If you can't read please download the document
Upload
henry-watson
View
229
Download
1
Embed Size (px)
DESCRIPTION
Terms to know Transgene: It is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another. Transgesis : The process of introducing an exogenous gene called a transgene into a living organisms so that the organism will exhibit a new property and transmit that property to its offspring. Transgenic Plants : The plants which expresses the characters coded by the transgene are called Transgenic plants.
Citation preview
Chapter 27 Genetic engineering of plants Terms to know
Transgene: It is a gene or genetic material that has been
transferred naturally or by any of a number of genetic engineering
techniques from one organism to another. Transgesis : The process
of introducing an exogenous genecalled a transgene into a living
organisms so that the organism will exhibit a new property and
transmit that property to its offspring. Transgenic Plants : The
plants which expresses the characters coded by the transgene are
called Transgenic plants. History of Plant Breeding
Selective Breeding used in the History Genetics studies started
with Mendel Cross pollination : Pollen from one plant to stigma of
another plant. Found dominate characteristics in plants Uses of
Traditional Breeding: Increase crop yield Increase Resistance to
pests and diseases Drought tolerance Disadvantages of Traditional
breeding: Long process Lot of man power Limited possibility of
improved traits. The Reproductive Organs of a Typical Plant: Pollen
grains are the male reproductive cells of the plant. They are made
in the anther (orange), the top portion of the stamen. The female
reproductive cells, the ova, are sequestered in the ovary. Pollen
reaches the ova via the stigma, which is attached to the ovary by
the pistil. Mutation Breeding Treat seeds with mutagens or expose
to X rays or gamma rays. Disadvantages Less predictable results Lot
of man power Successful in the flower world. Eg; new colours, more
petals. UV Treatment or Mutagens
Seeds Killed Alive Planted Tested for Improvements Found desirable
traits Test for Progeny heritable Sold to Markets Transgenic plant
: Insertion of a foreign gene into a specific plant.
Difference between Trangenic Technology and traditional Breeding:
Trangenic Technology : Transform gene from any source. Eg: animals,
bacteria, virus etc Traditional Breeding : Move genes only between
members of a particular genus of plants. Plant Tissue Culture
Totipotency : Ability of a cell to divide into any type of cell.
Explant : Mass of tissue or cells Solid medium Callus culture.
Tissue can be immature embryo, apical meristem, root tip Liquid
medium suspension culture Tissue should be protoplast (cells with
no cell wall), micro or macrospores. Nutrients and hormones are
used for growth and development. Eg : 2,4 dichlorophenoxyacetic
acid (analogous to auxin) Callus : Undifferentiated cell which form
a crystalline white layer on solid medium. I. Move callus to other
medium with reduced hormones which allows shoot to develop. II.
Move the callus to other medium with no hormone which allows root
hairs to grow. Temporary Physiological change Epigenetic change
True genetic changes
The process of regenerating a plant from a single cell may cause
three types of alterations, Temporary Physiological change
Epigenetic change True genetic changes An Entire Plant Can Be
Regenerated from a Single Cell Small samples of tissue, or even
single plant cells may be cultured in vitro. Under appropriate
conditions, these may regenerate into complete plants. Callus or
Liquid Culture of Plant Cells Can Regenerate Entire Plants
FIGURE 14.3 Callus or Liquid Culture of Plant Cells Can Regenerate
Entire Plants In callus culture a mass of undifferentiated cells
grows on a solid surface. In liquid culture, separated single cells
are grown. Both types of cultures can develop shoots and roots with
appropriate manipulation of plant hormone levels. Gene transfer in
plants
Why gene transfer? Crop improvement Disease resistance Stress
tolerance Improved performance Value-added traits Basic studies
Gene expression Reverse genetics - understanding functioning of
unknown genes Biochemistry and metabolism Gene transfer strategies:
Systems and vectors Agro bacterium Direct DNA uptake Virus-based
vectors Plant transformatIon wIth the Ti plasmid of AgrobacterIum
tumefacIens
