HMW Glutenin Genes Research Articles

Survey and new insights in the application of PCR‐based molecular markers for identification of HMW‐GS at the Glu‐B1 locus in durum and bread wheat

AbstractWheat, among all cereal grains, possesses unique characteristics conferred by gluten; in particular, high molecular weight glutenin subunits (HMW‐GS) are of considerable interest as they strictly relate to bread‐making quality and contribute to strengthening and stabilizing dough. Thus, the identification of allelic composition, in particular at the Glu‐B1 locus, is very important to wheat quality improvement. Several PCR‐based molecular markers to tag‐specific HMW glutenin genes encoding Bx and By subunits have been developed in recent years. This study provides a survey of the molecular markers developed for the HMW‐GS at the Glu‐B1 locus. In addition, a selection of molecular markers was tested on 31 durum and bread wheat cultivars containing the By8, By16, By9, Bx17, Bx6, Bx14 and Bx17 Glu‐B1 alleles, and a new assignation was defined for the ZSBy9_aF1/R3 molecular marker that was specific for the By20 allele. We believe the results constitute a practical guide for results that might be achieved by these molecular markers on populations and cultivars with high variability at the Glu‐B1 locus.

Advances in the regulation of HMW glutenin genes of wheat

Advances in the regulation of HMW glutenin genes of wheat

Characterization of HMW-GSs and their gene inaction in tetraploid wheat

In this study, we report the expression of HMW-GSs in 87 accessions of tetraploid wheat, the characterization of three inactive and one active HMW glutenin genes, and the functional verification of HMW-GSs by promoter-GUS expression. SDS-PAGE profiles revealed that tetraploid wheat has many different combinations of HMW-GSs and the number of subunits varies from 1 to 4. HMW glutenin genes at the Glu-A1x, Glu-A1y and Glu-B1y loci exhibited different frequencies of inaction while the Glu-B1x allele was expressed in all 87 accessions. Gene cloning showed that only 1Bx (Tdu-e) could express a full-length protein and its deduced protein sequence has the typical primary structure but with fewer cysteine residues. The expression of the other three HMW glutenin genes has been disrupted by stop codons in their repetitive domains. Besides short indels or mutations of one or more bases, an 85-bp deletion and a 185-bp insertion were found in the promoter regions of 1Ay (Tdu-s) and 1Bx (Tdu-e). The transient expression of promoter-GUS constructs indicated that the 1Ay promoter can drive expression of the GUS gene. We conclude that defects (stop codons or the insertion of large transposon-like elements) in the coding regions may be the most probable cause for the inaction of the HMW glutenin genes.

Novel HMW glutenin genes from Aegilops tauschii and their unique structures

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx5.3t and 1Dy12.1**t were revealed and characterized from Ae. tauschii accession PI554324. SDS-PAGE band of 1Dx5.3t was between those of 1Bx6 and 1Bx7, while 1Dy12.1**t with slightly faster migration rate than that of 1Dy12. The lengths of 1Dx5.3t and 1Dy12.1**t were 2115 bp and 1986 bp, encoding 703 and 660 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. 1Dx5.3t is the shortest of the known Dx-type alleles. 1Dy12.1**t is also a special subunit since it has an additional cysteine in the front of the central repetitive domain. This cysteine that is not existed in previously reported Dy-type genes may be useful for improving bread wheat quality. Median-joining Network analysis indicated that 1Dy12.1**t may be a key site in the genealogy of the Glu-Dy.

Novel and ancient HMW glutenin genes from Aegilops tauschii and their phylogenetic positions

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx3.1t and 1Dy11*t were revealed and characterized from Aegilops tauschii Coss. subspecies tauschii accession AS60. SDS-PAGE band of 1Dx3.1t was between those of 1Dx2 and 1Dx3, while 1Dy11*t was between 1Dy11 and 1Dy12. The lengths of 1Dx3.1t and 1Dy11*t were 2,514 bp and 1,968 bp, encoding 836 and 654 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. Network analysis indicated that 1Dx3.1t together with other five rare alleles only detected in Asia common wheat populations represented the ancestral sequences in Glu-D1 locus. Neighbor-joining tree analysis of previously cloned x-type and y-type alleles in the Glu-D1 locus supported the hypothesis that more than one Ae. tauschii genotypes were involved in the origin of hexaploid wheat and that different Ae. tauschii accessions contributed the D genome to common wheat and Ae. cylindrical Host, respectively. An Ae. tauschii accession with 1Dx3.1t or a closely related allele probably have involved in the origin of common wheat. Since accession AS60 used in this study belonged to typical ssp. tauschii, present results suggested the possibility that ssp. tauschii was involved in the evolution of common wheat.

