techniques-of-biotechnology-mcclean-good

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Biotechnology: Principles, Applications, and Social Implications From Protein to Product Phil McClean Department of Plant Science North Dakota State University The techniques used by the biotechnology industry to modify genes and introduce them into transgenic organisms

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What is Biotechnology? How about some definitions

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But we know nature does not have all of the traits we need Nature has a rich source of variation These definitions imply biotechnology is needed because:

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But nature does not contain all the genetic variation man desires

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What controls this natural variation? Allelic differences at genes control a specific trait Definitions are needed for this statement:

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What is the difference between genes and alleles for Mendel’s Traits? Mendel’s Genes Plant height Seed shape Tall Short Allele Smooth Wrinkled Allele

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This Implies a Genetic Continuum A direct relationship exists between the gene, its alleles, and the phenotypes (different forms ) of the trait

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Allelic Differences for Mendel’s Genes Plant Height Gene Gene: gibberellin 3--hydroxylase Function: adds hydoxyl group to GA20 to make GA1 Role of GA1: regulates cell division and elongation Mutation in short allele: a single nucleotide converts an alanine to threonine in final protein Effect of mutation: mutant protein is 1/20 as active

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Gene: strach branching enzyme (SBE) isoform 1 Function: adds branch chains to starch Mutation in short allele: transposon insertion Effect of mutation: no SBE activity; less starch, more sucrose, more water; during maturation seed looses more water and wrinkles Allelic Differences for Mendel’s Seed Shape Gene

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Central Dogma of Molecular Genetics (The guiding principle that controls trait expression)

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In General, Plant Biotechnology Techniques Fall Into Two Classes

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Gene Manipulation Starts At the DNA Level Source: Access Excellence

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DNA Is Packaged Source: Access Excellence is condensed into

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Chromosomes Contain Genes Source: Access Excellence

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Genes Are Cloned Based On:

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Homology Cloning

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Complementary Genetics

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3. Use PCR to amplify gene fragment Complementary Genetics (cont.)

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Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated PCR Animation

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PCR Again

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Complementary Genetics (cont.) 4. Gene fragment used to screen library

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Map-based Cloning

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Gene Manipulation It is now routine to isolate genes But the target gene must be carefully chosen Target gene is chosen based on desired phenotype Function: Glyphosate (RoundUp) resistance EPSP synthase enzyme Increased Vitamin A content Vitamin A biosynthetic pathway enzymes

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The RoundUp Ready Story Glyphosate is a broad-spectrum herbicide Active ingredient in RoundUp herbicide Kills all plants it come in contact with Inhibits a key enzyme (EPSP synthase) in an amino acid pathway Plants die because they lack the key amino acids A resistant EPSP synthase gene allows crops to survive spraying

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+ Glyphosate X RoundUp Sensitive Plants X X Without amino acids, plant dies X

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Bacterial EPSP synthase Shikimic acid + Phosphoenol pyruvate 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) Aromatic amino acids RoundUp Resistant Plants + Glyphosate With amino acids, plant lives RoundUp has no effect; enzyme is resistant to herbicide

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The Golden Rice Story Vitamin A deficiency is a major health problem Causes blindness Influences severity of diarrhea, measles >100 million children suffer from the problem For many countries, the infrastructure doesn’t exist to deliver vitamin pills Improved vitamin A content in widely consumed crops an attractive alternative

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-Carotene Pathway in Plants

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The Golden Rice Solution Daffodil gene Single bacterial gene; performs both functions Daffodil gene -Carotene Pathway Genes Added

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Metabolic Pathways are Complex and Interrelated Understanding pathways is critical to developing new products

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Modifying Pathway Components Can Produce New Products Modified Lipids = New Industrial Oils Turn On Vitamin Genes = Relieve Deficiency Increase amino acids = Improved Nutrition

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Trait/Gene Examples RoundUp Ready Bacterial EPSP Golden Rice Complete Pathway Plant Virus Resistance Viral Coat Protein Male Sterility Barnase Plant Bacterial Resistance p35 Salt tolerance AtNHX1 Trait Gene

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Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants

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Transformation Cassettes Contains

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Gene of Interest

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Selectable Marker

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Effect of Selectable Marker Transgenic = Has Kan or Bar Gene Non-transgenic = Lacks Kan or Bar Gene X

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Insertion Sequences

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Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette)

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Transformation cassettes are developed in the lab They are then introduced into a plant Two major delivery methods Delivering the Gene to the Plant

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Plant Tissue Culture A Requirement for Transgenic Development A plant part Is cultured Callus grows Shoots develop Shoots are rooted; plant grows to maturity

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Agrobacterium A natural DNA delivery system A plant pathogen found in nature Hormone genes expressed and galls form at infection site Delivers DNA that encodes for plant hormones Infects many plant species DNA incorporates into plant chromosome

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The Galls Can Be Huge

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Natural Infection Process Is Complex

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But Nature’s Agrobacterium Has Problems Infected tissues cannot be regenerated (via tissue culture) into new plants Transferred DNA (T-DNA) modified by Removing phytohormone genes Retaining essential transfer sequences Adding cloning site for gene of interest Phytohormone balance incorrect regeneration Solution? Why?

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The Gene Gun DNA vector is coated onto gold or tungsten particles Particles are accelerated at high speeds by the gun Particles enter plant tissue DNA enters the nucleus and incorporates into chromosome Integration process unknown

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Transformation Steps Prepare tissue for transformation Introduce DNA Culture plant tissue Field test the plants Agrobacterium or gene gun Multiple sites, multiple years

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The Lab Steps

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Lab Testing The Transgenics Transgene= Bt-toxin protein Transgene= CBF transcription factors

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More Modern Examples Transgene= Glyoxylase I Transgene= Mercuric ion reductase

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The Next Test Is The Field Herbicide Resistance

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Final Test Consumer Acceptance RoundUp Ready Corn

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The Public Controversy Should we develop transgenics? Should we release transgenics? Are transgenics safe? Are transgenics a threat to non-transgenic production systems? Are transgenics a threat to natural eco-systems?