A. tumefaciens is a gram-negative soil bacterium which naturally
transforms plant cells, resulting in crown gall (cancer) tumors
Tumor formation is the result of the transfer, integration and
expression of genes on a specific segment of A. tumefaciens plasmid
DNA called the T-DNA (transferred DNA) The T-DNA resides on a large
plasmid called the Ti (tumor inducing) plasmid found in
A.tumefaciens 11 Agrobacterium-mediated gene transfer
The keys To make a segment of DNA that contains a selectable marker
and a gene of interest to look like a T-DNA To get this T-DNA into
an Agrobacterium cell so that it can be mobilized by the vir genes
To produce and find transformed plant cells that can be regenerated
into normal, fertile plants Requirements A transfer cassette
bounded by functioning borders Ways to get this cassette into
Agrobacterium Disarmed Ti plasmids that retain functional vir genes
Advantages Technically simple Yields relatively uncomplicated
insertion events (low copy number, minimal rearrangements)
Unlimited size of foreign DNA Efficient (for most plants) Adaptable
to different cell types, culture procedures (protoplasts, tissue
sections, non-culture methods) Transformants are mitotically and
meiotically stable Disadvantages Host range is limited: not all
plants may be susceptible to Agrobacterium With susceptible plants,
accessible culture/regeneration systems must be adaptable to
Agrobacterium-mediated gene transfer The Infection process Wounded
plant cell releases phenolics and nutrients. Phenolics and
nutrients cause chemotaxic response of A. tumefaciens Attachment of
the bacteria to the plant cell. Certain phenolics (e.g.,
acetosyringone, hydroxyacetosyringone) induce vir gene
transcription and allow for T-DNA transfer and integration into
plant chromosomal DNA. Transcription and translation of the T-DNA
in the plant cell to produce opines (food) and tumors (housing) for
the bacteria. The opine permease/catabolism genes on the Ti plasmid
allow A. tumefaciens to use opines as a C, H, O, and N source. 14
Agrobacterium Transfers Plasmid DNA into Infected Plants
FIGURE 14.4 Agrobacterium Transfers Plasmid DNA into Infected
Plants Agrobacterium carrying a Ti plasmid is attracted by
acetosyringone to a wounded plant stem. The Ti plasmid is cut by
endonucleases to release single-stranded T-DNA, which is covered
with protective proteins, and transported into the plant cell
through a conjugation-like mechanism. The T-DNA enters the plant
nucleus where it integrates into plant chromosomal DNA. The Ti
plasmd of Agrobacterum tumafacens and the transfer of ts T-DNA to
the plant nuclear genome 16 Infecton of a plant wth A. tumefacens
and formaton of crown galls
Crown Gall on Tobacco Infecton of a plant wth A. tumefacens and
formaton of crown galls 17 Clone YFG (your favorite gene) or the
target gene in the small T-DNA plasmid in E. coli, isolate the
plasmid and use it to transform A. tumefaciens containing the
disarmed Ti plasmid Ti plasmid structure & function
Essential Elements for Carrying a Transgene on Ti Plasmids The
T-DNA segment contains both a transgene and a selective marker or
reporter gene. These have separate promoters and termination
signals. The marker or reporter gene must be expressed all the
time, whereas the transgene is often expressed only in certain
tissues or under certain circumstances and usually has a promoter
that can be induced by appropriate signals. Ti plasmid structure
& function Transfer of Modified Ti Plasmid into a Plant
FIGURE 14.6 Transfer of Modified Ti Plasmid into a Plant
Agrobacterium carrying a Ti plasmid is added to plant tissue
growing in culture. The T-DNA carries an antibiotic resistance gene
(neomycin in this figure) to allow selection of successfully
transformed plant cells. Both callus cultures (A) and liquid
cultures (B) may be used in this procedure. Gene Gun or Biolistic
Method
A gene gun is used for injecting cells with genetic information, it
is also known as biolistic particle delivery system.
Biotechnological Applications of Plant Breeding Genetically
modified crops
All plant characteristics, such as size, texture, and sweetness,
are determined on the genetic level. Also: The hardiness of crop
plants. Their drought resistance. Rate of growth under different
soil conditions. Dependence on fertilizers. Resistance to various
pests and diseases. Used to do this by selective breeding 24 24 Why
would we want to modify an organism?