Characterization of a novel variant HMW-glutenin gene from Elymus canadensis

High molecular weight (HMW) glutenin subunits (GS) play a key role in the determination of end-use quality of wheat and other cereal crops. In this study, we report the isolation and characterization of both promoter region and ORF of novel HMW-GS allele 1St1.3 from a perennial Triticeae species, Elymus canadensis. The amino acid (AA) sequences of E. canadensis 1St1.3 were deduced as 434 aa. Its protein primary structure comprises a signal peptide with a conserved N-terminal domain, a central repetitive domain and a C-terminal domain. E. canadensis 1St 1.3 possesses several distinct characteristics which are different from those of wheat HMW-GSs. The N-terminal domains of E. canadensis 1St 1.3 resemble that of y-type subunits, while their C-terminal domains are more similar to x-type subunits. The deletion of 85 bp fragment has been observed in promoter region of 1St 1.3, however which has not interrupted the expression of this gene. Our results indicate that 1St 1.3 is novel HMW-GS variants which will be valuable for enhancing our understanding of structural differentiation and the evolutionary relationship among HMW-GSs in Triticeae species.

Screening Selected Spring Wheat Cultivars for Productivity, Protein Content and the Glu-B1 Allele Using PCR

Spring wheat cultivars (Sama, Yocra Rojeo, Sids 1, Sakha 8, Sakha 61, Giza 164, Giza 168, Bani Swaf 1, Bani Swaf 3, and Shandawel) were evaluated for their productivity in two field experiments during 2005/2006 and 2006/2007 winter seasons. The results indicated that cultivars (Sids 1 and Giza 164) produced the highest grain yield. While, cultivars (Bani Swaf 3, and Bani Swaf 1) produced the lowest grain yield. Also, the last cultivars gave the lowest harvest index. Wheat cultivars Sama, Giza-164 and Sids 1 were the tallest plants. Moreover, there were significant differences in yield components among wheat cultivars. The highest protein cultivar Sakha 61 had a mean protein percentage of 15.65%. Cultivar Sakha 61 had genetic potential to produce grain with higher protein content than the other cultivars. Bani Swaf 3 cv. had the highest protein content and the lowest grain yield. A set of PCR-based markers for specific HMW glutenin genes encoding By-subunits were used to identify wheat genotypes carrying By genes at the Glu-B1 locus for its bread-making quality. The presence of primer pair ZSBy9aF1/R3 in the reaction mixture, which is an allele-specific marker for the wheat HMW glutenin By9 allele, showed one band of 650 bp in wheat cultivars Sama, Yocra Rojeo, Sids 1, Sakha 8, Sakha 61, and Giza 168. Also, primer set ZSBy9F2/R2, specific for By16 and By-null subunits in wheat cultivars, amplified three different banding patterns for wheat cultivars. Wheat cultivars Sama, Sids 1, and Sakha 61 produced three PCR fragments for the By16 gene, which exists in Glu-B1f (Bx13 + By16). Cultivars Yecora Rojo, Sakha 8, Giza 164, Giza 168, and Bani Swaf 3 produced two PCR fragments. These two PCR fragments were found to be specific for By8, By9, By18 and By15 genes. Cultivars Bani Swaf 1, and Shandawel did not produce any PCR product that was found to be specific for By-null or 20 gene. It was concluded that wheat genotypes Sakha 61 produced the highest grain yield and had the highest protein content By16 gene, which are associated with a superior bread-making quality.

Isolation and Molecular Characterization of High Molecular Weight Glutenin Subunit Genes 1Bx13 and 1By16 from Hexaploid Wheat

The high molecular weight glutenin subunit (HMW-GS) pair 1Bx13 + 1By16are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames (ORFs) were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2,220 bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2,385 bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripeptides motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide (PGQGQQ) insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.

