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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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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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Protein
Trait
(or phenotype)
Translation
Seed shape
DNA
(gene)
Transcription
RNA
Plant height
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Gene Introduction
Introduces that gene into an organism
Technique called transformation
Forms transgenic organisms
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The nucleus
contains DNA
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DNA Is Packaged
Double-stranded
DNA
is condensed
into
Chromosomes
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Chromosome
Gene
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Homology Cloning
Clones transferred
to filter
Human clone
library
Mouse probe
added to filter
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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
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Complementary Genetics
(cont.)
3. Use PCR to amplify gene fragment
a. template DNA is melted (94C)
3
5
5
3
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PCR Animation
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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
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PCR fragment
probe added to filter
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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
Gene Marker
Gene/Marker
Gene/Marker
Gene/Marker
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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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Plant
EPSP synthase
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Aromatic
amino acids
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Aromatic
amino acids
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Phytoene
Problem:
Rice lacks
these enzymes
Phytoene desaturase
-carotene desaturase
Lycopene
Lycopene-beta-cyclase
-carotene
(vitamin A precursor)
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Normal
Vitamin A
Deficient
Rice
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Vitamin A
Pathway
is complete
and functional
Phytoene synthase
Phytoene
Single bacterial gene;
performs both functions
Phytoene desaturase
-carotene desaturase
Lycopene
Daffodil gene
-carotene
(vitamin A precursor)
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Golden
Rice
Lycopene-beta-cyclase
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Trait/Gene Examples
Gene
Trait
RoundUp Ready
Bacterial EPSP
Golden Rice
Complete Pathway
Male Sterility
Barnase
p35
Salt tolerance
AtNHX1
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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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X
Transgenic = Has Kan or Bar Gene
Plant grows in presence
of selective compound
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Insertion Sequences
TL
TR
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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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Tissue culture
required to generate
transgenic plants
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A plant part
Is cultured
Callus
grows
Shoots
develop
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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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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
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Cold Tolerance
Transgene=
Bt-toxin protein
Transgene=
CBF transcription factors
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Mercury Resistance
Transgene=
Glyoxylase I
Transgene=
Mercuric ion reductase
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Transgenics
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Final Test
Consumer Acceptance
RoundUp Ready Corn
After
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