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1.
Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene 总被引:5,自引:0,他引:5
Masci S. D'Ovidio R. Scossa F. Patacchini C. Lafiandra D. Anderson O.D. Blechl A.E. 《Molecular breeding : new strategies in plant improvement》2003,12(3):209-222
The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer. 相似文献
2.
Functional properties of flours from field grown transgenic wheat lines expressing the HMW glutenin subunit 1Ax1 and 1Dx5 genes 总被引:4,自引:0,他引:4
Barro Francisco Barceló Pilar Lazzeri Paul A. Shewry Peter R. Ballesteros Juan Martín Antonio 《Molecular breeding : new strategies in plant improvement》2003,12(3):223-229
The high molecular weight glutenin subunits (HMW-GS) of wheat are major determinants of the viscoelastic properties of gluten and dough. The bread making quality of field grown transgenic lines of bread wheat expressing the HMW-GS 1Ax1 or 1Dx5 genes were evaluated over a two year period. Subunit 1Ax1 represented about 29% and 48% of the total HMW-GS in lines 1-2 and 2-2, respectively, while subunit 1Dx5 represented 65.4% and 62% of the total HMW-GS in transgenic lines 6-2 and 9, respectively. The expression of subunits 1Ax1 or 1Dx5 in transgenic wheat led to corresponding decreases in the proportions of endogenous HMW-GS. HMW-GS 1Ax1 and 1Dx5 had contrasting effects on dough quality determined by the Alveograph and sedimentation test. Subunit 1Ax1 increased the tenacity (P), extensibility (L), deformation work (W), and sedimentation value, with the increase being related to the level of expression. In contrast, subunit 1Dx5 led to a smaller increment in the tenacity (P), but to drastic decrease in both extensibility (L), deformation work (W), and the sedimentation value. Expression of subunit 1Ax1 in transgenic wheat resulted in lines with improved rheological properties whereas the lines expressing subunit 1Dx5 resulted in unsuitable breadmaking-related characteristics. 相似文献
3.
McIntire TM Lew EJ Adalsteins AE Blechl A Anderson OD Brant DA Kasarda DD 《Biopolymers》2005,78(2):53-61
The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II. 相似文献
4.
Huayan Yin Xuye Du Biao Wang Xin Ma Cunyao Bo Anfei Li Xiaocun Zhang Lingrang Kong 《Molecular breeding : new strategies in plant improvement》2017,37(8):97
High-molecular-weight glutenin subunits (HMW-GS) in wheat grain are the major determinants of dough elasticity and viscosity and thus of bread-making quality. PCR-based molecular markers designed based on DNA polymorphisms were used to analyze HMW-GS genes in wheat. The loop-mediated isothermal amplification (LAMP) assay is a simple and rapid method for specific detection of genomic DNA target sequences. In the present study, we designed a set of LAMP markers by targeting the unique sequences of 1Dx2 and 1Dx5 genes. The primers could effectively distinguish the 1Dx2 and 1Dx5 genes from other genes at the Glu-1 locus. The results were confirmed by agarose gel electrophoresis. For visualization, ethidium bromide was used, and fluorescence only appeared in the positive samples. Under optimal conditions, the detection could be finished in 1 h. Thirty-eight wheat cultivars with known HMW-GS were used to validate LAMP markers for 1Dx2 and 1Dx5 genes. Only DNA samples with target genes could be amplified, and the results could be read easily using this method. The tests using LAMP were easy to perform, rapid, and sensitive. Thus, the current study results have the potential to be a powerful tool for the detection of HMW-GS genes in wheat. 相似文献
5.
