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Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

ABSTRACT: High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ? 4 and Glu-1Aiy1 ? 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

SUBMITTER: Cao S

PROVIDER: S-EPMC3913593 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

Cao Shuanghe S Li Zhixin Z Gong Caiyan C Xu Hong H Yang Ran R Hao Shanting S Wang Xianping X Wang Daowen D Zhang Xiangqi X

PloS one 20140204 2

High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven ge ...[more]

PMID: 24503781

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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

Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

Publications

Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

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