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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: 5090-21430-009-00
Project Type: Appropriated

Start Date: Oct 01, 2010
End Date: Dec 31, 2014

Objective:
Objective 1: Develop transformation expression vectors to target transgene expression specifically to tissues that are initially infected by Fusarium graminearum. Objective 2: Develop antifungal candidate genes and gene constructs that can be used in targeted expression system to develop Fusarium-resistant barley. Objective 3: Identify components of the GA response that can be used as predictors of malting.

Approach:
Produce gene macroarrays from our lemma-specific gene library and a new epicarpspecific 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.

Last Modified: 10/24/2014
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