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Biotechnology:

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Transformation Cassettes. Contains. 1. Gene of interest ... Transformation cassettes are developed in the lab. They are then introduced into a plant ... – PowerPoint PPT presentation

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Title: Biotechnology:


1
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
2
What is Biotechnology?
How about some definitions
3
These definitions imply biotechnology is needed
because
  • Nature has a rich source of variation

But we know nature does not have all of the
traits we need
4
But nature does not contain all the genetic
variation man desires
5
What controls this natural variation?
Allelic differences at genes control a specific
trait
Definitions are needed for this statement
6
What is the difference between genes and alleles
for Mendels Traits?
Mendels Genes Plant height
Seed shape
Smooth Wrinkled Allele
Tall Short Allele
7
This Implies a Genetic Continuum
A direct relationship exists between the gene,
its alleles, and the phenotypes (different forms
) of the trait
8
Allelic Differences for Mendels 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
9
Allelic Differences for Mendels 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
10
Central Dogma of Molecular Genetics
(The guiding principle that controls trait
expression)
11
In General, Plant Biotechnology Techniques Fall
Into Two Classes
12
Gene Manipulation Starts At the DNA Level
Source Access Excellence
13
DNA Is Packaged
is condensed into
Source Access Excellence
14
Chromosomes Contain Genes
Source Access Excellence
15
Genes Are Cloned Based On
16
Homology Cloning
17
Complementary Genetics
18
Complementary Genetics (cont.)
3. Use PCR to amplify gene fragment
19
PCR Animation
Denaturation DNA melts Annealing Primers
bind Extension DNA is replicated
20
PCR Again
21
Complementary Genetics (cont.)
4. Gene fragment used to screen library
22
Map-based Cloning
23
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
24
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

25
RoundUp Sensitive Plants
Glyphosate
X
X
Without amino acids, plant dies
X
X
26
RoundUp Resistant Plants
Shikimic acid Phosphoenol pyruvate
Glyphosate
RoundUp has no effect enzyme is resistant to
herbicide
Bacterial EPSP synthase
3-enolpyruvyl shikimic acid-5-phosphate (EPSP)
With amino acids, plant lives
Aromatic amino acids
27
The Golden Rice Story
  • Vitamin A deficiency is a major health problem
  • Causes blindness
  • Influences severity of diarrhea, measles
  • gt100 million children suffer from the problem
  • For many countries, the infrastructure doesnt
    exist
  • to deliver vitamin pills
  • Improved vitamin A content in widely consumed
    crops
  • an attractive alternative

28
?-Carotene Pathway in Plants
29
The Golden Rice Solution
?-Carotene Pathway Genes Added
Daffodil gene
Single bacterial gene performs both functions
Daffodil gene
30
Metabolic Pathways are Complex and Interrelated
Understanding pathways is critical to
developing new products
31
Modifying Pathway Components Can Produce New
Products
Turn On Vitamin Genes Relieve Deficiency
Modified Lipids New Industrial Oils
Increase amino acids Improved Nutrition
32
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
33
Introducing the Gene or Developing Transgenics
Steps
1. Create transformation cassette
2. Introduce and select for transformants
34
Transformation Cassettes
Contains
35
Gene of Interest
36
Selectable Marker
37
Effect of Selectable Marker
Non-transgenic Lacks Kan or Bar Gene
X
Transgenic Has Kan or Bar Gene
38
Insertion Sequences
39
Lets Build A Complex Cassette
pB19hpc (Golden Rice Cassette)
40
Delivering the Gene to the Plant
  • Transformation cassettes are developed in the lab
  • They are then introduced into a plant
  • Two major delivery methods

41
Plant Tissue Culture A Requirement for Transgenic
Development
Callus grows
A plant part Is cultured
Shoots develop
Shoots are rooted plant grows to maturity
42
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

43
The Galls Can Be Huge
44
Natural Infection Process Is Complex
45
But Natures Agrobacterium Has Problems
Infected tissues cannot be regenerated (via
tissue culture) into new plants
Why?
  • Phytohormone balance incorrect regeneration

Transferred DNA (T-DNA) modified by
Solution?
  • Removing phytohormone genes
  • Retaining essential transfer sequences
  • Adding cloning site for gene of interest

46
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

47
Transformation Steps
Prepare tissue for transformation
Introduce DNA
  • Agrobacterium or gene gun

Culture plant tissue
Field test the plants
  • Multiple sites, multiple years

48
The Lab Steps
49
Lab Testing The Transgenics
Transgene Bt-toxin protein
Transgene CBF transcription factors
50
More Modern Examples
Transgene Glyoxylase I
Transgene Mercuric ion reductase
51
The Next Test Is The Field
Herbicide Resistance
52
Final Test Consumer Acceptance
RoundUp Ready Corn
53
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?
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