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United States Department of Agriculture

Agricultural Research Service

Research Project: PRODUCTION OF WHEAT GERMPLASM WITH ENHANCED BAKING QUALITY Title: Variant high-molecular-weight glutenin subunits arising from biolistic transformation of wheat

Authors
item Blechl, Ann
item Vensel, William

Submitted to: Journal of Cereal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 18, 2013
Publication Date: May 1, 2013
Citation: Blechl, A.E., Vensel, W.H. 2013. Variant high-molecular-weight glutenin subunits arising from biolistic transformation of wheat. Journal of Cereal Science. 57:496-503.

Interpretive Summary: Wheat flour proteins called high-molecular-weight (HMW)-glutenins provide strength and elasticity for bread and noodle-making. In order to improve dough strength, biotechnologists have used the biolistic method (“gene gun”) of genetic transformation to introduce more copies of the natural genes that encode these proteins into wheat varieties with low bread-making potential. Some of the seeds from the genetically engineered plants contain proteins that are bigger or smaller than the natural proteins. To determine the identity of those new proteins, we used tandem mass spectrometry to detect peptide fragments produced after digesting them with the enzyme trypsin. We found that the new proteins were HMW-glutenins that had lost or gained part of their repeating amino acid domains. These bigger and smaller HMW-glutenins were formed by an unknown process after introduction of the DNA into the wheat embryo cells. The proteins participate in the gluten polymer that forms the backbones of doughs. Further investigation is required to determine whether or not they improve dough strength.

Technical Abstract: Genetic transformation via the biolistic method has been used to introduce genes encoding natural and novel high-molecular-weight glutenin subunits (HMW-GS) into wheat. The appearance of new seed proteins of sizes not predicted by the transgene coding sequences has been noted in some of these experiments. In this report, the identities of thirteen of these novel proteins were determined by tandem mass spectrometry (MS/MS). In two lines transformed with a plasmid carrying the gene encoding subunit 1Dx5, six different proteins larger than and one protein smaller than the native protein were shown to contain peptides from 1Dx5. A novel protein found in some progeny of crosses between a plant transformed with the plasmid encoding 1Dy10 and Great Plains winter wheats was larger than but contained several peptides from 1Dy10. Four lines derived from transformations that included both plasmids were analyzed. In each of two lines, a protein larger than native 1Dx5 was shown to have peptides from 1Dx5. In one line, a protein larger than and a protein smaller than HMW-GS each contained peptides from the N- and C-terminus of 1Dx5 as well as from the repeat region of 1Dy10. In the fourth line derived from transformation with the plasmids encoding 1Dx5 and 1Dy10, the native Bx7 gene was apparently replaced by a gene that encodes a larger version of 1Bx7. The variant proteins accumulate in the polymeric protein fraction that is insoluble in 50% propanol, indicating that they can form inter-molecular disulfide bonds. These results show that the novel proteins found in some transformants are encoded by altered versions of either the transforming or endogenous HMW-GS genes. Such size expansions and contractions are fairly common during biolistic transformation with these genes; they are detected in one fifth (6 of 30) of the events made in this laboratory. The altered HMW-GS transgenes are potential sources of new genetic variability for these proteins that play crucial roles in wheat end-use properties.

Last Modified: 12/20/2014
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