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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF BARLEY SEED QUALITY THROUGH MOLECULAR AND FUNCTIONAL GENOMIC ANALYSIS OF GENE EXPRESSION

Location: Cereal Crops Research

Project Number: 3655-21430-008-00
Project Type: Appropriated

Start Date: Jan 09, 2007
End Date: Sep 30, 2010

Objective:
Develop transformation expression vectors to target transgene expression to tissues initially infected by Fusarium graminearum. Identify genes upregulated during infection of seed lemma and epicarp; clone and test their promoters for inducibility and tissue-specificity. Develop antifungal genes to use in targeted expression system to develop Fusarium-resistant barley. Identify components of the GA response to predictors of malting quality. Determine whether the gene for a barley aleurone GA receptor can be used as an indicator of malting quality. Analyze gene responses to GA in malting barley and GA response mutants under malting conditions.

Approach:
Produce gene macroarrays from our lemma-specific gene library and a new epicarp-specific library. Probe libraries with cDNA from Fusarium-infected lemma and epicarp. Clone and identify the upregulated genes, and confirm tissue-specificity with RNA blots. A modified inverse PCR will be used to clone their promoters from barley. Promoter (upstream) regions will be ligated upstream of the green fluorescent protein gene in an expression vector and functionally confirmed in transient bombardment assays where tissues will be examined for fluorescence before and after infection with Fusarium. If successful, barley will be stably transformed with antifungal protein genes driven by these promoters using the Agrobacterium vector pRSHyg. These genes (cloned in this lab) include lemma thionin, Ltp, and germin. Transformants will be tested for Fusarium resistance. The gene for the barley gibberellin (GA) hormone receptor will be cloned using homologies to the rice receptor. The gene will be compared in GA response mutants. Receptor sequence and mRNA levels will be analyzed in barleys of varying malting qualities. The Barley1 GeneChip will be used to examine transcripts in 7 malting barleys and GA response mutants. The differences in transcript profiles will provide insights into the relationship of the GA signal transduction pathway and malting quality. The new biological safefy registration protocol number is SC04-149R. It expires 10/6/07.

Last Modified: 8/27/2014
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