What is Biotechnology ?

1. 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

2. 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

3. Fruits with vaccines • Grains with improved nutrition But nature does not contain all the geneticvariation man desires

4. Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene What controls this natural variation? Allelic differences at genes control a specific trait Definitions are needed for this statement:

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

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

7. 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

8. 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

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

10. 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 In General, Plant Biotechnology Techniques Fall Into Two Classes

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

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

13. Chromosome Gene Chromosomes Contain Genes Source: Access Excellence

14. 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:

15. Clones transferred to filter Human clone library Mouse probe added to filter Hot-spots are human homologs to mouse gene Homology Cloning

16. 1. Protein sequence is related to gene sequence NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO- ATG 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’ 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 Complementary Genetics

17. a. template DNA is melted (94C) 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ b. primers anneal to complementary site in melted DNA (55C) c. two copies of the template DNA made (72C) Complementary Genetics (cont.) 3. Use PCR to amplify gene fragment

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

19. PCR Again

20. Clones transferred to filter Human clone library PCR fragment probe added to filter Hot-spots are human gene of interest Complementary Genetics (cont.) 4. Gene fragment used to screen library

21. Gene Marker 1. Use genetic techniques to find marker near gene Gene/Marker 2. Find cosegregating marker 3. Discover overlapping clones (or contig) that contains the marker Gene/Marker Gene/Marker 4. Find ORFs on contig 5. Prove one ORF is the gene by transformation or mutant analysis Mutant + ORF = Wild type? Yes? ORF = Gene Map-based Cloning

22. 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

23. 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

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

25. 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

26. 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

27. 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

28. 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

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

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

31. 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

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

33. 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

34. 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

35. 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

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

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

38. 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)

39. 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

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

41. 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

42. The Galls Can Be Huge

43. Natural Infection Process Is Complex

44. 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

45. 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

46. 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

47. The Lab Steps

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

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

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