GENE EXPRESSION AND BASIC TRANSFORMATION WHY DO WE

GENE EXPRESSION AND BASIC TRANSFORMATION

WHY DO WE NEED BIOTECHNOLOGY ? • Nature has a rich source of variation • Here we see bean has many seedcoat colors and patterns in nature But we know nature does not have all of the traits we need

BUT NATURE DOES NOT CONTAIN ALL THE GENETIC VARIATION MAN DESIRES • Fruits with vaccines • Grains with improved nutrition

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

WHAT IS THE DIFFERENCE BETWEEN GENES AND ALLELES FOR MENDEL’S TRAITS? Mendel’s Genes Plant height Seed shape Smooth Wrinkled Allele Tall Short Allele

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

ALLELIC DIFFERENCES FOR MENDEL’S GENES PLANT HEIGHT GENE Gene: gibberellin 3 - -hydroxylase Function: adds hydoxyl group to GA 20 to make GA 1 Role of GA 1: 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

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

CENTRAL DOGMA OF MOLECULAR GENETICS (The guiding principle that controls trait expression) Trait (or phenotype) Protein Translation Seed shape DNA RNA Transcription (gene) Plant height

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

GENE MANIPULATION STARTS AT THE DNA LEVEL The nucleus contains DNA Source: Access Excellence

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

CHROMOSOMES CONTAIN GENES Chromosome Gene Source: Access Excellence

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)

HOMOLOGY CLONING Clones transferred to filter Human clone library Mouse probe added to filter Hot-spots are human homologs to mouse gene

COMPLEMENTARY GENETICS 1. Protein sequence is related to gene sequence NH 3+-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’ 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

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

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

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

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

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 (Round. Up) resistance EPSP synthase enzyme Increased Vitamin A content Vitamin A biosynthetic pathway enzymes

THE ROUNDUP READY STORY • Glyphosate is a broad-spectrum herbicide • Active ingredient in Round. Up 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

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

ROUNDUP RESISTANT PLANTS Shikimic acid + Phosphoenol pyruvate + Glyphosate Bacterial EPSP synthase Round. Up has no effect; enzyme is resistant to herbicide 3 -enolpyruvyl shikimic acid-5 -phosphate (EPSP) With amino acids, plant lives Aromatic amino acids

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

-CAROTENE PATHWAY IN PLANTS IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Problem: Rice lacks these enzymes Phytoene desaturase ξ-carotene desaturase Lycopene-beta-cyclase Normal Vitamin A “Deficient” Rice -carotene (vitamin A precursor)

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

METABOLIC PATHWAYS ARE COMPLEX AND INTERRELATED Understanding pathways is critical to developing new products

MODIFYING PATHWAY COMPONENTS CAN PRODUCE NEW PRODUCTS Turn On Vitamin Genes = Relieve Deficiency Modified Lipids = New Industrial Oils Increase amino acids = Improved Nutrition

TRAIT/GENE EXAMPLES Trait Gene Round. Up Ready Bacterial EPSP Golden Rice Complete Pathway Plant Virus Resistance Viral Coat Protein Male Sterility Barnase Plant Bacterial Resistance p 35 Salt tolerance At. NHX 1

INTRODUCING THE GENE OR DEVELOPING TRANSGENICS Steps 1. Create transformation cassette 2. Introduce and select for transformants

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

GENE OF INTEREST Promoter Region • Controls when, where and how much the gene is expressed ex. : Ca. MV 35 S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development) Transit Peptide • Targets protein to correct organelle ex. : Rb. CS (RUBISCO small subunit; choloroplast target Coding Region • Encodes protein product ex. : EPSP -carotene genes

SELECTABLE MARKER Promoter Coding Region Promoter Region • Normally constitutive ex. : Ca. MV 35 s (Cauliflower Mosaic Virus 35 S RNA promoter Coding Region • Gene that breaks down a toxic compound; non-transgenic plants die ex. : npt. II [kanamycin (bacterial antibiotic) resistance] aph. IV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance]

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

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

Let’s Build A Complex Cassette p. B 19 hpc (Golden Rice Cassette) TL T-DNA Border aph. IV 35 S Gt 1 Hygromycin Resistance Insertion Selectable Sequence Marker psy Phytoene Synthase Gene of Interest 35 S rbc. S crtl Phytoene Desaturase Gene of Interest TR T-DNA Border Insertion Sequence

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 Tissue culture required to generate transgenic plants

PLANT TISSUE CULTURE A REQUIREMENT FOR TRANSGENIC DEVELOPMENT A plant part Is cultured Callus grows Shoots develop Shoots are rooted; plant grows to maturity

AGROBACTERIUM A NATURALDNA 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 Gall on leaf

THE GALLS CAN BE HUGE

NATURAL INFECTION PROCESS IS COMPLEX

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

TRANSFORMATION STEPS Prepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos Introduce DNA • Agrobacterium Culture plant tissue • Develop shoots • Root the shoots Field test the plants • Multiple sites, multiple years

THE LAB STEPS

WHAT NEXT ? Lab test Field test Consumer acceptance

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