High Molecular Weight Glutenin Genes Research Articles

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.

Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat

Specific amplification of the complete coding region of all six high-molecular-weight (HMW) glutenin genes present in hexaploid wheat was obtained by the polyerase chain reaction (PCR). Primers specific for the N-terminal region of the 1Dx gene and for the repetitive domain of the y-type HMW glutenin genes were also developed. Although the primers were constructed on the basis of the nucleotide sequences of HMW glutenin genes present in T. aestivum L. cv 'Cheyenne', they were very efficient in amplifying HMW glutenin genes of diploid and tetraploid wheat species. PCR analysis of HMW glutenin genes of T. urartu Tuman., T. longissimum (Schweinf. & Muschl.) Bowden and T. speltoides (Tausch) Gren. ex Richt, showed a high degree of length polymorphism, whereas a low degree of length variation was found in accessions of T. tauschii (Coss.) Schmal. Furthermore, using primers specific for the repetitive regions of HMW genes, we could demonstrate that the size variation observed was due to a different length of the central repetitive domain. The usefulness of the PCR-based approach to analyze the genetic polymorphism of HMW glutenin genes, to isolate new allelic variants, to estimate their molecular size and to verify the number of cysteine residues is discussed.

PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus

Genes encoding high-molecular-weight (HMW) glutenin subunits, present in bread-wheat lines and cultivars, were studied by RFLP (restriction fragment length polymorphism) and PCR (polymerase chain reaction) analyses. In particular, allelic subunits of the x-or y-type, encoded at the Glu-D1 locus present on the long arm of chromosome 1D, were investigated. The variation in size, observed in different allelic subunits, is mainly due to variation in the length of the central repetitive domain, typical of these proteins. Deletions or duplications, probably caused by unequal crossingover, have given rise to the size heterogeneity currently observed. The possibility of using the PCR technique for a detailed analysis of HMW glutenin genes in order to obtain a more accurate estimation of the molecular weight of their encoded subunits, and the detection of unexpressed genes, is also described.

High Molecular Weight Glutenin Genes: Effects on Quality in Wheat

To utilize electrophoretic variants of wheat (Triticum aestivum L.) storage proteins as rapid screening criteria in selection of parents or progeny for end‐use quality, breeders should know the relative effects of the alternate alleles at the storage‐protein loci on which selection will act. The objective of this study was to determine the direct effects of high molecular weight (HMW) glutenin genes on quality in wheat, without the confounding effects of linkage disequilibrium. Genetic analyses of the direct effects of loci coding for glutenin subunits on mixing time, mixing tolerance, absorption, and protein concentration were conducted with 135 random wheat selections from a randomly mated population. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS‐PAGE) was used to screen for HMW glutenin subunits. Significant differences were observed among alleles within loci for some of the quality traits measured. Some of the epistatic interactions also were significant, indicating that the effects of some subunits depend partly on which of the snbunits coded by other genomes are present. The Subunit 5 + 10 combination at the Glu‐Dl locus had the largest positive effects on dough mixing traits, and the 6 + 8 combination at Glu‐Bl had large negative effects on most traits. Alleles at Glu‐Al had no significant effects on any traits. Glutenin‐based selection for increased mixing time and tolerance gave lower predicted gains than phenotypic selection.

Tissue-Specific Expression of a Wheat High Molecular Weight Glutenin Gene in Transgenic Tobacco

Tissue-Specific Expression of a Wheat High Molecular Weight Glutenin Gene in Transgenic Tobacco

Tissue-specific expression of a wheat high molecular weight glutenin gene in transgenic tobacco.

The expression of a wheat genomic clone containing the entire coding sequence of the high molecular weight glutenin subunit 12 gene flanked by 2.6 kilobases of 5' and 1.5 kilobases of 3' sequences has been studied after introduction into tobacco. Seeds of different tobacco plants containing the full-length wheat genomic clone accumulated different amounts of intact high molecular weight glutenin subunit mRNA and of a polypeptide displaying the solubility, molecular weight, and antigenic properties of the high molecular weight glutenin subunit 12. The wheat protein accumulated without obvious degradation products and constituted up to approximately 0.1% of the total tobacco endosperm protein. Restriction fragments corresponding to 2.6 kilobases, 1.4 kilobases, and 433 base pairs of high molecular weight glutenin 5' upstream sequence were fused to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene in the vector polyCATter and transferred into tobacco. Chloramphenicol acetyltransferase enzyme activity was detected only in the seed endosperm tissue of the transformed plants. It was detected in tobacco seeds 8 days after anthesis and persisted until seed maturity. It is concluded that 433 base pairs of high molecular weight glutenin upstream sequence are sufficient to confer endosperm-specific expression of this monocot gene in the dicot tobacco.

DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat.

Restriction enzyme digests of DNA from nullisomic-tetrasomic and intervarietal chromosome substitution lines of wheat were probed with a high molecular weight (HMW) glutenin cDNA. Three restriction endonucleases were used to investigate restriction-fragment differences among five wheat varieties. The results suggest that the hybridizing fragments contain single gene copies and permit the identification of the subunit encoded by each gene. Restriction-fragment variation associated with previously established allelic differences between varieties was observed. Also, there is a clear relationship between the electrophoretic mobility of a HMW subunit and the length of the central repetitive section of the gene encoding it. These results are discussed with reference to the evolution of the HMW glutenin gene family and the uses of restriction-fragment variation in plant breeding and genetics.

Nucleotide sequence of a gene from chromosome 1D of wheat encoding a HMW-glutenin subunit.

A high molecular weight glutenin gene in hexaploid wheat has been isolated by cloning in bacteriophage lambda and characterized. The gene corresponds to polypeptide 12 encoded by chromosome 1D in the variety "Chinese Spring". The coding sequence predicted contains seven cysteine residues six of which flank a central repetitive region comprising more than 70% of the polypeptide. These findings are related to the role of high molecular weight subunits in the viscoelastic theory of gluten structure.