Ikeda TM Nagamine T Fukuoka H Yano H 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(4):680-687
To clarify the composition of low-molecular-weight glutenin subunits (LMW-GSs) in a soft wheat cultivar, we cloned and characterized
LMW-GS genes from a cDNA library and genomic DNA in Norin 61. Based on alignment of the conserved N- and C- terminal domains
of the deduced amino-acid sequences, these genes are classified into 12 groups. One of these groups (group 5), the corresponding
gene of which has not been reported previously, contains two additional hydrophobic amino-acid clusters interrupting the N-terminal
repetitive domain. Other groups (groups 11 and 12), which were not identified in other cultivars as a protein product, showed
all eight cysteines in the C-terminal conserved domain. With specific primer sets for these groups it was revealed that Glu-D3 and Glu-A3 encoded the former and the latter, respectively. Both groups of genes were expressed in immature seeds. The presence of these
groups of LMW-GSs may affect the dough strength of soft wheat.
Received: 26 March 2001 / Accepted: 16 July 2001 相似文献
6.
Variation and classification of B low-molecular-weight glutenin subunit alleles in durum wheat 总被引:7,自引:0,他引:7
M. T. Nieto-Taladriz M. Ruiz M. C. Martínez J. F. Vázquez J. M. Carrillo 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,95(7):1155-1160
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 相似文献
7.
Ta1小麦轮选群体高分子量谷蛋白亚基组成分析 总被引:2,自引:0,他引:2
利用Ta1小麦 Ms2 创建改良小麦面包品质的优质群体,采用SDS-PAGE法对其2次互交轮回群体C2的HMW-GS组成进行了分析.结果表明:在供试的193个样品中各HMW-GS及其组成模式的频率不尽相同,Glu-A1、Glu-B1和Glu-D1位点上产生频率最高的亚基分别是1、14+15和2+12,各为54.40%、35.75%和60.10%,优质亚基5+10的频率为17.6%; null、14+15、2+12 模式产生频率最高,为13.47%,并有 14+15,5+10 的优质亚基聚合体出现,占5.2%;该群体也产生了亲本不具有的13、16、22亚基及19种新的HMW-GS组成模式.说明利用Ta1小麦轮回选择技术是创造新亚基类型的一个有效途径. 相似文献
8.
He G.Y. Rooke L. Steele S. Békés F. Gras P. Tatham A.S. Fido R. Barcelo P. Shewry P.R. Lazzeri P.A. 《Molecular breeding : new strategies in plant improvement》1999,5(4):377-386
Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T2 plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making. 相似文献
9.
This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops. 相似文献
10.
11.
小麦高分子量谷蛋白亚基及其基因的研究进展 总被引:12,自引:2,他引:12
主要介绍了小麦高分子量谷蛋白亚基(HMW-GS)及其基因的研究进展情况,目前,转基因小麦的技术已经逐渐成熟,由于分子生物学领域分子标记技术的迅速发展,尤其是PCR技术的广泛应用,为实现外源优良储藏蛋白基因导入改良品种提供了可能,利用已知小麦品种的基因序列设计引物,从众多的未知小麦品种中扩增出新基因加以研究并做外源优质HMW-GS基因的转入已成为一种趋势。 相似文献
12.
M. L. Alvarez S. Guelman N. G. Halford S. Lustig M. I. Reggiardo N. Ryabushkina P. Schewry J. Stein R. H. Vallejos 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(2):319-327
Wheat HMW glutenin subunit genes 1Ax1 and 1Dx5 were introduced, and either expressed or overexpressed, into a commercial wheat
cultivar that already expresses five subunits. Six independent transgenic events were obtained and characterized by SDS-PAGE
and Southern analysis. The 1Dx5 gene was overexpressed in two events without changes in the other endosperm proteins. Overexpression
of 1Dx5 increased the contribution of HMW glutenin subunits to total protein up to 22%. Two events express the 1Ax1 subunit
transgene with associated silencing of the 1Ax2* endogenous subunit. In the SDS-PAGE one of them shows a new HMW glutenin
band of an apparent Mr lower than that of the 1Dx5 subunit. Southern analysis of the four events confirmed transformation and suggest that the transgenes
are present in a low copy number. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic
wheat expressing the 1Ax1 subunit transgene and overexpressing the Dx5 gene. Transgenes and expression patterns were stably
transmitted to the progenies in all the events except one where in some of the segregating T2 seeds the silencing of all HMW glutenin subunits was reverted associated with a drastic lost of transgenes from a high to
a low copy number. The revertant T2 seeds expressed the five endogenous subunits plus the 1Ax1 transgene.
