lines and a line expressing the wild type subunit 1Dx5 transgene showed that two of the transgenic lines had poor mixing properties and that this was associated with co-suppression of HMW subunit gene expression. The other two transgenic lines had improved mixing properties (measured as increased mixing time) and this was associated with increased proportions of large glutenin polymers. None of the transgenic lines expressing the extended form of the 1Dx5 subunit showed the ‘overstrong’ mixing properties exhibited by transgenic lines expressing the wild type 1Dx5 transgene.     

转基因小麦与普通小麦杂交后代中稳定株系的筛选

为了给小麦品质改良提供优异的种质,以小麦转1Dx5和1Ax1基因品系为父本,以长江中下游冬麦区小麦栽培品种为母本配制杂交组合,获得BC1F1、BC1F2、BC1F3和BC1F4代.在各杂交后代中,采用系谱选择法结合SDS-PAGE检测技术,鉴定各系的HMW-GS组成,获得了多个外源1Dx5或1Ax1基因稳定超表达的小麦新型纯系.     

Effect of Allelic Variation of Glutenin Subunits and Gliadins on Baking Quality in the Progeny of Two Biotypes of Bread Wheat cv. Ulla

During the determination of the HMW glutenin subunit composition of Finnish varieties, the variety Ulla was observed to contain two biotypes which differed from each other at two loci:Glu-A1andGlu-A3/Gli-A1. One of them, called Ulla 1, contained subunit 2* (Glu-A1b) andGlu-A3o/Gli-A1o, and Ulla 2 contained the null allele (Glu-A1c) andGlu-A3a/Gli-A1c. In order to determine the effect of this allelic variation on quality, the two biotypes were crossed and random lines were produced from the progeny by single seed descent. In total, 95 F6 lines were analysed from four bulked Ulla progeny lines. Significant interaction between the allelic variants of HMW glutenins and LMW gluten proteins affected the SDS-sedimentation volume at the mean flour protein level of 13·1% (dmb); the effect of LMW gluten variants was larger in the lines deficient of a HMW glutenin subunit than in lines having a HMW glutenin subunit (2*). At the higher flour protein levels (mean=15·1%, dmb) the effect on SDS-sedimentation volume was additive; progeny carrying alleles b (subunit 2*) and o/o atGlu-A1andGlu-A3/Gli-A1had significantly greater sedimentation volumes than the progeny carrying alleles c (no subunit) and a/c, respectively. The SDS-sedimentation volumes indicated differences in the quantities of the polymeric glutenins, gel proteins which have been shown to reflect dough strength. In the four bulked Ulla progeny lines, the variation in HMW glutenin subunits affected the dough strength values of the Extensigraph. However, the variation in LMW glutenin subunits did not affect Extensigraph dough strength values, as was predicted by SDS-sedimentation volumes. In the Ulla progeny, adding a HMW glutenin subunit affected Extensigraph dough strength more than adding a LMW glutenin subunit, although both increased the SDS-sedimentation volumes. Moreover, the variation in LMW gluten proteins affected the dough mixing stability in the Farinograph and test baking results of the Ulla progeny.     

Pasting properties of transgenic lines of a commercial bread wheat expressing combinations of HMW glutenin subunit genes

Seven transgenic lines of a commercial wheat (Triticum aestivum L.) cultivar expressing transgenic subunits 1Ax1, 1Dx5 and 1Dy10, alone or in combination have been developed. Pasting properties were determined in these transgenic lines using a Rapid Visco Analyser (RVA) in order to determine the possible impact of HMW-GS transgene expression on the starch properties. Expression of the HMW-GS transgenes increased the proportions of the corresponding 1Ax, 1Dx and 1Dy subunits affecting significantly the ratios of HMW-GS:LMW-GS and x-type:y-type HMW-GS. Starch granule size distribution varied significantly among all transgenic lines, with the Anza control and transgenic line T616 (expressing subunits 1Ax1 and 1Dy10) showing the highest and the lowest percentage of B granules, respectively. All transgenic lines increased the water-binding capacities (WBC) at 25 °C and 90 °C. Line T606 (expressing subunits 1Ax1 and 1Dx5) and line T590 (expressing subunit 1Dy10) showed the lowest and the highest values for peak viscosity, respectively. Notably, lines expressing only transgenic x-type subunits (T580, T581 and T606), with high ratios of x-type:y-type HMW-GS, had low peak viscosities, final viscosities and breakdown viscosities. Line T590 had the highest breakdown viscosity while lines T606 and T581 had the lowest.     

