PowerPoint Presentation

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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

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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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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:

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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

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:

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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

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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 GA

to make GA

Role of GA

: 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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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

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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

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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

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Gene Manipulation Starts

At the DNA Level

The nucleus

contains DNA

Source: Access Excellence

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DNA Is Packaged

Source: Access Excellence

Double-stranded

DNA

Chromosomes

is condensed

into

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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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Human clone

library

Clones transferred

to filter

Mouse probe
added to filter

Hot-spots are human

homologs

to mouse gene

Homology Cloning

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Complementary Genetics

1. Protein sequence is related to gene sequence

-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

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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)

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Denaturation: DNA melts
Annealing: Primers bind
Extension: DNA is replicated

PCR Animation

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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

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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

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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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+ Glyphosate

RoundUp Sensitive Plants

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate

(EPSP)

Plant

EPSP synthase

Aromatic

amino acids

Without amino

acids, plant dies

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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

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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

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

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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

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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

Modified Lipids =

New Industrial Oils

Turn On Vitamin

Genes =

Relieve

Deficiency

Increase amino acids =

Improved Nutrition

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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

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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

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

Transit Peptide

•

Targets protein to correct organelle

ex.: RbCS (RUBISCO small subunit; choloroplast target

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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

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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

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Insertion Sequences

•

Used for Agrobacterium-transformation

ex.: Right and Left borders of T-DNA

Required for proper gene insertions

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Let’s Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

aphIV

35S Gt1

psy

35S rbcS

crtl

Hygromycin

Resistance

Phytoene

Synthase

Phytoene

Desaturase

T-DNA
Border

Selectable

Marker

Gene of
Interest

Insertion
Sequence

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• 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

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Plant Tissue Culture

A Requirement for Transgenic Development

A plant part

Is cultured

Callus

grows

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

• 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

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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?

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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

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

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Lab Testing The Transgenics

Insect Resistance

Transgene=

Bt-toxin protein

Cold Tolerance

Transgene=

CBF transcription factors

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Salt Tolerant

Mercury Resistance

More Modern Examples

Transgene=

Glyoxylase I

Transgene=

Mercuric ion reductase

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The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

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Final Test

Consumer Acceptance

RoundUp Ready Corn

Before

After

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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?

• Are transgenics a threat to natural
eco-systems?

Document Outline