Better crop yield, especially under harsh conditions. Herbicide or
disease resistance Nutrition or pharmaceuticals, vaccine delivery
In 2010, approximately 89% of soy and 69% of corn grown in the U.S.
were grown from Roundup Ready seed. OER open education resources
Roundup Ready Gene The glyphosate resistance gene protects food
plants against the broad-spectrum herbicide Glyphosate-
N-(phosphonomethyl) glycine [Roundup], which efficiently kills
invasive weeds in the field. The major advantages of the "Roundup
Ready system include better weed control, reduction of crop injury,
higher yield, and lower environmental impact than traditional weed
control systems. Notably, fields treated with Roundup require less
tilling; this preserves soil fertility by lessening soil run-off
and oxidation. Glyphosate - N-(phosphonomethyl) glycine
An aminophosphonic analogue of the natural amino acidglycine. It is
absorbed through foliage and translocated to actively growing
points. (Meristems!!!) Mode of action is to inhibit
anenzymeinvolved in the synthesis of thearomaticamino acids:
tyrosine, tryptophan phenylalanine Glyphosate Glycine Glyphosate -
N-(phosphonomethyl) glycine
It does this by inhibiting
theenzyme5-enolpyruvylshikimate-3-phosphate synthase (EPSPS),
whichcatalyzesthe reaction ofshikimate-3-phosphate (S3P) and
phosphoenol pyruvateto form 5-enolpyruvyl-shikimate-3-phosphate
(ESP). ESP subsequentlydephosphorylatedto chorismate, an essential
precursor in plants for these aromaticamino acids. Glyphosate
Glycine Roundup Ready Gene Glyphosate functions by occupying the
binding site of the phosphoenol pyruvate, mimicking an intermediate
state of the enzyme substrates complex. The "Roundup Ready system
introduces a stable gene alteration which prevents Glyphosate
binding and allowing the formation of the essential aromaticamino
acids Roundup Ready Gene Theshikimatepathway is not present in
animals, which instead obtain aromatic amino acids from their diet.
Glyphosate has also been shown to inhibit other plant enzymes Also
has been found to affect animal enzymes. TheUnited States
Environmental Protection Agencyconsiders glyphosate to be
relatively low in toxicity, and without carcinogenic or teratogenic
effects However, some farm workers have reported chemical burns and
contact skin burns Environmental degradation
When glyphosate comes into contact with the soil, it can be rapidly
bound to soil particles and be inactivated. Unbound glyphosate can
be degraded by bacteria. However, glyphosate has been shown to
increase the infection rate of wheat by fusarium head blight in
fields that have been treated with glyphosate. In soils, half-lives
vary from as little as 3 days at a site in Texas to 141 days at a
site in Iowa. In addition, the glyphosate metabolite amino methyl
phosphonic acid has been shown to persist up to 2 years in Swedish
forest soils. Glyphosate absorption varies depending on the kind of
soil. Insect Resistance B. thuringiensis (commonly known as 'Bt')
is an insecticidal bacterium, marketed worldwide for control of
many important plant pests - mainly caterpillars of the Lepidoptera
(butterflies and moths) but also mosquito larvae, and simuliid
blackflies that vector river blindness in Africa. Bt products
represent about 1% of the total agrochemical market (fungicides,
herbicides and insecticides) Genetically modified crops
1992- The first commercially grown genetically modified food crop
was a tomato - was made more resistant to rotting, by adding an
anti-sense gene which interfered with the production of the enzyme
polygalacturonase. The enzyme polygalacturonasebreaks down part of
the plant cell wall, which is what happens when fruit begins to
rot. Genetically modified crops
Need to build in a: Promoter Stop signal CODING SEQUENCE INTRON
poly A signal PROMOTER ON/OFF Switch Makes Protein stop sign
Genetically modified crops
So to modify a plant: Need to know the DNA sequence of the gene of
interest Need to put an easily identifiable maker gene near or next
to the gene of interest Have to insert both of these into the plant
nuclear genome Good screen process to find successful insertion
Building the Transgenes
ON/OFF Switch Makes Protein stop sign CODING SEQUENCE INTRON poly A
signal PROMOTER Plant Selectable Marker Gene Plasmid DNA Construct
Plant Transgene bacterial genes antibiotic marker replication
origin Cloning into a Plasmid The plasmid carrying genes for
antibiotic resistance, and a DNA strand, which contains the gene of
interest, are both cut with the samerestriction endonuclease. The
plasmid is opened up and the gene is freed from its parent DNA
strand. They have complementary "sticky ends." The opened plasmid
and the freed gene aremixed with DNA ligase, which reforms the two
pieces as recombinant DNA. Cloning into a Plasmid Plasmids + copies
of the DNA fragment producequantities of recombinant DNA. This
recombinant DNA stew is allowed to transform a bacterial culture,
which is then exposed to antibiotics. All the cells except those
which have been encoded by the plasmid DNA recombinant are killed,
leaving a cell culture containing the desired recombinant DNA. So,
how do you get the DNA into the Plant? Meristems Injections
REMEMBER!!!!!!!
