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Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars.

ABSTRACT: Two-dimensional gel electrophoresis (2-DGE) was used as a complement to SDS-PAGE to determine the allelic compositions of LMW-GS in 32 Korean wheat cultivars. Protein patterns generated by 2-DGE from each cultivar were compared to patterns from standard wheat cultivars for each allele. At the Glu-A3 locus, thirteen c, twelve d, three e (null), two g and two new alleles were identified. At the Glu-B3 locus, one b, nineteen d, four h, one i and five ad alleles were identified. At the Glu-D3 locus, twenty-three a, four b, four c and one l alleles were identified. When compared to results obtained previously using SDS-PAGE, there were discrepancies in the allelic designations of 10 of 32 cultivars (31%). While SDS-PAGE is a rapid and relatively simple method for assessing LMW-GS composition, the similar mobilities of the proteins makes it difficult to discriminate certain alleles. 2-DGE is a more complicated technique, but provides a more accurate picture of the complement of the LMW-GS in a given cultivar. In addition to providing essential information for wheat breeders, the 2-DGE reference maps generated in this study will make it possible to study the contributions of individual LMW-GS to flour quality.

SUBMITTER: Lee JY

PROVIDER: S-EPMC5654455 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars.

Lee Jong-Yeol JY Jang You-Ran YR Beom Hye-Rang HR Altenbach Susan B SB Lim Sun-Hyung SH Lee Choung Keun CK

Breeding science 20170811 4

Two-dimensional gel electrophoresis (2-DGE) was used as a complement to SDS-PAGE to determine the allelic compositions of LMW-GS in 32 Korean wheat cultivars. Protein patterns generated by 2-DGE from each cultivar were compared to patterns from standard wheat cultivars for each allele. At the Glu-A3 locus, thirteen c, twelve d, three e (null), two g and two new alleles were identified. At the Glu-B3 locus, one b, nineteen d, four h, one ...[more]

PMID: 29085250

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Project description:BackgroundWheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase.ResultsWe performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing.ConclusionsOur results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.

Project description:BackgroundHigh-molecular-weight glutenin subunits (HMW-GS) play important roles in the elasticity of dough made from wheat. The HMW-GS null line is useful for studying the contribution of HMW-GS to the end-use quality of wheat.MethodsIn a previous work, we cloned the Glu-1Ebx gene from Thinopyrum bessarabicum and introduced it into the wheat cultivar, Bobwhite. In addition to lines expressing the Glu-1Ebx gene, we also obtained a transgenic line (LH-11) with all the HMW-GS genes silenced. The HMW-GS deletion was stably inherited as a dominant and conformed to Mendel's laws. Expression levels of HMW-GS were determined by RT-PCR and epigenetic changes in methylation patterns and small RNAs were analyzed. Glutenins and gliadins were separated and quantitated by reversed-phase ultra-performance liquid chromatography. Measurement of glutenin macropolymer, and analysis of agronomic traits and end-use quality were also performed.ResultsDNA methylation and the presence of small double-stranded RNA may be the causes of post-transcriptional gene silencing in LH-11. The accumulation rate and final content of glutenin macropolymer (GMP) in LH-11 were significantly lower than in wild-type (WT) Bobwhite. The total protein content was not significantly affected as the total gliadin content increased in LH-11 compared to WT. Deletion of HMW-GS also changed the content of different gliadin fractions. The ratio of ω-gliadin increased, whereas α/β- and γ-gliadins declined in LH-11. The wet gluten content, sedimentation value, development time and stability time of LH-11 were remarkably lower than that of Bobwhite. Bread cannot be made using the flour of LH-11.ConclusionsPost-transcriptional gene silencing through epigenetic changes and RNA inhibition appear to be the causes for the gene expression deficiency in the transgenic line LH-11. The silencing of HMW-GW in LH-11 significantly reduced the dough properties, GMP content, wet gluten content, sedimentation value, development time and stability time of flour made from this wheat cultivar. The HMW-GS null line may provide a potential material for biscuit-making because of its low dough strength.

Dataset Information

Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars.

Publications

Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars.

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