F10 - BIOL115E.A - Plant Mol Genetics Biotech

Topic outline

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  General

  • News forum
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  Topic 1

  Course documents

  • Syllabus File
  • How to read primary literature File
  • How to give an academic talk URL
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  Topic 2

  Lab resources

  For amiRNA projects we will synthesize the amiRNAs as Gateway entry vectors by including truncated attL arms in the synthesized genes. As a group you need to think about a control for this approach - how will you know that this works? Have a look at this article for some ideas. Here are the amiRNA designer links for BOB2 alone and for BOB1 and BOB2 as targets.

  For sHSP:GFP projects we will PCR genomic sHSP clones with a CACC tail for directional cloning into pENTR/D-TOPO. A list of heat induced sHSPs can be found below. Use the Affy-ID to find a gene locus identifier using this tool. Once you have identified your gene, click through to the locus detail page and then click on the sequence viewer link (under the map links category). Click on the sequence ruler bar above your gene to retrieve a coded genomic sequence. Copy and paste the genomic region starting at the upstream gene into Primer3 (link below) and design primers which amplify the promoter and gene. Make sure that you add a CACC on the 5' end and that you remove the stop codon.

  Once entry vectors have been created they will be recombined into pFAST destination vectors using an LR reaction.

  Primers will be designed using Primer3. Adjust parameters so that the Tm is 65 degrees.

  • DNA sequencing at Yale URL
  • Gateway cloning URL
  • WMD3 amiRNA design tools URL
  • Gene silencing using amiRNAs File
  • pFAST vectors File
  • amiRNA gene synthesis scheme File
  • List of heat induced genes in Arabidopsis from TPJ 41:1 (2005) File
  • First round of sequencing results File
  • Summary of first round of sequencing File
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  Topic 3

  Week 13 - Phytoremediation and perennialization

  Ben will present a pair of paper investigation the use of plant for remediation of mercury and selenium pollution.

  Jesse will present an overview of perennialization and a pair of papers (to be posted soon) investigating the scientific issues involved in breeding for this trait.

  • Phytoremediation of Mercury and Organomercurials in Chloroplast Transgenic Plants: Enhanced Root Uptake, Translocation to Shoots, and Volatilization, Hussein et al., 2007 File
  • Transgenic Indian Mustard Overexpressing Selenocysteine Lyase or Selenocysteine Methyltransferase Exhibit Enhanced Potential for Selenium Phytoremediation under Field Conditions, Banuelos et al., 2007 File
  • Phytoremediation, Pilon-Smits 2005 File
  • Prospects for developing perennial grain crops, Cox et al., 2006 File
  • PEP1 regulates perennial flowering in Arabis alpina, Wang et al., 2009 File
  • A single chromosome addition from Thinopyrum elongatum confers a polycarpic, perennial habit to annual wheat, Lammer et al., 2004 File
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  Topic 4

  Week 12 - Glyphosate and glyphosate resistance and an investigation of mapping approaches which can be used for MAS

  Michael will present a pair of papers and a short review investigating the mechanism of glyphosate action and how plants evolve resistance to this important herbicide.

  Elan will present a pair of QTL papers and compare and contrast this approach to the NAM design from the Buckler lab.

  • Investigating the mechanisms of glyphosate resistance in Lolium multiflorum, Perez-Jones et al., 2007 File
  • The Site of the Inhibition of the Shikimate Pathway by Glyphosate, Amrhein et al., 1980 File
  • Evolved Glyphosate Resistance in Plants: Biochemical and Genetic Basis of Resistance, Powles and Preston, 2006 File
  • Genetic Analysis of the Morphological Differences Between Maize and Teosinte. Doebley and Stec, 1991 File
  • fw2.2: A Quantitative Trait Locus Key to the Evolution of Tomato Fruit Size, Frary et al., 2000 File
  • Status and Prospects of Association Mapping in Plants, Zhu et al., 2008 File
  • Genetic design and statistical power of nested association mapping in Maize, Yu et al., 2008 File
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  Topic 5

  Week 11 - Biofortification of Vitamin A

  Rosalie and Camille will discuss a variety of approaches for Vitamin A biofortification. Technologies used include both transgenic as well as MAB strategies, which in turn require association mapping and QTL data.

