Chapter 17. Genetic Engineering of Plants: Methodology
I. Transformation of plants Ti Plasmid of Agrobacterium tumefaciens Physical Methods
I. Transformation of plants
Ti Plasmid of Agrobacterium tumefaciens Physical Methods
Ti Plasmid of Agrobacterium tumefaciens
Physical Methods
II. Manipulating gene expression in plants III. Selectable markers and reporter genes
II. Manipulating gene expression in plants
III. Selectable markers and reporter genes
World Food Challange
1973 4 Billion Human population on earth 1996 5.8 Billion 2003 6.3 Billion 2020 8.5 Billion estimated
1973 4 Billion Human population on earth
1996 5.8 Billion
2003 6.3 Billion
2020 8.5 Billion estimated
Improving Crop Plants for Higher Yields and Nutrition
1.) Resistance to: insects, herbicides, infection by bacterial, fungal and viral pathogens 2.) Delayed senescence (death) and fruit ripening 3.) Increased tolerance to environmental stress high salt, low moisture (drought), heat or frost damage 4.) High nutritional value 5.) Efficient uptake and utilization of soil nutrients (nitrogen, phosphorus)
1.) Resistance to: insects, herbicides, infection by bacterial, fungal and viral pathogens
2.) Delayed senescence (death) and fruit ripening
3.) Increased tolerance to environmental stress
high salt, low moisture (drought), heat or frost damage
4.) High nutritional value
5.) Efficient uptake and utilization of soil nutrients (nitrogen, phosphorus)
Ex. monoclonal antibodies, vaccines, blood clotting factors, b-carotene
Takes time: 10-15 years to develop a new variety Not as precise as molecular methods Limited by species barriers
Takes time: 10-15 years to develop a new variety
Not as precise as molecular methods
Limited by species barriers
Introduction of foreign genes from any source Rapid Precise Known genes are introduced Specific location in plant genome can be targeted
Introduction of foreign genes from any source
Rapid
Precise
Known genes are introduced Specific location in plant genome can be targeted
Known genes are introduced
Specific location in plant genome can be targeted
Contain foreign genes that produce new or improved trait Created by introducing foreign DNA into a totipotent plant cell Totipotent cells can regenerate an entire plant
Contain foreign genes that produce new or improved trait
Created by introducing foreign DNA into a totipotent plant cell
Totipotent cells can regenerate an entire plant
Global trade began in 1995 and has increased rapidly 2002 Area planted in GM crops was 58.7 million hectares 16 countries U.S, Canada, China, Argentina grew 99% U.S. 69%
Global trade began in 1995 and has increased rapidly
2002 Area planted in GM crops was 58.7 million hectares
16 countries
U.S, Canada, China, Argentina grew 99% U.S. 69%
U.S, Canada, China, Argentina grew 99%
U.S. 69%
1. Herbicide tolerance 2. Insect resistance
1. Herbicide tolerance
2. Insect resistance
Soybeans 62% Corn 12% Canola 5%
Soybeans 62%
Corn 12%
Canola 5%
Agrobacterium tumefaciens
Gram negative, soil bacterium Plant pathogen, infects plants (dicots) at site of wound -Agent of crown gall disease (See Fig. 17.1) -Causes uncontrolled plant cell division and formation of a gall (tumor) on the plant Ti (tumor induction) plasmid is required for infection -Ti plasmid naturally transforms plant cells -Has been engineered as a vector for introduction of foreign DNA into plants
Gram negative, soil bacterium
Plant pathogen, infects plants (dicots) at site of wound
-Agent of crown gall disease (See Fig. 17.1) -Causes uncontrolled plant cell division and formation of a gall (tumor) on the plant
-Agent of crown gall disease (See Fig. 17.1)
-Causes uncontrolled plant cell division and formation of a gall (tumor) on the plant
Ti (tumor induction) plasmid is required for infection
-Ti plasmid naturally transforms plant cells -Has been engineered as a vector for introduction of foreign DNA into plants
-Ti plasmid naturally transforms plant cells
-Has been engineered as a vector for introduction of foreign DNA into plants
Ti Plasmid.