Received: 16 June 1999 / Accepted: 29 July 1999 相似文献
13.
S. Masci R. D’Ovidio D. Lafiandra D. D. Kasarda 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(3-4):396-400
Received: 25 May 1999 / Accepted: 22 June 1999 相似文献
14.
Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity 总被引:7,自引:0,他引:7
The expression of foreign phytoalexins in a new host is thought to increase fungal resistance, since host-specific pathogens have not experienced selection for detoxifying or metabolising the novel antifungal compounds. Two resveratrol synthase genes vst1 and vst2 from grapevine (Vitis vinifera L.) and the pinosylvin synthase gene pss from pine (Pinus sylvestris L.) were stably transformed into bread wheat. The expression of the target genes is regulated by stress-inducible grapevine promoters. The vst1 and vst2 promoters were functional in wheat and retained their expression profiles described for grapevine. ALL vst and pss transgenic lines accumulated stilbene derivatives upon induction by UV light. The detected stilbenes showed a remarkable similarity to resveratrol and pinosylvin, however were found to be more hydrophilic than resveratrol and pinosylvin. Upon inoculation with the biotrophic pathogen Puccinia recondita f.sp. tritici several vst expressing wheat lines showed a significant reduction of disease symptoms (19 +/- 9% to 27 +/- 8%) compared to wild-type plants. The reduction of disease symptoms was even more obvious after inoculation with the facultative biotrophic pathogen Septoria nodorum Berk. and ranged from 42 +/- 13% to 71 +/- 4%. None of the four tested pss expressing lines showed a reduction in disease incidence. 相似文献
15.
Selecting a promoter for driving transgene expression is one of the most important factors to consider in a transformation project. Information about the native regulation of the promoter activity is important, but it is also necessary to consider how that activity will be affected when integrated into the genome of the transformed plants. Study of a promoter performance in individually transformed lines provides useful information in this area. The maize ubiquitin 1 (Ubi‐1) promoter has been widely used to drive constitutive transgene expression in monocotyledonous plants. However, lack of data on its activity in individual transformed wheat lines constitutes a gap in the understanding and predictability of this promoter's performance. In this paper, we began addressing this problem by examining the expression of the marker gene uidA, coding for β‐glucuronidase (GUS), under the control of the maize Ubi‐1 promoter in individual transgenic wheat (Triticum aestivum L.) lines from different wheat varieties. The expression of uidA driven by this promoter depended to a great extent on the specific transformation event. Whilst expression was strong and constitutive in all tissues in some of the lines analysed, there were also transgenic lines in which GUS activity was restricted to only a few tissues. In general the maize Ubi‐1 promoter had strong activity in young, metabolically active tissues and in pollen grains. 相似文献
16.
Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin 总被引:10,自引:0,他引:10
P. I. Payne L. M. Holt A. J. Worland C. N. Law 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1982,63(2):129-138
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. 相似文献
17.
Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin 总被引:12,自引:2,他引:12
P. I. Payne L. M. Holt C. N. Law 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1981,60(4):229-236
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. 相似文献
18.
19.
Xin Gao Qisen Zhang Marcus P. Newberry Ken J. Chalmers Diane E. Mather 《Amino acids》2013,44(3):1061-1071
The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue, which may interfere with glutenin polymerisation through cross-linkage within the Bx7.1 subunit, causing this subunit to act as a chain terminator. 相似文献
20.
The thirty-three 5' flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety 'Chinese Spring' was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5' flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5' flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs. 相似文献