Plant promoter driven heterologous expression of HMW glutenin gene(s) subunit in E. coli

Study of the structure of high molecular weight (Mr) glutenin subunits and their interaction has been limited by the difficulty of isolating single subunits in large amounts from the complex mixture of gluten wheat endosperm. One way to overcome this problem is the expression of high Mr glutenin subunits in heterologous systems. These systems have the additional advantages that, specific gene modifications can be made and the new gene constructs can quickly be expressed. HMW glutenin gene subunits, associated with better dough quality, were isolated from wheat (T. aestivum var. PBW343) using allele specific primers (AS-PCR) designed on allelic gene sequences. Three glutenin genes 1Ax1, Dx5 and Dy10 of sizes 1.8, 2.0 and 1.47 kb were cloned and sequenced. Probable secondary structure of HMW glutenin protein was predicted using online ExPASy server. HMW glu Dx5 and Dy10 gave significant similarity (more than 90%) to the available database but, 1Ax1 didn't show significant similarity. Glutenin genes were further subcloned in expression vector pCAMBIA-1304 driven by CaMV35S promoter and their expression was observed in Escherichia coli. The transcription was confirmed by Reverse transcriptase PCR (Rt-PCR) and sequencing. The sequences of HMW glu genes showed 100% homology with cloned HMW glutenin genes. Although initial western blotting using polyclonal antibodies against glutenin protein has strongly indicated its expression at the translation level, it is needed to be further confirmed through purification and sequencing of proteins.

Molecular characterization and phylogenetic analysis of a novel glutenin gene (Dy10.1t) fromAegilops tauschii

A novel y-type high molecular mass glutenin subunit (HMM-GS) possessing a mobility that is slightly slower than that of the subunit Dy10 obtained by SDS-PAGE, named Dy10.1t, in the wild wheat Aegilops tauschii was identified by 1- and 2-dimensional gel electrophoresis, capillary electrophoresis, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The gene encoding the HMM subunit Dy10.1t was amplified with allele-specific PCR primers, and the amplified products were cloned and sequenced. The coding domain of the Dy10.1t subunit gene consisted of 1980 bp encoding a protein of 658 residues with an M rs of 68 611 Da, which was similar to the M rs determined by MALDI-TOF-MS. The deduced amino acid sequence indicated that Dy10.1t subunit displayed a greater similarity to the Dy12 subunit, differing by only 8 amino acid substitutions. Six coding region single-nucleotide polymorphisms were discovered in the Dy10.1t gene by multiple alignments (1 per 330 bp), 1 in the N-terminal domain and the others in the central repeats. Five of them resulted in residue substitutions, whereas 3 created enzyme site changes. The homology and neighbour-joining trees constructed from code domain sequences of 20 x- and y-type glutenin genes from different Triticum species separated into 2 halves, which corresponded to the x-type and y-type HMM glutenin alleles. Phylogenetic analysis revealed that the Glu-1 gene duplication event probably occurred at about 16.83 million years ago, whereas the divergence times of A, B, and D genomes within x-type and y-type halves were before 7.047 and 10.54 million years ago, respectively.

Y-type gene specific markers for enhanced discrimination of high-molecular weight glutenin alleles at the Glu-B1 locus in hexaploid wheat

A number of primers were designed which target DNA sequence variation of the coding and /or promoter regions of wheat HMW glutenin y-type genes located at the Glu-B1 locus. This allowed the development of a set of PCR-based markers for specific HMW glutenin genes encoding By-subunits for which no markers were previously available. Markers were validated using test cultivars containing specific Glu-B1 alleles confirmed by SDS-PAGE and RP-HPLC analysis. Among the specific markers developed, primer pair ZSBy8F5/R5 was specific for the By8 gene, which exists in Glu-B1b (Bx7+By8) and Glu-B1u (Bx7*+By8) alleles. This marker allows discrimination of alleles containing By8 and By8* that are usually difficult to distinguish using SDS-PAGE. Since the over-expressed Glu-B1 allele ( Glu-Bl al.) contains the By8* subunit, it is possible to use this marker in breeding programs for selecting for the over-expression of subunit Bx7 in crosses that segregate between normal Bx7 and over-expressed Bx7 subunits. This marker also represents an alternative for distinguishing two common Glu-B1 alleles: Glu-B1i (Bx17+By18) and Glu-B1b (Bx7+By8). Two primer pairs ZSBy9aF1/R3 and ZSBy9F7/R6 both gave characteristic banding patterns for Glu-B1c (Bx7+By9) and can therefore be used to discriminate By9 - containing alleles from non - By9 alleles. Primer pair ZSBy9F2/R2 produced amplicons with a diagnostic banding pattern for allele Glu-B1f (Bx13+By16) and also permitted the discrimination of Glu-B1h (Bx14+By15) and Glu-B1e (Bx20) that have opposing genetic effects on wheat quality and are difficult to discriminate by SDS-PAGE.

