Download Gene Expression and Basic Transformation

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 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
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)
Protein
Trait
(or phenotype)
Translation
Seed shape
DNA
RNA
(gene) Transcription
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
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’
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 oC)
3’
5’
5’
3’
3’
5’
5’
3’
b. primers anneal to complementary site in melted DNA (55 oC)
3’
5’
5’
3’
c. two copies of the template DNA made (72 oC)
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. Discover overlapping clones
(or contig) that contains the marker
4. Find ORFs on contig
Gene Marker
Gene/Marker
Gene/Marker
Gene/Marker
5. Prove one ORF is the gene by
Mutant + ORF = Wild type?
transformation or mutant analysis 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 (RoundUp) 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 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
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
RoundUp 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
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
Single bacterial gene; Phytoene desaturase
Pathway
performs both functions
is complete
ξ-carotene desaturase
and functional
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
RoundUp Ready
Bacterial EPSP
Golden Rice
Complete Pathway
Plant Virus Resistance
Viral Coat Protein
Male Sterility
Barnase
Plant Bacterial Resistance
p35
Salt tolerance
AtNHX1
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.: 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
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]
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
pB19hpc (Golden Rice Cassette)
TL
T-DNA
Border
aphIV
35S Gt1
Hygromycin
Resistance
Insertion Selectable
Sequence Marker
psy
Phytoene
Synthase
Gene of
Interest
35S rbcS
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 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
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
THANK YOU