Present in pathogenic strains of A. tumefaciens Natural vector, transfers DNA from bacteria to plant cells. (See Fig. 17.3) 200 to 800 kbp (a large plasmid)
Present in pathogenic strains of A. tumefaciens
Natural vector, transfers DNA from bacteria to plant cells. (See Fig. 17.3)
200 to 800 kbp (a large plasmid)
Genetic elements present on the Ti plasmid
1. T-DNA -Transferred to plant
a) Left and right border regions For recombination with plant DNA b) Genes for synthesis of plant growth hormones Auxin and cytokinin (See Fig. 17.5) Cause gall formation => where A. tumefaciens lives in the plant c) Genes for opine synthesis: octopine, nopaline and agropine (See Fig. 17.6) C & N source that only A. tumefaciens with Ti plasmid can use
a) Left and right border regions
For recombination with plant DNA
b) Genes for synthesis of plant growth hormones
Auxin and cytokinin (See Fig. 17.5) Cause gall formation => where A. tumefaciens lives in the plant
Auxin and cytokinin (See Fig. 17.5)
Cause gall formation => where A. tumefaciens lives in the plant
c) Genes for opine synthesis: octopine, nopaline and agropine (See Fig. 17.6)
C & N source that only A. tumefaciens with Ti plasmid can use
-Not transferred to plant
2. vir. 25 virulence genes
Induced by plant wound-response compounds (See Fig. 17.2) Required for transfer of T-DNA from plasmid into plant cell
Induced by plant wound-response compounds (See Fig. 17.2)
Required for transfer of T-DNA from plasmid into plant cell
3. Opine catabolic genes. Allow A. tumefaciens to use opines for growth
4. ori. For replication of Ti plasmid in A. tumefaciens Genetic Engineering of the Ti Plasmid
To produce a vector useful for introducing foreign DNA into plant cells
1. Reduction of large size, to increase transformation efficiency
Genes not necessary for function as a vector were deleted -Biosynthesis of plant hormones -Biosynthesis and utilization of opines -vir
Genes not necessary for function as a vector were deleted
-Biosynthesis of plant hormones -Biosynthesis and utilization of opines -vir
-Biosynthesis of plant hormones
-Biosynthesis and utilization of opines
-vir
2. Addition of E. coli origin of replication
For use as shuttle vector between E. coli and A. tumefaciens
3. Addition of plant and bacterial selectable markers
Antibiotic resistance genes (neomycin, kanamycin)
4. Addition of multiple cloning site within T-DNA region
Under transcriptional control of promoter that functions in plant cells Ex. 35S promoter of cauliflower mosaic virus Termination-polyadenylation sequence
Under transcriptional control of promoter that functions in plant cells
Ex. 35S promoter of cauliflower mosaic virus
Termination-polyadenylation sequence
Vectors Used to Introduce Foreign DNA Into Plant Cells
Two different sytems have been developed
I. Binary vector system i.e. a 2 plasmdi vector system (See Fig. 17.7)
1. Binary cloning vector Cloning steps performed in E. coli Foreign DNA ligated into multiple cloning site in T-DNA region Recombinant vector transfered to A. tumefaciens containing a disarmed Ti plasmid 2. Disarmed Ti plasmid Lacks T-DNA region, but has vir genes Mobilizes T-DNA containing foreign DNA, from cloning vector in Agrobacterium into plant cell
1. Binary cloning vector
Cloning steps performed in E. coli Foreign DNA ligated into multiple cloning site in T-DNA region Recombinant vector transfered to A. tumefaciens containing a disarmed Ti plasmid
Cloning steps performed in E. coli
Foreign DNA ligated into multiple cloning site in T-DNA region
Recombinant vector transfered to A. tumefaciens containing a disarmed Ti plasmid
2. Disarmed Ti plasmid
Lacks T-DNA region, but has vir genes Mobilizes T-DNA containing foreign DNA, from cloning vector in Agrobacterium into plant cell
II. Cointegrate cloning vector
-Similar to binary vector, but:
Doesn't have an ori for replication in A. tumefaciens This forces the vector to recombine (integrate into) with the disarmed Ti plasmid Sequence for homologous recombination with the disarmed Ti plasmid Results in formation of a single plasmid in A. tumefaciens
Doesn't have an ori for replication in A. tumefaciens
This forces the vector to recombine (integrate into) with the disarmed Ti plasmid
