1Dx5 Genes Research Articles

Molecular cloning and sequence analysis of 1Dx-type HMW-GS genes from different wheat varieties

AbstractHigh-molecular-weight glutenin subunits (HMW-GS) contribute to dough elasticity and bread baking quality in wheat. In this study, wheat varieties were classified based on their HMW-GS composition into three groups: 1Dx5 (5 + 10, Gaoyou 8901, Xinmai 28, Xinmai 19, Xinmai 26 and Jinbaoyin), 1Dx2 (2 + 12, Zhoumai 24, Xinmai 9 and Yumai) and 1Dx4 (4 + 12, Aikang 58). Sequence analysis showed that 1Dx-GY8901, 1Dx-XM28, 1Dx-XM19 and 1Dx-XM26 were similar to the 1Dx5 gene and clustered on the same branch, while 1Dx-AK58, 1Dx-ZM24, 1Dx-JBY, 1Dx-YM, 1Dx-XM9 and 1Dx-JBY were more similar to the 1Dx2 gene and clustered on the same branch with 1Dx.2.2. There was a mutation of Ser to Cys at position S2, for an extra Cys in the repeat regions of 1Dx-XM19, 1Dx-XM26, 1Dx-XM28 and 1Dx-GY8901. The wheat HMW-GS genes exhibited similar percentages of α-helix, extended strand, β-turn and random coil structure, with ranges of 13.33–13.59, 4.77–5.78, 7.08–9.18 and 72.3–73.94%, respectively. Sequence conservation and the composition of HMW-GS subunits were also analysed for a series of strong gluten wheat varieties, Xinmai 9 (1, 7 + 8, 2 + 12), Xinmai 19 (1, 7 + 9, 5 + 10), Xinmai 26 (1, 7 + 8, 5 + 10) and Xinmai 28 (1, 7 + 9, 5 + 10). The results of this work should facilitate future breeding efforts and provide the theoretical basis for wheat quality improvement.

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.

Biolistic transformation of wheat using the HMW-GS 1Dx5 gene without selectable markers.

Genetic manipulation using linear DNA was applied to the common wheat variety Xindong No. 26 via particle bombardment with the aim to improve bread-making quality of flour. Initially, 2 biolistic parameters, helium pressure and target distance, were optimized using plasmid pAHC25. We transformed wheat immature embryo scutella with the linear 1Dx5 gene without selectable markers. The highest transient β-glucuronidase expression was obtained when scuttles were bombarded at 1100 psi with a 9-cm target distance. Using the optimized parameters, the transformation of the common wheat variety Xindong No. 26 was carried out using the linear 1Dx5. Three transgenic plants were identified from 1003 transgenic plants, yielding a transformation frequency of 0.4%. A sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis confirmed that the 1Dx5 gene was expressed in 4 T1 seeds of the transgenic plants. These experiments indicate the possibility of obtaining marker-free transgenic wheat plants via particle bombardment using the minimal gene cassettes with the particle bombardment parameters.

GENETIC TRANSFORMATION OF EGYPTIAN WHEAT WITH 1DX5 HIGH-MOLECULAR-WEIGHT GLUTENIN SUBUNIT GENE

Variation in the composition of the high-molecular-weight glutenin subunits (HMW-GS) of wheat is associated with large differences in the bread-making properties. The primary aim of this work was to produce transgenic Egyptian wheat cultivar (Giza 164) with HMW-GS gene (1DX5) to improve bread-making quality. Immature embryo-derived calli were cotransformed with pK-DX5 and pACH25 plasmids containing gus and bar genes using biolistic bombardment. Stable integration and expression of marker genes were detected in seven plants. However, the expression of 1DX5 gene was confirmed in five plants using SDS-PAGE analysis.

