N
D
S
U
Extension
Biotechnology:
Principles, Applications,
and Social Implications
From Protein to Product
Phil McClean
Department of Plant Science
North Dakota State University
The techniques used by the biotechnology industry
to modify genes and introduce them into transgenic organisms
N
D
S
U
Extension
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
N
D
S
U
Extension
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 biotechnology
is needed because:
N
D
S
U
Extension
But nature does not contain all the
genetic variation man desires
•Fruits with vaccines
•Grains with improved nutrition
N
D
S
U
Extension
What controls this natural variation?
Allelic differences at genes control a specific trait
Gene -
a piece of DNA that controls the
expression of a trait
Allele -
the alternate forms of a gene
Definitions are needed for this statement:
N
D
S
U
Extension
What is the difference between
genes and alleles for Mendel’s Traits?
Mendel’s Genes
Plant height Seed shape
Tall Short
Allele
Smooth Wrinkled
Allele
N
D
S
U
Extension
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
N
D
S
U
Extension
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
N
D
S
U
Extension
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
Allelic Differences for Mendel’s
Seed Shape Gene
N
D
S
U
Extension
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
N
D
S
U
Extension
In General, Plant Biotechnology Techniques
Fall Into Two Classes
• 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
N
D
S
U
Extension
Gene Manipulation Starts
At the DNA Level
The nucleus
contains DNA
Source: Access Excellence
N
D
S
U
Extension
DNA Is Packaged
Source: Access Excellence
Double-stranded
DNA
Chromosomes
is condensed
into
N
D
S
U
Extension
Chromosomes Contain Genes
Chromosome
Gene
Source: Access Excellence
N
D
S
U
Extension
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)
N
D
S
U
Extension
Human clone
library
Clones transferred
to filter
Mouse probe
added to filter
Hot-spots are human
homologs
to mouse gene
Homology Cloning
N
D
S
U
Extension
Complementary Genetics
1. Protein sequence is related to gene sequence
NH
3
+
-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
N
D
S
U
Extension
3. Use PCR to amplify gene fragment
Complementary Genetics
(cont.)
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’
3’ 5’
5’ 3’
c. two copies of the template DNA made (72C)
N
D
S
U
Extension
Denaturation: DNA melts
Annealing: Primers bind
Extension: DNA is replicated
PCR Animation
N
D
S
U
Extension
PCR Again
N
D
S
U
Extension
Human clone
library
Clones transferred
to filter
PCR fragment
probe added to filter
Hot-spots are
human gene
of interest
Complementary Genetics
(cont.)
4. Gene fragment used to screen library
N
D
S
U
Extension
Map-based Cloning
1. Use genetic techniques to
find marker near gene
GeneMarker
2. Find cosegregating marker
Gene/Marker
3. Discover overlapping clones
(or contig) that contains the marker
Gene/Marker
4. Find ORFs on contig
Gene/Marker
5. Prove one ORF is the gene by
transformation or mutant analysis
Mutant +
ORF
= Wild type?
Yes?
ORF
= Gene
N
D
S
U
Extension
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
N
D
S
U
Extension
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
N
D
S
U
Extension
+ Glyphosate
X
RoundUp Sensitive Plants
X
X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Plant
EPSP synthase
Aromatic
amino acids
Without amino
acids, plant dies
X
N
D
S
U
Extension
Bacterial
EPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Aromatic
amino acids
RoundUp Resistant Plants
+ Glyphosate
With amino
acids, plant lives
RoundUp has no effect;
enzyme is resistant to herbicide
N
D
S
U
Extension
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
N
D
S
U
Extension
-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
Normal
Vitamin A
“Deficient”
Rice
N
D
S
U
Extension
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 A
Pathway
is complete
and functional
Golden
Rice
N
D
S
U
Extension
Metabolic Pathways are Complex
and Interrelated
Understanding
pathways is
critical
to developing new
products
N
D
S
U
Extension
Modifying Pathway Components
Can Produce New Products
Modified Lipids =
New Industrial Oils
Turn On Vitamin
Genes =
Relieve
Deficiency
Increase amino acids =
Improved Nutrition
N
D
S
U
Extension
Trait/Gene Examples
RoundUp Ready
Bacterial EPSP
Golden Rice
Complete Pathway
Plant Virus ResistanceViral Coat Protein
Male Sterility
Barnase
Plant Bacterial Resistance
p35
Salt tolerance
AtNHX1
Trait
Gene
N
D
S
U
Extension
Introducing the Gene or
Developing Transgenics
Steps
1. Create transformation cassette
2. Introduce and select for transformants
N
D
S
U
Extension
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
N
D
S
U
Extension
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 Region
TP
Transit Peptide
•
Targets protein to correct organelle
ex.: RbCS (RUBISCO small subunit; choloroplast target
N
D
S
U
Extension
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
N
D
S
U
Extension
Effect of Selectable Marker
Transgenic =
Has Kan or Bar Gene
Plant grows in presence
of selective compound
Plant dies in presence
of selective compound
Non-transgenic =
Lacks Kan or Bar Gene
X
N
D
S
U
Extension
Insertion Sequences
•
Used for Agrobacterium-transformation
ex.: Right and Left borders of T-DNA
Required for proper gene insertions
T
L
T
R
N
D
S
U
Extension
Let’s Build A Complex Cassette
pB19hpc (Golden Rice Cassette)
T
L
T
R
aphIV
35S Gt1
psy
35S rbcS
crtl
Hygromycin
Resistance
Phytoene
Synthase
Phytoene
Desaturase
T-DNA
Border
T-DNA
Border
Selectable
Marker
Gene of
Interest
Gene of
Interest
Insertion
Sequence
Insertion
Sequence
N
D
S
U
Extension
• Transformation cassettes are developed in the lab
• They are then introduced into a plant
• Two major delivery methods
Delivering the Gene
to the Plant
•
Agrobacterium
•
Gene Gun
Tissue culture
required to generate
transgenic plants
N
D
S
U
Extension
Plant Tissue Culture
A Requirement for Transgenic Development
A plant part
Is cultured
Callus
grows
Shoots
develop
Shoots are rooted;
plant grows to maturity
N
D
S
U
Extension
Agrobacterium
A 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 on
stem
Gall on
leaf
• DNA
incorporates
into plant chromosome
N
D
S
U
Extension
The Galls Can Be Huge
N
D
S
U
Extension
Natural Infection Process Is Complex
N
D
S
U
Extension
But Nature’s Agrobacterium
Has 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?
N
D
S
U
Extension
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
N
D
S
U
Extension
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
N
D
S
U
Extension
The Lab Steps
N
D
S
U
Extension
Lab Testing The Transgenics
Insect Resistance
Transgene=
Bt-toxin protein
Cold Tolerance
Transgene=
CBF transcription factors
N
D
S
U
Extension
Salt Tolerant
Mercury Resistance
More Modern Examples
Transgene=
Glyoxylase I
Transgene=
Mercuric ion reductase
N
D
S
U
Extension
The Next Test Is The Field
Non-transgenics
Transgenics
Herbicide Resistance
N
D
S
U
Extension
Final Test
Consumer Acceptance
RoundUp Ready Corn
Before
After
N
D
S
U
Extension
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?