2013 Annual Report
1a.Objectives (from AD-416):
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.
1b.Approach (from AD-416):
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.
Temporal studies on barley seed storage proteins and beta-amylase are underway to determine if there is a difference between the accumulation of transcript and protein in seed storage proteins and beta-amylase. Beta-amylase is an important enzyme to the malting and brewing industry and is the main contributor to diastatic power (indicator of malt quality). Beta-amylase activity is positively correlated with grain protein. However, grain and malt protein levels have to stay within an acceptable range due to downstream problems in brewing. Identifying temporal accumulation differences between hordein (main seed storage protein in barley) and beta-amylase will give insight into breeding for high beta-amylase lines without increasing grain and malt protein levels above the acceptable range. Collection has begun on developing grains at numerous time points throughout development. Beta-amylase and hordein extractions have been optimized. These extractions have been completed for one biological repetition and collections for three biological repetitions. Analysis of the barley beta-amylase 1 (Bmy1) promoter has begun and substantial progress has been made. A series of eight vectors has been created to study the Bmy1 promoter. The first vector contains the entire known Bmy1 promoter region from an elite malting barley cultivar (Legacy) upstream of the green fluorescent protein. The subsequent seven vectors contain sequentially shortened versions of the full Bmy1 promoter ranging from 503 base pairs to 22 base pairs. Additionally, two control vectors using the ubiquitin promoter from maize and the D hordein promoter from barley have been created. The vectors are inserted into developing barley grains via microparticle bombardment. Optimization of this technique is currently underway and parameters are being adjusted. Disproportionating enzymes can utilize a variety of sizes of short chain sugars as its substrate. Interestingly, it has the potential to generate substrate for beta-amylase during mashing (process of mixing milled grain with water to solubilize storage compounds into a nutrient rich broth used to make beer). The gene for disproportionating enzyme 1 (DPE1) has not been completely sequenced in barley. We have begun sequencing the DPE1 gene and have extended the previously reported sequence of 300 base pairs to 2700 base pairs out of the estimated 4000 base pairs. Sequencing the entire DPE1 gene will enable further studies that will determine the importance of DPE1 to the malting and brewing industry.
Analysis of giberellic acid (GA) mutants in barley. GA-mutants were created using ethyl methanesulfonate. These GA-mutants were able to germinate and produce alpha-amylase and therefore appeared to circumvent GA-dependent germination. These two mutants were analyzed for bioactive GA as well as non-bioactive GA precursors. One GA-mutant was found to have GA synthesis arrested at the beginning of the GA synthesis pathway, whereas the other mutant was found to have a mutation in a precursor thus preventing synthesis of bioactive GA. These mutants are extremely novel and will enable researchers to study and further understand the germination and malting processes.
Regulation of a malting quality gene. Beta-amylase is one of the most important starch degrading enzymes involved with fermentable sugar production during mashing. Beta-amylase kinetics has been extensively studied in the past, however, very little is known about the regulation of the gene (Bmy1) during grain development. ARS researchers at the Cereal Crops Research Unit in Madison, WI sequenced the Bmy1 promoter from four diverse genotypes. Gene promoters contain the information needed to turn genes on and off. A series of deletions in the Bmy1 promoter have been created to identify important sequences that may be involved in turning the Bmy1 gene on and/or off. Understanding the regulation of Bmy1 will give breeders the knowledge and tools to successfully increase the amount of beta-amylase without increasing grain protein levels to unacceptable industry values.