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United States Department of Agriculture

Agricultural Research Service

Title: Cloning of the Promoter for a Novel Barley Gene, Lem1, and Its Organ Specific Promotion of Gfp Expression in Lemma and Palea

Authors
item Skadsen, Ronald
item Sathish, Puthigae - UNIVERSITY OF WISCONSIN
item Federico, Maria - UNIVERSITY OF WISCONSIN
item Kaeppler, Heidi - UNIVERSITY OF WISCONSIN

Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 26, 2001
Publication Date: N/A

Interpretive Summary: The fungal pathogen, Fusarium graminearum, is a major cause of poor barley and wheat grain quality U.S. There are no barley or wheat varieties that produce proteins or chemicals that can provide a barrier to colonization by this pathogen. The fungus colonizes the leafy scale (the lemma) that surrounds the developing floral structures and, later, the seed. As it grows inward, it destroys the seed and deposits toxins. The interior of barley and wheat seeds produces dietary proteins that can rapidly destroy the fungus. If these proteins can also be produced in the lemma, they may provide an antifungal barrier. To do this, it is necessary to discover genes that are preferentially turned on in the lemma. Then, the section of DNA that controls this specificity must be cloned. We have discovered such a gene (Lem1) and cloned the adjoining section of DNA, its promoter, which causes it to be active only in the lemma. To show that it works as expected, the promoter was attached to a gene that encodes a green fluorescent protein (GFP) from jellyfish. This recombinant DNA was delivered into lemmas and vegetative leaves of barley and wheat. The tissues were viewed under blue light. In the microscope, hundreds of bright green fluorescent cells could be seen in the lemma, but not in leaves, proving that the promoter works. By combining this promoter to antifungal protein genes, it is possible to produce barley that is resistant to Fusarium. This will be of benefit to farmers, malting companies and brewers.

Technical Abstract: The differential display method was used to identify a novel barley gene, Lem1, expressed primarily in the outer leafy organs (lemma and palea) that enclose developing florets and seeds. The promoter was isolated from a BAC genomic clone and used in a translational fusion with a green fluorescent protein gene (Gfp) to produce a transient expression vector. Following particle bombardment, Gfp was expressed only in lemmas, paleas and awns of developing spikes. Lem1 did not promote Gfp expression in vegetative leaves or in mature spikes, although expression of co-bombarded uidA (GUS) occurred under the regulation of a ubiquitin promoter. This reproduced the developmentally regulated pattern of mRNA accumulation. Lem1 was found to be a single intronless gene encoding an acidic 102 amino acid protein. Its deduced signal peptide, hydrophobic nature and possible transmembrane domains indicate that it is a membrane-associated protein. The promoter of Lem1 contains previously identified auxin and ethylene response elements, which may participate in its developmental regulation. In a two-row barley, in which lateral spikelets fail to develop, Lem1 mRNA was absent in the lateral spiketlets and present only in the developing median spikelets. This suggests that Lem1 may play a role in the normal development of the spikelet.

Last Modified: 10/25/2014
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