Tunica-Corpus modelof the apical meristem (growing tip). The
epidermal (L1) and subepidermal (L2) layers form the outer layers
called thetunica. The inner L3 layer is called the corpus. Cells in
the L1 and L2 layers divide in a sideways fashion which keeps these
layers distinct, while the L3 layer divides in a more random
fashion. REMEMBER!!!!!!! Thetissuein mostplantsconsisting of
undifferentiated cells (meristematic cells), found in zones of the
plant where growth can take place. Meristematic cells are analogous
in function tostem cellsinanimals, are incompletely or not
differentiated, and are capable of continued cellular division.
First method of DNA transfer to a plant. Inject DNA into the tip
containing the most undifferentiated cells more chance of DNA being
incorporated in plant Genome Worked about 1 in 10,000 times!
Particle Bombardment 41 Particle Bombardment Particle-Gun
Bombardment DNA- or RNA-coated gold/tungsten particles are loaded
into the gun and you pull the trigger. Selected DNA sticks to
surface of metal pellets in a salt solution (CaCl2). 42 Particle
Bombardment 2. A low pressure helium pulse delivers the coated
gold/tungsten particles into virtually any target cell or tissue.
3. The particles carry the DNA cells do not have to be removed from
tissue in order to transform the cells 4. As the cells repair their
injuries, they integrate their DNA into their genome, thus allowing
for the host cell to transcribe and translate the transgene. 43
Particle Bombardment The DNA sometimes was incorporated into the
nuclear genome of the plant Gene has to be incorporated into cells
DNA where it will be transcribed Also inserted gene must not break
up some other necessary gene sequence 44 Agrobacterium tumefaciens
Overall process Uses the natural infection mechanism of a plant
pathogen Agrobacterium tumefaciens naturally infects the wound
sites in dicotyledonous plant causing the formation of the crown
gall tumors. Capable to transfer a particular DNA segment (T-DNA)
of the tumor-inducing (Ti) plasmid into the nucleus of infected
cells where it is integrated fully into the host genome and
transcribed, causing the crown gall disease. So the pathogen
inserts the new DNA with great success!!! Overall process The vir
region on the plasmid inserts DNA between the T-region into plant
nuclear genome Insert gene of interest and marker in the T-region
by restriction enzymes the pathogen will then infect the plant
material Works fantastically well with all dicot plant species
tomatoes, potatoes, cucumbers, etc Does not work as well with
monocot plant species - corn As Agrobacterium tumefaciens do not
naturally infect monocots Overview of the Infection Process Ti
plasmids and the bacterial chromosome act in concert to transform
the plant
1.Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA
required for initial binding of the bacterium to the plant cell and
code for polysaccharide on bacterial cell surface. 2.Virulence
region (vir) carried on pTi, but not in the transferred region
(T-DNA).Genes code for proteins that prepare the T-DNA and the
bacterium for transfer. 49 3. T-DNA encodes genes for opine
synthesis and for tumor production.
4. occ (opine catabolism) genes carried on the pTi and allows the
bacterium to utilize opines as nutrient. 50 for transfer to the
plant
Agrobacterium chromosomal DNA pscA chvA chvB T-DNA-inserts into
plant genome tra bacterial conjugation for transfer to the plant
pTi vir genes opine catabolism oriV 51 Agrobacterium tumafaciens
senses Acetosyringone via a 3-component-like system
3 components: ChvE, VirA, VirG