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Biotechnology:Principles and Products
From Protein to Product
Phil McCleanDepartment of Plant Science
North Dakota State University
The techniques used by the biotechnology industryto modify genes and introduce them into transgenic organisms
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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 thebiological function of an organism by adding genesfrom another organism
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But we know nature does not have all of the traits we need
• Here we see bean has many seedcoat colors and patterns in nature
•Nature has a rich source of variation
These definitions imply biotechnologyis needed because:
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Nature does not contain all thegenetic variation man desires
•Fruits with vaccines
•Grains with improved nutrition
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What controls this natural variation?
Allelic differences for a gene controlvariation for a specific trait
Gene - a piece of DNA that controls the expression of a trait
Allele - the alternate forms of a gene
Important definitions for this statement:
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What is the difference betweengenes and alleles for Mendel’s Traits?
Mendel’s GenesPlant height Seed shape
Tall ShortAllele
Smooth WrinkledAllele
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This Implies aGenetic 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 phenotypic differences for that trait
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Allelic Differences for Mendel’s GenesPlant Height Gene
Gene: gibberellin 3--hydroxylaseFunction: adds hydoxyl group to GA20 to make GA1
Role of GA1: regulates cell division and elongationMutation in short allele: a single nucleotide converts an alanine to threonine in final proteinEffect of mutation: mutant protein is 1/20 as active
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Gene: strach branching enzyme (SBE) isoform 1Function: adds branch chains to starchMutation in short allele: transposon insertionEffect 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)
DNA(gene)
RNA
Protein Trait(or phenotype)
Transcription
Translation
Plant height
Seed shape
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Plant Biotechnology Requires Two Steps
• Identify a gene from another species which controls a trait of interest• Or modify an existing gene (create a new allele)
Gene Manipulation
• Introduces that gene into an organism• Technique called transformation• Forms transgenic organisms
Gene Introduction
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Gene Manipulation StartsAt the DNA Level
The nucleus
contains DNA
Source: Access Excellence
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DNA Is Packaged
Source: Access Excellence
Double-strandedDNA
Chromosomes
is condensedinto
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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)
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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 enzymeIncreased 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 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
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+ Glyphosate
X
Biochemical Response of RoundUp Sensitive Plants
X
X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)
Plant EPSP synthase
Aromaticamino acids
Without amino acids, plant dies
X
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BacterialEPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate(EPSP)
Aromaticamino 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 is an attractive alternative
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-Carotene Pathway in Plants
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
-carotene(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Problem:Rice lacks
these enzymes
NormalVitamin A
“Deficient”Rice
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The Golden Rice Solution
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
-carotene(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Daffodil gene
Single bacterial gene;performs both functions
Daffodil gene
-Carotene Pathway Genes Added
Vitamin APathway
is completeand functional
GoldenRice
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Metabolic Pathways are Complexand Interrelated
Understanding pathways is critical to developing
new products
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Modifying Pathway ComponentsCan 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 Geneor
Developing Transgenics
Steps
1. Create transformation cassette
2. Introduce and select for transformants
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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
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Gene of Interest
Coding Region• Encodes protein product
ex.: EPSP -carotene genes
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)
Promoter Coding RegionTP
Transit Peptide• Targets protein to correct organelle
ex.: RbCS (RUBISCO small subunit; choloroplast target
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Selectable Marker
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]
Promoter Region• Normally constitutive
ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter
Promoter Coding Region
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Effect of Selectable Marker
Transgenic = Has NptII or Bar Gene
Plant grows in presenceof selective compound
Plant dies in presenceof selective compound
Non-transgenic = Lacks NptII or Bar Gene
X
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Insertion Sequences
• Used for Agrobacterium-transformationex.: Right and Left borders of T-DNA
Required for proper gene insertions
TL TR
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Let’s Build A Complex Cassette
pB19hpc (Golden Rice Cassette)
TL TRaphIV 35S Gt1 psy 35S rbcS crtl
HygromycinResistance
PhytoeneSynthase
PhytoeneDesaturase
T-DNABorder
T-DNABorder
SelectableMarker
Gene ofInterest
Gene ofInterest
InsertionSequence
InsertionSequence
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• Transformation cassettes are developed in the lab
• They are then introduced into a plant
• Two major delivery methods
Delivering the Geneto the Plant
• Agrobacterium
• Gene GunTissue culturerequired to generatetransgenic plants
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Plant Tissue CultureA Requirement for Transgenic Development
A plant part Is cultured
Callusgrows
Shootsdevelop Shoots are rooted;
plant grows to maturity
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AgrobacteriumA 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
Gall onstem
Gall onleaf
• DNA incorporates into plant chromosome
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But Nature’s AgrobacteriumHas 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• Develop shoots• Root the shoots
Field test the plants
• Leaf, germinating seed, immature embryos
• Tissue must be capable of developing into normal plants
• Agrobacterium or gene gun
• Multiple sites, multiple years
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Lab Testing The Transgenics
Insect Resistance
Transgene=Bt-toxin protein
Cold Tolerance
Transgene=CBF transcription factors
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Salt Tolerant Mercury Resistance
More Modern Examples
Transgene=Glyoxylase I
Transgene=Mercuric ion reductase