Biotechnology: Principles, Applications, and Social Implications From Protein to Product
Biotechnology: Principles, Applications, and Social Implications From Protein to Product The techniques used by the biotechnology industry to modify genes and introduce them into transgenic organisms Phil McClean Department of Plant Science North Dakota State University NDSU Extension What is Biotechnology? How about some definitions General Definition The application of technology to improve a biological organism Detailed Definition The application of the technology to modify the biological function of an organism by adding genes from another organism NDSU Extension These definitions imply biotechnology is needed because: •Nature has a rich source of variation • Here we see bean has many seedcoat colors and patterns in nature NDSU But we know nature does not have all of the traits we need Extension But nature does not contain all the genetic variation man desires •Fruits with vaccines •Grains with improved nutrition NDSU Extension What controls this natural variation? Allelic differences at genes control a specific trait Definitions are needed for this statement: Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene NDSU Extension What is the difference between genes and alleles for Mendel’s Traits? Mendel’s Genes Plant height Seed shape Smooth Wrinkled Allele Tall NDSU Short Allele Extension This Implies a Genetic Continuum A direct relationship exists between the gene, its alleles, and the phenotypes (different forms ) of the trait Alleles must be: • similar enough to control the same trait • but different enough to create different phenotypes NDSU Extension 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 NDSU Extension Allelic Differences for Mendel’s Seed Shape Gene 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 NDSU Extension Central Dogma of Molecular Genetics (The guiding principle that controls trait expression) Protein Trait (or phenotype) Translation Seed shape DNA (gene) Transcription RNA Plant height NDSU Extension In General, Plant Biotechnology Techniques Fall Into Two Classes Gene Manipulation • Identify a gene from another species which controls a trait of interest • Or modify an existing gene (create a new allele) Gene Introduction • Introduces that gene into an organism • Technique called transformation • Forms transgenic organisms NDSU Extension Gene Manipulation Starts At the DNA Level The nucleus contains DNA NDSU Source: Access Excellence Extension DNA Is Packaged Double-stranded DNA is condensed into Chromosomes NDSU Source: Access Excellence Extension Chromosomes Contain Genes Chromosome Gene Source: Access Excellence NDSU Extension Genes Are Cloned Based On: Similarity to known genes Homology cloning (mouse clone used to obtain human gene) Protein sequence Complementary genetics (predicting gene sequence from protein) Chromosomal location Map-based cloning (using genetic approach) NDSU Extension Homology Cloning Clones transferred to filter Human clone library Mouse probe added to filter Hot-spots are human homologs to mouse gene NDSU Extension Complementary Genetics 1. Protein sequence is related to gene sequence NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COOATG GAT-GCT TGG-AGT-AAA C C C G A TCT G C A G 2. The genetic code information is used to design PCR primers Forward primer: 5’-ATGGAT/CGCN-3’ Reverse primer: 5’-T/CTTNC/GT/ACCA-3’ NDSU Notes: T/C = a mixture of T and C at this position; N = a mixture of all four nucleotides Reverse primer is the reverse complement of the gene sequence Extension Complementary Genetics (cont.) 3. Use PCR to amplify gene fragment a. template DNA is melted (94C) 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ b. primers anneal to complementary site in melted DNA (55C) 3’ 5’ 5’ 3’ c. two copies of the template DNA made (72C) 5’ 5’ 3’ NDSU 3’ Extension PCR Animation NDSU Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated Extension PCR Again NDSU Extension Complementary Genetics (cont.) 4. Gene fragment used to screen library Clones transferred to filter Human clone library PCR fragment probe added to filter NDSU Hot-spots are human gene of interest Extension Map-based Cloning 1. Use genetic techniques to find marker near gene 2. Find cosegregating marker 3. Discover overlapping clones (or contig) that contains the marker 4. Find ORFs on contig Gene Marker Gene/Marker Gene/Marker Gene/Marker NDSU 5. Prove one ORF is the gene by Mutant + ORF = Wild type? transformation or mutant analysis Yes? ORF = Gene Extension 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 NDSU Extension 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 NDSU Extension RoundUp Sensitive Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate Plant EPSP synthase X 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Without amino acids, plant dies X X X NDSU Aromatic amino acids Extension RoundUp Resistant Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate Bacterial EPSP synthase RoundUp has no effect; enzyme is resistant to herbicide 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) With amino acids, plant lives