Sequence for homologous recombination with the disarmed Ti plasmid
Results in formation of a single plasmid in A. tumefaciens
Comparison of Methods for Transfer of DNA to Plants
1. Vectors engineered from Ti plasmids
Good for dicots (e.g. soybeans) Not effective for major grain crops which are monocots rice, corn, wheat
Good for dicots (e.g. soybeans)
Not effective for major grain crops which are monocots
rice, corn, wheat
2. Microprojectile bombardment (Biolistics)
Good for ll plant types Coat DNA onto gold particles (~1 mm), shoot into cells Unknown mechanism for integration into plant genome Stable integration into genome is difficult to achieve
Good for ll plant types
Coat DNA onto gold particles (~1 mm), shoot into cells
Unknown mechanism for integration into plant genome
Stable integration into genome is difficult to achieve
3. Transformation of protoplasts
Digest cell wall with enzymes, introduce DNA by various methods Ex. Electroporation, liposome fusion, microinjection Allow cell to resynthesize cell wall and regenerate plant Totipotent. Ability of a cell to develop into an entire plant May be difficult or not possible with some plants
Digest cell wall with enzymes, introduce DNA by various methods
Ex. Electroporation, liposome fusion, microinjection
Allow cell to resynthesize cell wall and regenerate plant
Totipotent. Ability of a cell to develop into an entire plant May be difficult or not possible with some plants
Totipotent. Ability of a cell to develop into an entire plant
May be difficult or not possible with some plants
Manipulation of Gene Expression in Plants
Primarily at transcriptional leve via regulated or constitutive promoters
Cloned cDNA genes do not their original promoter
Provide the appropriate promoter prior to transfer to the plant
Use a strong constitutive promoter 35S cauliflower mosaic virus promoter
Use a strong constitutive promoter
35S cauliflower mosaic virus promoter
Use promoters for genes that encode proetins found at high levels in the tissue where expression of the transgene is wanted Ex. -Leaves Ribulosebisphosphate carboxylase promoter Enzyme that fixes CO2 during photosynthesis -Seeds Phaseolin gene promoter Seed-storage protein in beans Maize ubiquitin gene promoter Protein present in corn kernals -Roots Sporamin gene promoter Sweet potatoe tuber storage protein
Use promoters for genes that encode proetins found at high levels in the tissue where expression of the transgene is wanted
Ex.
-Leaves
Ribulosebisphosphate carboxylase promoter Enzyme that fixes CO2 during photosynthesis
Ribulosebisphosphate carboxylase promoter
Enzyme that fixes CO2 during photosynthesis
-Seeds
Phaseolin gene promoter Seed-storage protein in beans Maize ubiquitin gene promoter Protein present in corn kernals
Phaseolin gene promoter
Seed-storage protein in beans
Maize ubiquitin gene promoter
Protein present in corn kernals
-Roots
Sporamin gene promoter Sweet potatoe tuber storage protein
Sporamin gene promoter
Sweet potatoe tuber storage protein
Use promoters that are expressed in response to certain environmental conditions or the presence (or absence) of chemical signals Ex. 1. Expression during environmental stress Osmotin protein promoter Induced by dessication, salt or wounding Ex. 2 Nutrient limitation Acid phosphatase promoter Induced in roots by low P concentration
Use promoters that are expressed in response to certain environmental conditions or the presence (or absence) of chemical signals
Ex. 1. Expression during environmental stress
Osmotin protein promoter Induced by dessication, salt or wounding
Osmotin protein promoter
Induced by dessication, salt or wounding
Ex. 2 Nutrient limitation
Acid phosphatase promoter Induced in roots by low P concentration
Acid phosphatase promoter
Induced in roots by low P concentration
Other genetic elements that affect gene expression
Increase transcription of a gene up to several kilobases from a gene Usually located hundreds to thousands of nucleotides up- or downstream of a promoter
Increase transcription of a gene up to several kilobases from a gene
Usually located hundreds to thousands of nucleotides up- or downstream of a promoter
