Annual Progress Report on Malting Barley Research March, 2007

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ligated to GenomeWalker (Clontech Laboratories, Mountain View, CA) adaptors to produce GenomeWalker libraries. Using gene specific primers, based on the 5’ regions of cDNA sequences and two kinds of GenomeWalker adaptor primers, PCR amplifications were performed using general conditions. PCR products were cloned and sequenced. A search for putative cis-acting DNA elements with sequences homologous to the sequences of the cloned products was performed using the PLACE (Plant cis-acting Regulatory DNA Elements) database. Preparation of peptides and antisera Synthetic peptides were commercially prepared (Anaspec, Inc., San Jose, CA) to limited regions (about 15 amino acids) of deduced amino acid sequences of the four highly expressed α-glucosidases. We selected one region of each of the four proteins that we determined to be unique to that protein and that we predicted to be antigenic based on the extent of it’s hydrophobicity. Synthetic peptides were conjugated with keyhole limpet hemocyanin then used to produce antibodies specific to the four isozymes of α-glucosidase from barley. Polyclonal antibodies to those conjugated peptides were produced in rabbits. Western blots Crude protein extracts were made from barley tissues using the protocol of Tibbot et al. (1998). Briefly, the seeds or leaves were ground to a fine powder with liquid nitrogen in a mortar. Approximately one gram of ground tissue was suspended in 1 mL of buffer containing 50 mM Na2CO3/NaHCO3 (pH 9.0), 1 M NaCl, 1% Triton- X100, 2 mM β-mercaptoethanol and 1% protease inhibitor cocktail for plant extracts (Sigma, St. Louis, MO, USA). Suspensions were shaken gently for 1 hr at 4°C. The homogenates were centrifuged at 12,000xg for 10 min at 4°C. Four mL of 50 mM sodium succinate buffer (pH 4.5) were added to the supernatants, which were then dialyzed against the same buffer. After dialysis, extracts were centrifuged again to obtain samples for Western blot analysis. Proteins in these supernatants were resolved by SDS-PAGE, transferred to polyvinylidene fluoride membranes and probed with the rabbit antiserum. Detection of cross reacting proteins were determined using 118
horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology, Beverly, MA, USA) and an ECL-plus Western blotting detection reagent (Amersham Bioscience, Piscataway, NJ) on X-ray film (Roche Diagnostics) according to the manufacturer’s instructions. Results and Discussion To determine in which tissues and developmental stages the four major α- glucosidase genes were expressed, we investigated the temporal gene expression patterns in seeds and leaves. To accomplish this we used quantitative reverse transcriptase polymerase chain reaction (qtRT-PCR) to determine the relative abundance of mRNA transcripts for individual genes. To ensure accurate expression profiling, geometric averaging of transcripts of multiple (3 – 5) internal control genes has been conducted which allows normalization of transcript abundance. We have preliminary data for expression of three of the novel abundantly expressed genes, but we only have complete data for Agl2, upon which we focus in this report, and Agl1, which we have examined for comparative purposes. The results are that Agl1 expression is significantly higher in seeds than in leaves. In contrast, expression levels of Agl2 are the same in seeds and leaves. Furthermore, expression of Agl2 in both seeds and leaves is greatly reduced compared to Agl1 expression levels in seeds. Differences in the levels of expression in seeds of these two genes may result from interesting structural features of the genes or their promoters. We determined that Agl1 contains an amylase element (AE) in its first intron and that Agl2 has no AE anywhere in the structural gene. AE is a cis-element required for α-amylase expression during sugar starvation (Hwang et al., 1998). This difference may influence expression of Agl1 and Agl2 during early stages of germination when sugar levels are low. Additional influence on gene expression may result from differences we’ve found in the promoters of these two genes. The Agl1 promoter contained a gibberellic acid response element (GARE). The promoter of Agl2 did not contain a GARE, which renders it insensitive to the high levels of GA synthesized by the embryo during 119
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Annual Pro
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-i- INTRODUCTION The March 2007 <st
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USDA-ARS NORTHERN CROP SCIENCE LABO
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Spring Regional over two years at D
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Table 1. 2006 UCD Two-Rowed Malting
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environments was practiced, 2) elit
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Table 4. Selected six-rowed lines c
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Other Barley Research: Feed Program
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Executive Summary AMBA Prog
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Table 1. Agronomic performance of M
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Lines M122 and M124 were rated sati
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EARLY GENERATION LINES Forty F5 pop
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have evaluated a PCR based marker,
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Edward Schiefelbein, Research Scien
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small grains pathology input to thi
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Project Personnel Small Grains Path
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Investigations on Barley Diseases a
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Minnesota barley disease survey and
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pathotypes at the seedling stage in
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germplasm with the highest level of
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Mirlohi, A., Brueggeman, R., Steffe
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Developing Improved Malt Barley Var
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Table 2. Montana’s Statewide Tria
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As a Barley CAP collaborator, our C
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3.2. A barley nursery composed of d
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5. Related Barley Projects Barley P
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Barley Breeding - Advanced Testing
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Table 3. Agronomic comparisons of N
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Preliminary Malting Barley Yield Tr
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dwarf genotypes with the convention
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STUDIES ON BARLEY DISEASES AND THEI
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inoculated plots. The inoculum used
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much of North Dakota in 2006, few f
Page 67 and 68: 5. Collect isolates of barley patho
Page 69 and 70: oot rot. In collaboration with Scot
Page 71 and 72: 2. United States Wheat and Barley S
Page 73 and 74: 6. Lee, S. and Neate, S.M. (2006) P
Page 75 and 76: Objectives Met in the One-Year Fund
Page 77 and 78: These automated methods have been t
Page 79 and 80: Beta-limit dextrin Alpha-Amylase Al
Page 81 and 82: M w kDa 800 600 400 200 0 0.4 0 2 4
Page 83 and 84: Figure 7. Time course of I2-binding
Page 85 and 86: 10 9 8 7 6 5 4 3 2 1 0 1993 1994 19
Page 87 and 88: 2. Other Research (A) Predicting Se
Page 89 and 90: forms (through enzymolysis during m
Page 91 and 92: PUBLICATIONS (2006-07) Schwarz, P.B
Page 93 and 94: CI9214, PI552963 (Heartland), and N
Page 95 and 96: showing good levels of resistance t
Page 97 and 98: Germplasm Enhancement for RWA Resis
Page 99 and 100: Gary Hein in Colorado and Nebraska.
Page 101 and 102: Kevin Hicks, Research Leader, USDA-
Page 103 and 104: Detailed Report on
Page 105 and 106: Other Barley Research and Future Di
Page 107 and 108: Table 1. 2006/2007 Oregon winter ba
Page 109 and 110: Table 4. Agronomic data for OSU win
Page 111 and 112: MALTING QUALITY ANALYSIS OF NEW BAR
Page 113 and 114: We recently secured an industrial c
Page 115 and 116: 2006-2007 Progress
Page 117: enzyme it encodes. We found that se
Page 121 and 122: found in plant cell walls. The degr
Page 123 and 124: Establishing that sequence variatio
Page 125: Charles Karpelenia, technician Joe
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Annual Progress Report on Malting Barley Research March, 2007

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