Evaluation and characterization of high-molecular weight 1D glutenin subunits from Aegilops tauschii in synthetic hexaploid wheats

The high-molecular weight (HMW) glutenin subunits of bread wheat are major determinants of end-use quality. The objective of this study was to determine the 1Dx and 1Dy subunits present in 43 synthetic hexaploid wheat (SHW) lines derived by crossing durum ‘Langdon’ to 43 Aegilops tauschii accessions. Protein samples were initially electrophoresed multiple times on SDS-PAGE gels to arrange subunits into similar groups and then were electrophoresed on urea/SDS-PAGE gels. Initial results with SDS-PAGE gels indicated that there were six 1Dx and six 1Dy subunits in these SHW lines. However, results of the urea/SDS-PAGE indicated that some of the subunit groups could be further differentiated into additional subunits. A total of eleven 1Dx and eight 1Dy subunits including the newly designated subunits 1Dx2^t-1, 1Dx2^t-2, 1Dx2^t-3, 1Dx1.5^t-1, 1Dx2.1^t-1, 1Dy10^t-1, and 1Dy12^t-1 were identified, and they composed 17 1Dx and 1Dy combinations in the SHW lines. Eight of the combinations included at least one novel subunit and hence they were novel Glu-D1 alleles. Our results indicated that urea/SDS-PAGE can be very useful in identifying new HMW glutenin subunits. Quality testing of the SHW lines will determine if any of the alleles are useful in improving wheat-baking quality.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Effects of the replacement of Glu-A1 by Glu-D1 locus on agronomic performance and bread-making quality of the hexaploid wheat cv. Courtot

The aim of this study was to evaluate the cumulative and interactive effects on wheat (Triticum aestivum L.) gluten strength and mixing properties of dough associated with the duplication of the Glu-D1 locus. A partially isohomoeoallelic line RR240, in which a segment of the wheat chromosome 1D containing the Glu-D1 locus encoding the Dx2 + Dy12 subunits and translocated to the long arm of the chromosome 1A through homoeologous recombination, was assessed. Agronomic traits and yield components were studied in the translocated line RR240 and compared with the control line cv. Courtot. Both lines were evaluated under field conditions in two experimental years. Technological effects resulting from the duplication of HMW glutenin subunits Dx2 and Dy12 were evaluated using the Alveograph test, the Mixograph test and the baking test. The RR240 line was shown to have a lower agronomic performance for 1000-kernel weight and grain yield. However the duplication of the Glu-D1 allele was associated with a significant effect on dough strength and mixing resistance, and on the Zeleny sedimentation volume. Baking parameters were not significantly modified between both lines although the score values of the CNERNA test were observed to be slightly higher in RR240 than in Courtot.     

Genetic Transformation of a High Molecular Weight Glutenin (Glu-1Dx5) to Rice cv. Fatmawati

Abstract

In order to improve rice dough functionality, we co-transformed the Glu-1Dx5 gene encoding a high molecular weight (HMW) glutenin subunit Dx5 from bread wheat, Triticum aestivum L. and either bar gene conferring resistance to herbicide bialaphos or hpt gene conferring resistance to hygromycin B to rice callus cells of cv. Fatmawati. We molecularly characterized 9 plants regenerated from bialaphos-containing medium and 63 plants from hygromycin-containing medium. The Glu-1Dx5 gene was detected by PCR analysis in 15 transgenic T₀ plants. Further analysis of T₁ and T₂ plants revealed that some transgenic plants carried the Glu-1Dx5 gene. Analysis of the endosperm extracts of T₂ plants by SDS-PAGE revealed the existence of a protein similar in size to the wheat Glu-1Dx5 gene product, suggesting successful expression of the transgene. These plants will be incorporated into breeding program for further assessment of their benefits.     

小麦高分子量谷蛋白亚基1Ax 2*的AS-PCR鉴定

为了从DNA水平上快速鉴定Glu-A1位点编码的1Ax2^＊优质亚基，建立了稳定的检测1Ax2^＊优质亚基基因的PCR扩增体系。通过该体系，1Ax2^＊优质亚基基因扩增出2652bp特异片段，而其他亚基基因则不能扩增出该片段。验证材料的PCR鉴定结果与SDS-PAGE电泳结果一致，表明利用该体系鉴定1Ax2^＊亚基是可行的。利用此体系鉴定了44份外引小麦品种（系）的谷蛋白Glu-A1位点，有24个品种（系）含有1Ax2^＊亚基，1Ax2^＊亚基出现频率为54．5％。     

Expression and Functional Analysis ofMr58 000 Peptides Derived from the Repetitive Domain of High Molecular Weight Glutenin Subunit 1Dx5

A highly repetitiveM_r58 000 peptide based on residues 102 to 643 of subunit 1Dx5 and forms containing one to four cysteine residues were expressed inE. coliand purified to homogeneity. Incorporation into dough using a 2 g Mixograph showed that most peptides resulted in reduced strength, which was possibly due to dilution or chain termination of glutenin polymers. However, a form containing four cysteines (two each close to the N-terminus and C-terminus) resulted in increased strength, indicating that the repetitive domains of the HMW subunits are sufficient to contribute to dough strength when incorporated into glutenin polymers.     