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 (1D x5) 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 1D x5 HMW glutenin subunits, D x/D y-, 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.

Differentiation of the high molecular weight glutenin subunit D t x 2.1 of Aegilops tauschii indicated by partial sequences of its encoding gene and SSR markers

The high molecular weight glutenin subunits (HMW-GS) are important components of storage proteins in common wheat (Triticum aestivum L.). Conventional techniques for identifying the HMW-GS apply SDS-PAGE. To study differentiation of the HMW glutenin genes, partial DNA sequences of the concerned alleles were characterized from eight accessions of hexaploid synthetic wheat and common wheat varieties, and compared with the available sequences. SSR markers based on, and generated from, DNA sequences of the D genome of common wheat and Aegilops tauschii were analyzed to estimate genetic relationships of the accessions. Results showed that the HMW-GS Dtx2.1 determined by SDS-PAGE could be identified as two types at the DNA sequence level. One type, designated as Dtx2.1a, showed identical SNPs to the Dtx 1.5 allele and another type, designated as Dtx2.1b, had different characteristics. The SSR analysis revealed a relatively close genetic relationship of the two accessions containing the Dtx2.1a allele to the accessions containing the Dtx1.5 allele. It was concluded that the conventional SDS-PAGE has limitations in determining some HMW-GS, because the minor changes on the DNA sequence of a particular glutenin subunit cannot be detected at protein level, and that the AS-PCR method based on variation of DNA sequences of particular genes will provide a more powerful tool for identifying the HMW-GS. The differentiation of the genes encoding the HMW-GS is probably more rapid than expected. The close relationship between evolutionary changes of the microsatellite DNA and the HMW-GS genes detected in this study needs to be further investigated.

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat.

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F1-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.

The wheat D-genome HMW-glutenin locus: BAC sequencing, gene distribution, and retrotransposon clusters.

A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y-type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5' end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5' to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters.

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.

Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat

We used PCR to obtain phylogenetically informative sequences from the high molecular weight glutenin genes of wheat. The validity of partial sequence comparisons as a means of studying glutenin phylogenetics was established by constructing neighbour-joining trees from partial alignments of 12 published glutenin allele sequences. PCR was then used to obtain 20 novel glutenin allele sequences from various Triticum and Aegilops species, including a 3000 year old preserved wheat. A neighbour-joining tree derived from all known glutenin allele sequences had eight clades, representing the eight loci from which the allele sequences were derived, and was split into two halves, one comprising alleles from the Glu-1-1 loci and the other comprising Glu-1-2 alleles. The topology was compatible with the postulated relationships between the A, B, D, and G genomes. The Glu gene duplication event was tentatively dated at 7.2-10.0 million years ago (MYA), the origin of the four genomes at 5.0-6.9 MYA, and the split between the B and G genomes at 2.5-3.5 MYA. The Glu-B1-1 alleles in cultivated wheats fell into two subgroups that diverged 1.4-2.0 MYA, suggesting that emmer was domesticated twice. The D allele sequences were relatively diverse, indicating that the hybridization event that resulted in the hexaploid bread wheats might have occurred more than once.

Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus

The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences. Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from a transposition event.

Construction and expression of a synthetic wheat storage protein gene

A synthetic wheat high-molecular-weight (HMW) glutenin storage protein gene analog was constructed for expression in E. coli. This first synthetic HMW-glutenin gene and future modifications are intended to allow systematic dissection of the molecular basis of HMW-glutenin role in the visco-elastic properties critical for wheat product processing and utilization. The design of the gene included four features: different construction strategies for the separate assembly of major polypeptide domains, the inclusion of convenient restriction sites for modifications, use of a codon selection similar to E. coli highly expressed genes, and the ability to produce repetitive sequence domains of exact numbers of defined repeats. The complete synthetic HMW-glutenin construct was 1908 bp, and contained 32 identical copies of one of the HMW-glutenin repetitive domain motifs. The gene expressed the novel HMW-glutenin protein to relatively high levels in bacterial cultures and the protein exhibited the known anomalous behavior of HMW-glutenins in SDS-PAGE.