techniques of biotechnology mcclean good

background image

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

background image

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

background image

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:

background image

N

D

S

U

Extension

But nature does not contain all the

genetic variation man desires

•Fruits with vaccines

•Grains with improved nutrition

background image

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:

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

N

D

S

U

Extension

Gene Manipulation Starts

At the DNA Level

The nucleus

contains DNA

Source: Access Excellence

background image

N

D

S

U

Extension

DNA Is Packaged

Source: Access Excellence

Double-stranded

DNA

Chromosomes

is condensed

into

background image

N

D

S

U

Extension

Chromosomes Contain Genes

Chromosome

Gene

Source: Access Excellence

background image

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)

background image

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

background image

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

background image

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)

background image

N

D

S

U

Extension

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

PCR Animation

background image

N

D

S

U

Extension

PCR Again

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

N

D

S

U

Extension

Metabolic Pathways are Complex

and Interrelated

Understanding

pathways is

critical

to developing new

products

background image

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

background image

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

background image

N

D

S

U

Extension

Introducing the Gene or

Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

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

background image

N

D

S

U

Extension

The Galls Can Be Huge

background image

N

D

S

U

Extension

Natural Infection Process Is Complex

background image

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?

background image

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

background image

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

background image

N

D

S

U

Extension

The Lab Steps

background image

N

D

S

U

Extension

Lab Testing The Transgenics

Insect Resistance

Transgene=

Bt-toxin protein

Cold Tolerance

Transgene=

CBF transcription factors

background image

N

D

S

U

Extension

Salt Tolerant

Mercury Resistance

More Modern Examples

Transgene=

Glyoxylase I

Transgene=

Mercuric ion reductase

background image

N

D

S

U

Extension

The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

background image

N

D

S

U

Extension

Final Test

Consumer Acceptance

RoundUp Ready Corn

Before

After

background image

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?


Document Outline


Wyszukiwarka

Podobne podstrony:
2006 J of Biotechnology Start up C Trigo moje
2009 Smith (01 Nature of Biotec Nieznany
Investigation Techniques of A Homicide
Technika Cyfrowa, biotechnologia inż, sem3, TCiM, egzamin
Practical Analysis Techniques of Polymer Fillers by Fourier Transform Infrared Spectroscopy (FTIR)
Basic Techniques Of Kodokan Judo
Biological techniques of studying bacteria and fungi
039 Drying of Biotechnological Products
Richard Bandler And John Grinder Patterns Of The Hypnotic Techniques Of Milton Erickson
Chaos Kung Fu Techniques of Sexual Magick
Martial Arts Peters Michael E Knife Throwing Techniques of the Ninja
(eBook PDF Sex) The Top One Hundred Lovemaking Techniques Of All Time
The Techniques of Astral Projection by Dr Douglas M Baker
letter of complaint & tips good presentation
Bob Cassidy Techniques of Mentalism
Hine, Phil Techniques of Modern Shamanism vol 1

więcej podobnych podstron