Gene Expression and Basic Transformation

8/2/2019 Gene Expression and Basic Transformation

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GENE EXPRESSION AND BASIC

TRANSFORMATION


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WHY DO WE NEED BIOTECHNOLOGY?

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


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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 atgenes 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 MENDELS TRAITS?

Mendels Genes

Plant height Seed shape

Tall Short

Allele

Smooth Wrinkled

Allele


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THIS IMPLIES A

GENETIC CONTINUUM

Adirect 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 MENDELS GENES

PLANT HEIGHT GENE

Gene: gibberellin 3-F-hydroxylase

Function: adds hydoxyl group to GA20 to make GA1Role of GA1: regulates cell division and elongation

Mutation in short allele: a single nucleotide convertsan alanine to threonine in final protein

Effect of mutation: mutant protein is 1/20 as active


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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, moresucrose, more water; during maturation seed looses

more water and wrinkles


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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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PLANT BIOTECHNOLOGYTECHNIQUES

FALL INTO TWO CLASSES

Identify a gene from another species which

controlsa 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


Double-strandedDNA

Chromosomes

is condensed

into


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CHROMOSOMES CONTAIN GENES

Chromosome

Gene



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GENESARE 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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HOMOLOGYCLONING

Human clone

library

Clones transferred

to filter

Mouse probe

added to filter

Hot-spots are human

homologs to mouse gene


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COMPLEMENTARYGENETICS

1. Protein sequence is related to gene sequence

NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO-

ATG GAT-GCT TGG-AGT-AAA

C C C G

A TCTG 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

a. template DNA is melted (94 ooC)

3 5

5 3

3 5

5 3

b. primers anneal to complementary site in melted DNA (55 ooC)

3 5

5 3

3 5

5 3

c. two copies of the template DNA made (72 ooC)


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PCR ANIMATION

Denaturation: DNA melts

Annealing: Primers bind

Extension: DNA is replicated


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Human clone

library

Clones transferred

to filter

PCR fragment

probe added to filter

Hot-spots are human gene

of interest

4. Gene fragment used to screen library


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MAP-BASED CLONING

1. Use genetic techniques to

find marker near gene

Gene Marker

2. Find cosegregating markerGene/Marker

3. Discover overlapping clones

(or contig) that contains the marker Gene/Marker

4. Find ORFs on contigGene/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 READYSTORY

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 cropsto survive spraying


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ROUNDUP SENSITIVE PLANTS

+ Glyphosate

X

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate

(EPSP)

Plant

EPSPsynthase

Aromatic

amino acids

Without amino acids,

plant dies

X


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ROUNDUP RESISTANT PLANTS

Bacterial

EPSPsynthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate

(EPSP)

Aromatic

amino acids

+ 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 doesnt exist

to deliver vitamin pills

Improved vitamin A content in widely consumed crops

an attractive alternative


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F-CAROTENE PATHWAY IN PLANTSIPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

F -carotene

(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

-carotene desaturase

Problem:

Rice lacks

these enzymes

Normal

Vitamin A

Deficient

Rice


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THE GOLDEN RICE SOLUTIONF-Carotene Pathway Genes Added

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

F -carotene

(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

-carotene desaturase

Daffodil gene

Single bacterial gene;

performs both functions

Daffodil gene

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 PATHWAYCOMPONENTS

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 Resistance Viral Coat Protein

Male Sterility Barnase

Plant Bacterial Resistance p35

Salt tolerance AtNHX1

Trait Gene


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INTRODUCING THE GENE ORDEVELOPING 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

F-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)

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 dieex.: 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

X


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INSERTION SEQUENCES

Used for Agrobacterium-transformation

ex.: Right and Left borders of T-DNA

Required for proper gene insertions

TL TR


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Lets Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

TL TRaphIV 35S Gt1 psy 35S rbcS crtl

Hygromycin

Resistance

Phytoene

Synthase

Phytoene

Desaturase

T-DNA

Border

T-DNA

Border

SelectableMarker

Gene ofInterest

Gene ofInterest

InsertionSequence

InsertionSequence


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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 GunTissue culture

required to generate

transgenic plants


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PLANT TISSUE CULTUREAREQUIREMENT FOR TRANSGENIC DEVELOPMENT

A plant part

Is cultured

Callus

grows

Shoots

develop Shoots are rooted;plant grows to maturity


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AGROBACTERIUMANATURAL DNADELIVERY 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 onstem

Gall on

leaf

DNAincorporates into plant chromosome


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THE GALLS CAN BE HUGE


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NATURAL INFECTION PROCESS IS COMPLEX


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BUT NATURES AGROBACTERIUM

HAS PROBLEMS

Infected tissues cannot be regenerated (via tissue culture)

into new plants

TransferredDNA (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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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

Multiple sites, multiple years


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THE LAB STEPS


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WHAT NEXT ?

Lab test

Field test

Consumer

acceptance


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THANK YOU

Documents

Gene Expression and Basic Transformation