Inheritance and Expression of Copies of Transgenes <italic>1Dx5</italic> and <italic>1Ax1</italic> in Elite Wheat (<italic>Triticum aestivum</italic> L.) Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Emai18 were carried out. Chuan89-107x72-8-11b, Chuan89-107x102-1-2 and Emai18x72-8-11b, and F(1) plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F(1) and F(2) and backcross progenies of BC(1)F(1), BC(1)F(2), BC(1)F(3), BC(2)F(1), BC(2)F(2) and BC(2)F(3) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes 1Dx5 and 1Ax1 were expressed and segregated in the target wheat according to Mendelian laws. A range of 1Dx5 expression levels were observed in the progenies of Chuan89-107x72-8-11b and Emai18x72-8-11b, but the expression levels of 1Ax1 in progenies of Chuan89-107x102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign 1Dx5 gene of 5-10 copies had the more complicated expression mechanism than the 1Ax1 gene of 4-5 copies.

Multiplex polymerase chain reaction analysis of Glu-1 high-molecular-mass glutenin genes from wheat by capillary electrophoresis with laser-induced fluorescence detection

The unique bread-making properties of wheat are closely correlated with composition and quantity of high-molecular-mass (HMW) glutenin subunits encoded by the Glu-1 genes. We report the development of a multiplex polymerase chain reaction (PCR) method to identify bread wheat genotypes carrying HMW glutenin allele composition of Glu-1 complex loci ( Glu-A1, Glu-B1 and Glu-D1) by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Two triplex primer sets of HMW glutenin subunit genes were examined. An automated and rapid CE–LIF technique is helpful in the multiplex PCR optimization process. Two fluorescent intercalating dyes (EnhanCE, and YO-PRO-1) are compared for detection of DNA fragments. Amplified DNA fragments of HMW glutenin Glu-1 genes were well separated both by agarose slab-gel electrophoresis and CE, and revealed minor differences between the sequences of 1Ax2 * , 1Axnull, 1Bx6, 1Bx7, 1Bx17 and 1Dx5 genes. Moreover, CE technique requires samples of smaller volumes in comparison to slab-gel electrophoresis, and data can be obtained in less than 20 min. There was a very high concordance in the assessment of the molecular size of PCR-generated DNA markers. Fast and accurate identification of molecular markers of Glu-1 genes by CE–LIF can be an efficient alternative to standard procedure separation for early selection of useful wheat genotypes with good bread-making quality.

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.

Identification, Cloning and Characterisation of a HMW-Glutenin Gene from an Old Hungarian Wheat Variety, Bánkúti 1201

Despite its good functional properties, the variety Bankuti 1201 has been found to possess 2+12 or 3+12 allele composition on chromosome 1D. In order to determine the reason for its quality traits a gene-specific PCR technique was applied in preliminary experiments to examine the HMW glutenin allele composition of the variety. In the course of the analysis a fragment characteristic of Bankuti 1201 was identified and the nucleotide sequence was determined. This showed the presence of a 1Ax2* gene variant which, despite near homology, differed from the original 1Ax2* gene at one important point. At 1181 bp of the 1Ax2* sequence nucleotide exchange was observed which is the middle nucleotide of the TCT-TGT base triplet, involving the exchange of serine for cysteine. The gene was designated 1Ax2*B. The presence of an extra sulphydryl group, like that of the extra cysteine in the 1Dx5 gene, facilitates the formation of further disulphide bonds, might lead to an improvement in gluten quality characters.

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 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.

PCR analysis to distinguish between alleles of a member of a multigene family correlated with wheat bread-making quality

Good or poor wheat bread-making quality is associated with two allelic pairs at theGlu-D1 complex locus, designated 1Dx5-1Dy10 and 1Dx2-1Dy12, respectively. The polymerase chain reaction (PCR) verified the presence of the HMW-glutenin 1Dx5 gene from genomic DNA extracted from part of the endosperm of a single dry seed, or a small amount of leaf or root tissue, of several bread-wheat cultivars. This easy, quick, and non-destructive PCR-based approach is proposed as a very efficient and safe alternative to standard procedures for selecting bread-wheat genotypes with good bread-making properties.