Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

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.     

Functional properties of flours from field grown transgenic wheat lines expressing the HMW glutenin subunit 1Ax1 and 1Dx5 genes

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.     

Detection of high-molecular-weight glutenin subunit genes for 1Dx2 and 1Dx5 using loop-mediated isothermal amplification assay

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.     

Identification of new low-molecular-weight glutenin subunit genes in wheat

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     

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

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

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

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.     

Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits

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 M_r 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     

A 1B-coded low-molecular-weight glutenin subunit associated with quality in durum wheats shows strong similarity to a subunit present in some bread wheat cultivars

Received: 25 May 1999 / Accepted: 22 June 1999     

Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity

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.     

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

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

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

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

A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough

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.     

盐逆境下转基因耐盐小麦与其受体呼吸途径的动态变化

以转基因耐盐品种８９１２２和其受体陇春１３号两种小麦为实验材料,研究科幼苗在不同盐浓度胁迫下呼吸途径动态变化。结果表明,８９１２２出现盐呼吸明显迟于其受体;两听Ｖａｌｔ与ρＶａｌｔ变化并不同步,且Ｖａｌｔ均受高盐浓度的抑制,但低盐浓度能诱导其受体的Ｖａｌｔ;两品种的ρＶａｌｔ与ρ′Ｖｃｙｔ彼此协同调节适应盐境,且ρ′Ｖｃｙｔ仍是盐胁迫过程中线粒体电子传递的主要途径。同时讨论了盐逆境下抗氰呼吸的一些     

Isolation and identification of mRNA for the high-molecular-weight storage proteins of wheat endosperm

The prolamin storage proteins of the wheat endosperm contain a sub-class of high-molecular-weight (HMW) polypeptides which have been implicated in determining breadmaking quality. Membrane-bound polysomes isolated from developing wheat endosperms contain mRNA for these HMW components. Although unfractionated polyadenylated RNA derived from the polysomes did not direct the synthesis of these components in an in-vitro wheat-germ system, it did when incubated with a rabbit reticulocyte lysate system. Identification of the translation products as HMW prolamins was based on their large incorporation of [³H]leucine and [³H]glycine relative to [³H]lysine, their mobility on polyacrylamide-gel electrophoresis and the observation that the changes of mobility in response to change in wheat genotype were the same as those observed for the authentic protein. The mRNA was fractionated by electrophoresis and density-gradient centrifugation. The mRNA for the HMW prolamins was found to have a relative molecular mass of about 1.6·10⁶.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - poly(A)⁺RNA polyadenylated RNA - SDS sodium dodecyl sulphate     

Analysis and validation of genome-specific DNA variations in 5' flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

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.