Transformation of wheat with high molecular weight subunit genes results in improved functional properties

Barro, Francisco; Rooke, L.; Békés, F.; Gras, P. W.; Tatham, Arthur S.; Fido, Roger J.; Lazzeri, P. A.; Shewry, Peter R.; Barceló, P.

doi:10.1038/nbt1197-1295

Cited by 289 publications

(140 citation statements)

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“…For Western blot, prolamins derived from equal amount of endosperms were diluted 10 times, resolved with SDS-PAGE, and transferred to a PVDF membrane with a BioRad Mini Trans-blot cell. Dx5 was detected using a 1:5,000 dilution of a monoclonal antibody that has previously been described (Barro et al 1997;Sangtong et al 2002). Blotting and detection were done with GE Healthcare ECL Western blotting detection system (Piscataway, NJ, USA).…”

Section: Western Blot Analysis Of Dx5 Expressionmentioning

confidence: 99%

Divergent properties of prolamins in wheat and maize

Zhang

Sangtong

Peterson

et al. 2013

Planta

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Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of c-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires c-zeins for its proper subcellular organization in maize.

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Section: Western Blot Analysis Of Dx5 Expressionmentioning

confidence: 99%

Divergent properties of prolamins in wheat and maize

Zhang

Sangtong

Peterson

et al. 2013

Planta

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“…In wheat, expression of the Glu-1Dx5 gene has not been reported in any tissue other than endosperm and has not been associated with differential transmission to our knowledge. Further, expression of the Glu-1Dx5 gene has been confined to seeds in both transgenic wheat (Barro et al 1997) and transgenic tobacco . Consistent with these observations, we failed to detect 1Dx5 HMW glutenin expression by SDS-PAGE and Western blotting in embryos, leaves, pollen and immature anthers of our transgenic maize.…”

Section: Discussionmentioning

confidence: 99%

“…Proteins were detected using a monoclonal antibody that specifically binds to the N-terminal end of the repetitive regions of the 1Dx and 1Ax HMW glutenins (Barro et al 1997). …”

Section: Immuno-blot Analysismentioning

confidence: 99%

“…Wheat flour is different from other cereal flours, including maize, because it contains gluten that gives it the elasticity and extensity required for breadmaking (Barro et al 1997). Gluten consists mainly of two types of seedstorage proteins, the glutenins and the gliadins.…”

Section: Introductionmentioning

confidence: 99%

“…Cloned HMW glutenin genes have been shown to be functional when introduced into Escherichia coli (Galili 1989), tobacco (Roberts et al 1989), wheat (Altpeter et al 1996;Blechl and Anderson 1996;Barro et al 1997;Alvarez et al 2000) and tritordeum (Rooke et al 1999). Detailed inheritance studies of these transgenes have not been reported, although it has been reported that the transgene is stable through three generations in wheat (Altpeter et al 1996).…”

Section: Introductionmentioning

confidence: 99%

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Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

et al. 2002

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We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the high-molecularweight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and self-pollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events. Abstract We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the highmolecular-weight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and selfpollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events.

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Storage Proteins and their Metabolism

Hagan

Higgins

2004

Handbook of Plant Biotechnology

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The identification and cloning of storage protein genes over the last 20 years has provided new tools to improve grain and forage crops. To date, storage protein biotechnology has been used to address deficiencies of essential amino acids in important crop species, improve the digestibility of forage crops for ruminant animals and improve quality traits such as the bread making quality of wheat dough. High lysine maize is already available through the breeding of storage protein mutants, whilst transgenic crops containing storage proteins that impart a nutritional benefit are currently undergoing field trials. However, despite recent advances in this area, a number of limitations remain. In some cases, the rate of synthesis of particular amino acids limits protein accumulation. In other cases, the introduction of a new storage protein has altered the expression levels of endogenous proteins, potentially affecting desirable traits. In addition, some of the storage proteins targeted for use in transgenic plants have been identified as allergens.

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Transformation of wheat with high molecular weight subunit genes results in improved functional properties

Cited by 289 publications

References 14 publications

Divergent properties of prolamins in wheat and maize

Divergent properties of prolamins in wheat and maize

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

Storage Proteins and their Metabolism

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