Transcript Biotechnology: Principles, Applications

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
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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 the
biological function of an organism by adding genes
from another organism
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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
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But we know nature does not have
all of the traits we need
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But nature does not contain all the
genetic variation man desires
•Fruits with vaccines
•Grains with improved nutrition
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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
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What is the difference between
genes and alleles for Mendel’s Traits?
Mendel’s Genes
Plant height
Seed shape
Smooth Wrinkled
Allele
Tall
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Short
Allele
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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
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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
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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
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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
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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
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Gene Manipulation Starts
At the DNA Level
The nucleus
contains DNA
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Source: Access Excellence
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DNA Is Packaged
Double-stranded
DNA
is condensed
into
Chromosomes
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Source: Access Excellence
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Chromosomes Contain Genes
Chromosome
Gene
Source: Access Excellence
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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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Homology Cloning
Clones transferred
to filter
Human clone
library
Mouse probe
added to filter
Hot-spots are human
homologs to mouse gene
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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’
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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
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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’
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3’
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PCR Animation
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Denaturation: DNA melts
Annealing: Primers bind
Extension: DNA is replicated
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PCR Again
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Complementary Genetics
(cont.)
4. Gene fragment used to screen library
Clones transferred
to filter
Human clone
library
PCR fragment
probe added to filter
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Hot-spots are human gene
of interest
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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
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5. Prove one ORF is the gene by
Mutant + ORF = Wild type?
transformation or mutant analysis Yes? ORF = Gene
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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 enzyme
Increased 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 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
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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
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Aromatic
amino acids
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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
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Aromatic
amino acids
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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
an attractive alternative
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-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)
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Normal
Vitamin A
“Deficient”
Rice
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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)
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Golden
Rice
Lycopene-beta-cyclase
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Metabolic Pathways are Complex
and Interrelated
Understanding pathways
is critical to developing
new products
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Modifying Pathway Components
Can Produce New Products
Turn On Vitamin Genes =
Relieve Deficiency
Modified Lipids =
New Industrial Oils
Increase amino acids =
Improved Nutrition
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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
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Introducing the Gene or
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
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
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ex.: EPSP
-carotene genes
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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]
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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
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Insertion Sequences
TL
TR
Required for proper gene insertions
• Used for Agrobacterium-transformation
ex.: Right and Left borders of T-DNA
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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
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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
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Tissue culture
required to generate
transgenic plants
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Plant Tissue Culture
A Requirement for Transgenic Development
Callus
grows
A plant part
Is cultured
Shoots
develop
Shoots are rooted;
plant grows to maturity
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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
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Gall on
leaf
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The Galls Can Be Huge
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Natural Infection Process Is Complex
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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
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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
• 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
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The Lab Steps
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Lab Testing The Transgenics
Insect Resistance
Cold Tolerance
Transgene=
Bt-toxin protein
Transgene=
CBF transcription factors
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More Modern Examples
Salt Tolerant
Mercury Resistance
Transgene=
Glyoxylase I
Transgene=
Mercuric ion reductase
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The Next Test Is The Field
Herbicide Resistance
Non-transgenics
Transgenics
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Final Test
Consumer Acceptance
RoundUp Ready Corn
After
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Before
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The Public Controversy
• Should we develop transgenics?
• Should we release transgenics?
• Are transgenics safe?
• Are transgenics a threat to non-transgenic
production systems?
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• Are transgenics a threat to natural
eco-systems?
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