NDSU Aromatic amino acids Extension 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 NDSU Extension -Carotene Pathway in Plants IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Problem: Rice lacks these enzymes Phytoene desaturase ξ-carotene desaturase Lycopene Lycopene-beta-cyclase -carotene (vitamin A precursor) NDSU Normal Vitamin A “Deficient” Rice Extension The Golden Rice Solution -Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Daffodil gene Phytoene synthase Phytoene Vitamin A Pathway is complete and functional Phytoene desaturase Single bacterial gene; performs both functions ξ-carotene desaturase Lycopene Daffodil gene -carotene (vitamin A precursor) NDSU Golden Rice Lycopene-beta-cyclase Extension Metabolic Pathways are Complex and Interrelated Understanding pathways is critical to developing new products NDSU Extension Modifying Pathway Components Can Produce New Products Turn On Vitamin Genes = Relieve Deficiency Modified Lipids = New Industrial Oils Increase amino acids = Improved Nutrition NDSU Extension Trait/Gene Examples Gene Trait RoundUp Ready Bacterial EPSP Golden Rice Complete Pathway Plant Virus Resistance Viral Coat Protein Male Sterility Barnase Plant Bacterial Resistance p35 Salt tolerance AtNHX1 NDSU Extension Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants NDSU Extension Transformation Cassettes Contains 1. Gene of interest • The coding region and its controlling elements 2. Selectable marker • Distinguishes transformed/untransformed plants 3. Insertion sequences • Aids Agrobacterium insertion NDSU Extension Gene of Interest Promoter TP Coding Region Promoter Region • Controls when, where and how much the gene is expressed ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development) Transit Peptide • Targets protein to correct organelle ex.: RbCS (RUBISCO small subunit; choloroplast target Coding Region • Encodes protein product NDSU ex.: EPSP -carotene genes Extension Selectable Marker Promoter Coding Region Promoter Region • Normally constitutive ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter Coding Region • Gene that breaks down a toxic compound; non-transgenic plants die ex.: nptII [kanamycin (bacterial antibiotic) resistance] aphIV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance] NDSU Extension Effect of Selectable Marker Non-transgenic = Lacks Kan or Bar Gene Plant dies in presence of selective compound X Transgenic = Has Kan or Bar Gene Plant grows in presence of selective compound NDSU Extension Insertion Sequences TL TR Required for proper gene insertions • Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA NDSU Extension Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette) TL aphIV 35S Gt1 psy 35S rbcS crtl TR T-DNA Border Hygromycin Resistance Phytoene Synthase Phytoene Desaturase T-DNA Border Insertion Sequence Selectable Marker Gene of Interest Gene of Interest Insertion Sequence NDSU Extension Delivering the Gene to the Plant • Transformation cassettes are developed in the lab • They are then introduced into a plant • Two major delivery methods • Agrobacterium • Gene Gun NDSU Tissue culture required to generate transgenic plants Extension Plant Tissue Culture A Requirement for Transgenic Development Callus grows A plant part Is cultured Shoots develop Shoots are rooted; plant grows to maturity NDSU Extension Agrobacterium A natural DNA delivery system • A plant pathogen found in nature • Infects many plant species • Delivers DNA that encodes for plant hormones • DNA incorporates into plant chromosome • Hormone genes expressed and galls form at infection site Gall on stem NDSU Gall on leaf Extension The Galls Can Be Huge NDSU Extension Natural Infection Process Is Complex NDSU Extension But Nature’s Agrobacterium Has Problems Infected tissues cannot be regenerated (via tissue culture) into new plants Why? • Phytohormone balance incorrect regeneration Solution? Transferred DNA (T-DNA) modified by • Removing phytohormone genes • Retaining essential transfer sequences • Adding cloning site for gene of interest NDSU Extension 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 NDSU Extension Transformation Steps Prepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos Introduce DNA • Agrobacterium or gene gun Culture plant tissue • Develop shoots • Root the shoots Field test the plants • Multiple sites, multiple years NDSU Extension The Lab Steps NDSU Extension Lab Testing The Transgenics Insect Resistance Cold Tolerance Transgene= Bt-toxin protein Transgene= CBF transcription factors NDSU Extension More Modern Examples Salt Tolerant Mercury Resistance Transgene= Glyoxylase I Transgene= Mercuric ion reductase NDSU Extension The Next Test Is The Field Herbicide Resistance Non-transgenics Transgenics NDSU Extension Final Test Consumer Acceptance RoundUp Ready Corn After NDSU Before Extension The Public Controversy • Should we develop transgenics? • Should we release transgenics? • Are transgenics safe? • Are transgenics a threat to non-transgenic production systems? NDSU • Are transgenics a threat to natural eco-systems? Extension
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