57份陕西新育成小麦品种（系）HMW-GS组成及品质分析

为探究陕西关中地区小麦HMW-GS亚基与品质性状间的关系,采用SDS-PAGE法对57份陕西关中地区小麦品种（系）HMW-GS亚基组成及相关品质性状进行了分析。结果表明,供试品种（系）中共检测出7种HMW-GS亚基类型和8种HMW-GS亚基组合;Glu-A1位点上有3种亚基类型,分别为1、2^*和Null,以1亚基为主（78.95%）;Glu-B1位点上检测到7+8（61.40%）与7+9（38.60%）两个类型;Glu-D1位点上检测到5+10（70.18%）和2+12（29.82%）两个类型。3个HMW-GS基因位点编码亚基共组成8种亚基组合,品质得分6～10分,其中1/7+8/5+10组合品质得分10分,出现频率最高。就HMW-GS不同位点对品质性状效应进行分析发现,Glu-D1位点对b^*值、形成时间、稳定时间、弱化度和粉质质量指数的影响达到极显著水平（P＜0.01）;对面团流变学特性的影响,Glu-D1>Glu-B1。不同类型亚基对小麦品质的效应存在差异,7+8亚基对蛋白质含量、湿面筋含量和容重具有正效应,7+9和5+10亚基对形成时间和稳定时间的影响显著高于其他亚基（P＜0.05）;携带1/7+8/5+10亚基组合小麦的蛋白质、湿面筋含量和容重最高;携带1/7+9/5+10亚基组合具有较高面粉L^*值和面团流变学特性指标值。     

Identification of SNPs and development of allelic specific PCR markers for high molecular weight glutenin subunit Dtx1.5 from Aegilops tauschii through sequence characterization

The high molecular weight (HMW) glutenin subunits D^tx1.5+D^ty10 from Aegilops tauschii are a novel pair of subunits not detected previously in common wheat (Triticum aestivum). The partial DNA sequences of the x-type HMW glutenin alleles from A. tauschii and synthetic hexaploid wheat samples with different HMW glutenin subunits were charcterised. Five samples were found to contain the HMW glutenin subunit D^tx1.5 that may affect bread-making quality. Polymorphisms of the DNA sequences were detected among alleles encoding different HMW glutenin subunits and within an allele encoding the same HMW glutenin subunit, such as the D^tx1.5 subunit. Three single nucleotide polymorphisms (SNPs) that can distinguish the Dx^t1.5 from D^tx2, D^tx5, Dx2 and Dx5 alleles were identified. Allelic specific (AS)-PCR primers were developed based the SNPs located at the non-repetitive N-terminal of the HMW glutenin subunits. The AS-PCR primers can accurately identify accessions containing the D^tx1.5 subunit from those containing other studied subunits by PCR analysis, suggesting the usefulness of AS-PCR for identifying different HMW glutenin subunits of A. tauschii and synthetic hexaploid wheat. The AS-PCR primers developed based on SNPs in this study are valuable in wheat breeding for effective selection of special HMW glutenin subunits.     

Overexpression of a Gluten Protein in Transgenic Wheat Results in Greatly Increased Dough Strength

The transgenic wheat line B73-6-1 contains additional genes encoding a gluten protein called HMW subunit 1Dx5, resulting in a four-fold increase in the proportion of this component in the seed proteins and corresponding increases in the proportions of total HMW subunits and total glutenins. This is associated with a dramatic increase in dough strength, as measured using a small scale Mixograph, with dough produced from B73-6-1 being too strong to be used in conventional breadmaking.     

A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used “minimal gene cassettes” and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA from regenerated plants identified 15 T₀ plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T₁ to T₃ generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T₃ generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1Dy10 transgene were identified in the T₄ generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.     

Technological quality of transgenic wheat expressing an increased amount of a HMW glutenin subunit

The transgenic line B73-6-1 (developed and characterized by UK scientists) contains 10–15 extra copies of a native HMW glutenin gene (1Dx5) with an approximately four-fold increase in the amount of the encoded protein. The technological and rheological properties of this line and the non-transformed control line were studied in a continental type, arid climatic environment (Martonvásár, Hungary, 2000–2002). The results show that in the three years of the field experiment, the transgene and associated functional properties were stably inherited over several generations. No difference was found in yield between the transgenic line and the original genotype, but clear genotypic differences were found in kernel hardness and kernel size. The transgenic line had an increased kernel hardness and a smaller kernel size. The transgenic line B73-6-1 tended to have a higher protein content than the control L88-6, but this difference was not statistically significant. In contrast there was a significant, 400%, increase in the amount of 1Dx5 HMW glutenin subunits, Dx/Dy-, HMW/LMW- and glutenin/gliadin ratio. At the same time there was a decrease in wet gluten content and SDS sedimentation. Thus, significant changes in the structure of the protein matrix caused by the altered x to y ratio of HMW-GS and as a result of this alteration, flour functional properties changed. Parameters that characterize the stability and strength of the dough confirmed that B73-6-1 was stronger, but the extensibility was reduced. This flour would be suitable for blending with flour of lower grade.