Noncoding DNA within eukaryotic genes May stabilize mRNA (prevent degradation by nucleases prior to translation) Not present in cDNA, an intron must be provided , or Clone gene directly so that introns will be present
Noncoding DNA within eukaryotic genes
May stabilize mRNA (prevent degradation by nucleases prior to translation)
Not present in cDNA, an intron must be provided , or
Clone gene directly so that introns will be present
Located at the end of a gene Necessary for efficient expression of genes Not present in prokaryotic genes Can be provided along with the promoter on the vector
Located at the end of a gene
Necessary for efficient expression of genes
Not present in prokaryotic genes
Can be provided along with the promoter on the vector
Selectable Marker and Reporter Genes For Transgenic Plants
Introduced into plant genome along with the gene of interest
Used to identify and isolate transgenic cells and plants
Allow growth of cells containing foreign DNA Ex Antibiotic resistance genes npt encodes neomycin phosphotransferase often used Inactivates neomycin, kanamycin and G418 by phosphorylating them
Allow growth of cells containing foreign DNA
Ex Antibiotic resistance genes
npt encodes neomycin phosphotransferase often used
Inactivates neomycin, kanamycin and G418 by phosphorylating them
Identify transformed plant cells and may allow the level of expression of a foreign gene to be measured Ex. Gene encoding an enzyme that can be assayed uidA encodes b-glucuronidase (GUS) Hydrolyzes 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-GlcU) to a colored product (Analogous to hydrolysis of X-Gal by b-galactosidase)
Identify transformed plant cells and may allow the level of expression of a foreign gene to be measured
Ex. Gene encoding an enzyme that can be assayed
uidA encodes b-glucuronidase (GUS)
Hydrolyzes 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-GlcU) to a colored product (Analogous to hydrolysis of X-Gal by b-galactosidase)
Hydrolyzes 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-GlcU) to a colored product
(Analogous to hydrolysis of X-Gal by b-galactosidase)
Production of Transgenic Plants Without Reporter and Marker Genes
Protein may be toxic or cause allergies Antibiotic resistance gene might be transferred to pathogens
Protein may be toxic or cause allergies
Antibiotic resistance gene might be transferred to pathogens
I. Cotransform plant cells using two vectors
1) One vector with a reporter gene 2) Other contains the target gene Some cells with reporter will also contain the target gene, but integrated at different chromosomal locations (the two genes are now unlinked) Eliminate reporter from plant by traditional breeding methods Cross transgenic plant with nontransgenic plant Identify progeny that have the target gene but not the reporter gene Propagate the transgenic plants lacking the reporter
1) One vector with a reporter gene
2) Other contains the target gene
Some cells with reporter will also contain the target gene, but integrated at different chromosomal locations (the two genes are now unlinked)
Eliminate reporter from plant by traditional breeding methods
Cross transgenic plant with nontransgenic plant Identify progeny that have the target gene but not the reporter gene Propagate the transgenic plants lacking the reporter
Cross transgenic plant with nontransgenic plant
Identify progeny that have the target gene but not the reporter gene
Propagate the transgenic plants lacking the reporter
II. Place the reporter gene between plant transposable sequences (Ds elements) (Fig. 17.18)
Ds elements are mobile segments of DNA that can move from one chromosomal location to another Transposase gene also present on the T-DNA Enzyme involved in movement of Ds element from one location to another The reporter and target genes become separated on the plant chromosome Reporter is eliminated by traditional breeding methods (see above)
Ds elements are mobile segments of DNA that can move from one chromosomal location to another
Transposase gene also present on the T-DNA
Enzyme involved in movement of Ds element from one location to another
The reporter and target genes become separated on the plant chromosome
Reporter is eliminated by traditional breeding methods (see above)
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