Biotechnology: Principles and Products

1. Biotechnology: Principles and Products 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

2. 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 What is Biotechnology? How about some definitions

3. Here we see bean has many • seedcoat colors and patterns • in nature These definitions imply biotechnology is needed because: • Nature has a rich source of variation But we know nature does not have all of the traits we need

4. Fruits with vaccines Nature does not contain all the geneticvariation man desires • Grains with improved nutrition

5. Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene What controls this natural variation? Allelicdifferences for a gene control variation for a specific trait Important definitions for this statement:

6. What is the difference between genes and alleles for Mendel’s Traits? Mendel’s Genes Plant heightSeed shape SmoothWrinkled Allele TallShort Allele

7. Alleles must be: • similar enough to control the same trait • but different enough to create phenotypic • differences for that trait This Implies a Genetic Continuum A direct relationship exists between the gene, its alleles, and the phenotypes (different forms ) of the trait

8. 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

9. 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

10. Protein Trait (or phenotype) Translation DNA (gene) RNA Transcription Central Dogma of Molecular Genetics (The guiding principle that controls trait expression) Seed shape Plant height

11. 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 Plant Biotechnology Requires Two Steps

12. The nucleus contains DNA Gene Manipulation Starts At the DNA Level Source: Access Excellence

13. Double-stranded DNA Chromosomes DNA Is Packaged is condensed into Source: Access Excellence

14. Chromosome Gene Chromosomes Contain Genes Source: Access Excellence

15. 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) Genes Are Cloned Based On:

16. 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

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

18. Biochemical Response of RoundUp Sensitive Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate X Plant EPSP synthase X 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Without amino acids, plant dies X X Aromatic amino acids

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

20. 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 • is an attractive alternative

21. IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Problem: Rice lacks these enzymes Phytoene desaturase ξ-carotene desaturase Lycopene Lycopene-beta-cyclase Normal Vitamin A “Deficient” Rice  -carotene (vitamin A precursor) -Carotene Pathway in Plants

22. IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Vitamin A Pathway is complete and functional Phytoene desaturase ξ-carotene desaturase Lycopene Lycopene-beta-cyclase  -carotene (vitamin A precursor) Golden Rice The Golden Rice Solution -Carotene Pathway Genes Added Daffodil gene Single bacterial gene; performs both functions Daffodil gene

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

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

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

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

27. 1. Gene of interest • The coding region and its controlling elements 2. Selectable marker • Distinguishes transformed/untransformed plants 3. Insertion sequences • Aids Agrobacterium insertion Transformation Cassettes Contains

28. 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 ex.: EPSP -carotene genes Gene of Interest

29. Coding Region Promoter 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] Selectable Marker

30. Plant dies in presence of selective compound Plant grows in presence of selective compound Effect of Selectable Marker Non-transgenic = Lacks NptII or Bar Gene X Transgenic = Has NptII or Bar Gene

31. TL TR Required for proper gene insertions • Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA Insertion Sequences

32. TL TR aphIV 35S Gt1 psy 35S rbcS crtl T-DNA Border Hygromycin Resistance Phytoene Synthase Phytoene Desaturase T-DNA Border Insertion Sequence Selectable Marker Gene of Interest Gene of Interest Insertion Sequence Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette)

33. Agrobacterium Tissue culture required to generate transgenic plants • Gene Gun Delivering the Gene to the Plant • Transformation cassettes are developed in the lab • They are then introduced into a plant • Two major delivery methods

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

35. Gall on stem Gall on leaf 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

36. The Galls Can Be Huge

37. Natural Infection Process Is Complex

38. 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

39. 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

40. Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos • Develop shoots • Root the shoots Transformation Steps Prepare tissue for transformation Introduce DNA • Agrobacterium or gene gun Culture plant tissue Field test the plants • Multiple sites, multiple years

41. The Lab Steps

42. Insect Resistance Cold Tolerance Lab Testing The Transgenics Transgene= Bt-toxin protein Transgene= CBF transcription factors

43. Salt Tolerant Mercury Resistance More Modern Examples Transgene= Glyoxylase I Transgene= Mercuric ion reductase

44. Non-transgenics Transgenics The Next Test Is The Field Herbicide Resistance

45. Before After Final Test Consumer Acceptance RoundUp Ready Corn

46. 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?