  • Improving the nutritional value of Golden Rice through increased pro-vitamin A content, Paine et al., 2005 File
  • Natural Genetic Variation in Lycopene Epsilon Cyclase Tapped for Maize Biofortification, Harjes et al., 2008 File
  • Seed-Specific Overexpression of an Endogenous Arabidopsis Phytoene Synthase Gene Results in Delayed Germination and Increased Levels of Carotenoids, Chlorophyll, and Abscisic Acid, Lindgren et al., 2003 File
  • Provitamin A Accumulation in Cassava (Manihot esculenta) Roots Driven by a Single Nucleotide Polymorphism in a Phytoene Synthase Gene, Welsch et al., 2010 File
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  Topic 6

  Week 10 - Abiotic stress Julia and Omer will present a series of papers investigating the role of the DREB family of TFs in responses to abiotic stresses including drought, salt, and high and low temperatures. Background reading about abiotic stress in plants and the role of the plant hormone ABA can be found in Plant Physiology by Taiz and Zeiger.

  • Two Transcription Factors, DREB1 and DREB2, with an EREBP/AP2 DNA Binding Domain Separate Two Cellular Signal Transduction Pathways in Drought- and Low- Temperature-Responsive Gene Expression, Respectively, in Arabidopsis. Liu et al., 1998 File
  • Arabidopsis CBF3/DREB1A and ABF3 in Transgenic Rice Increased Tolerance to Abiotic Stress without Stunting Growth, Oh et al., 2005 File
  • Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression. Sakuma et al., 2006 File
  • STRESS RESPONSE SUPPRESSOR1 and STRESS RESPONSE SUPPRESSOR2, Two DEAD-Box RNA Helicases That Attenuate Arabidopsis Responses to Multiple Abiotic Stresses., Kant et al., 2007 File
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  Topic 7

  Week 9 - Energy II

  Rosalie will present a pair of papers looking at lignin modification in trees and the effects of these modifications on the feeding preference and growth performance of insect herbivores.

  Camille will present a pair of papers about photosynthesis - the source of all of the biological energy we are discussing in the course. The first paper discusses increasing photosynthetic efficiency by optimizing the amount of light harvesting complexes produced in green algae. The second paper involves using nanotechnology to directly harvest high-energy electrons from the thylakoid membrane. It's been a while since Bio25, so you may be thinking to yourself "What is a thylakoid membrane?". A good review of the light reactions of photosynthesis can be found in chapter 7 of Plant Physiology by Taiz and Zeiger (QK711.2 .T35 2006 in the oversize section). I also have a copy of this book which you can borrow from me.

  • Does lignin modification affect feeding preference or growth performance of insect herbivores in transgenic silver birch, Tiimonen et al., 2005. File
  • Combinatorial modification of multiple lignin traits in trees through multigene cotransformation, Li et al., 2003. File
  • Direct Extraction of Photosynthetic Electrons from Single Algal Cells by Nanoprobing System, Ryu et al., 2010 File
  • Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion, Mussgnug et al., 2007 File
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  Topic 8

  Week 8 (after fall break) - Energy I

  Jesse will present an overview of current and next generation biofuels (Gressel) .  He will focus on Jatropha as a next generation oil seed plant and how Jatropha yields might be improved by manipulating BR signaling.  Please read the review as well as the two research papers - one is quite short and straightforward.

  Elan will present a pair of papers showing how modification of lignin biosynthesis can improve biofuel yields.

  The Sticklen review provides a good overview of the technical problems involved in producing cellulosic biofuels. This is an optional reading which I would strongly encourage you to read if you have any questions about cellulosic biofuels.

  • Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol, Sticklen 2008. (optional) File
  • Lignin modification improves fermentable sugar yields for biofuel production, Chen and Dixon, 2007 File
  • Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wallbound ferulic acid in alfalfa (Medicago sativa L.), Chen et al., 2006 File
  • Transgenics are imperative for biofuel crops, Gressel 2008. File
  • Plant regeneration through the direct induction of shoot buds, Kumar and Reddy, 2010. File
  • An Arabidopsis Brassinosteroid-Dependent Mutant Is Blocked in Cell Elongation, Azpiroz et al., 1998. File
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  Topic 9

  Week 5 - Nitrogen

  Ben will tell us about one of the normal functions of alanine aminotransferases (AlaAT) as well as about improving NUE by expressing AlaAT in the root, where nitrogen is absorbed from the ground.

  Omer will tell us about increasing NUE by expressing and early nodulation gene. What is an early nodulation gene (ENOD) you may ask? He'll tell us about that too.

  • Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana, Miyashita et al., 2007 File
  • Engineering nitrogen use efficiency with alanine aminotransferase, Good et al., 2007 File
  • Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling, Bi et al., 2009 File
  • Dg93, a Nodule-Abundant mRNA of Datisca glomerata with Homology to a Soybean Early Nodulin Gene, Okabura et al., 2000 File
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  Topic 10

  Week 4 - Biotic stress

  Michael will present a pair of papers about resistance to the brown planthopper, a very serious rice pest. The resistance is mediated by callose deposition and requires Bph14, a gene which confers planthopper resistance as a transgene.

  Julia will present a pair of papers about resistance to plum pox virus (discussed in Tomorrow's Table pp. 165-167).  The first paper (Tang et al.) investigates the molecular events that mediate PTGS, the second (Hily et al.) shows how PTGS can be used to engineer virus resistant plum strains.

  • Herbivore-Induced Callose Deposition on the Sieve Plates of Rice: An Important Mechanism for Host Resistance, Hao et al., 2008 File
  • Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice, Du et al., 2009 File
  • A biochemical framework for RNA silencing in plants, Tang et al., 2003 File
  • Stability of gene silencing-based resistance to Plum pox virus in transgenic plum (Prunus domestica L.) under field conditions, Hily et al., 2004 File
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  Topic 11

  Week 3 - The devil is in the details - the Monarch butterfly story

  This week we will discuss the Monarch butterfly/Bt corn story as a way of investigating the importance of the specific details when trying to assess the possible ecological effects of transgenic technologies.

  - Everyone should read Losey et al., Jesse and Obrycki, and Gatehouse et al.
  - Everyone should bring in an example of popular press coverage of this issue from 1999 for discussion.
  - Come prepared to present the key points of two of the shared papers. The shared papers are:
      Hellmich et al. (Omer, Rosalie, Camille),
      Oberhauser et al. (Camille, Amy, Elan, Michael),
      Pleasants et al. (Jesse, Elan, Julia),
      Sears et al. (Amy, Ben),
      Stanley-Horn et al. (Ben, Jesse, Rosalie), and
      Zangerl et al. (Omer, Julia, Michael)
  - If you are interested in reading more about risk assessment, you can find more information in Andow and Zwahlen (completely optional reading).

  • Transgenic pollen harms monarch larvae. Losey et al., 1999 File
  • Field deposition of Bt transgenic corn pollen: lethal effects on the monarch butterfly. Jesse and Obrycki, 2000 File
  • The case of the monarch butterfly: a verdict is returned. Gatehouse et al., 2002 File
  • Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillar under field conditions. Zangerl et al., 2001 File
  • Assessing the impact of Cry1Ab-expressing corn pollen on monarch butterfly larvae in field studies. Stanley-Horn et al., 2001 File
  • Impact of Bt corn pollen on monarch butterfly populations: A risk assessment. Sears et al., 2001 File
  • Corn pollen deposition on milkweeds in and near cornfields. Pleasants et al., 2001 File
  • Temporal and spatial overlap between monarch larvae and corn pollen. Oberhasuer et al., 2001 File
  • Monarch larvae sensitivity to Bacillus thuringiensis-purified proteins and pollen. Hellmich et al., 2001 File
  • Assessing environmental risks of transgenic plants. Andow and Zwahlen, 2006 File
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  Topic 12

  Week 2 - Short talks on a variety of topics
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  Topic 13

  Week 1 - Biotechnology and sustainability overview (Nick)

  • Greenhouse gas mitigation by agricultural intensification. Burney et al., 2010 File
  • Increasing crop productivity to meet global needs for feed, food, and fuel. Edgerton 2009. File
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  Topic 14

  An evolving list of possible topics for your presentations during the semester. Each topic has an attached review to get you started finding the relevant literature. If you have something to add to this list please email it to Nick and I will add more as I find them.

  • Marker assisted selection File
  • Besting Johnny Appleseed - using biotechnology in fruit breeding File
  • Food and ecosystem security via perennial grains File
  • Cell walls as biofuel feedstocks File
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  Topic 15