Chapter 18 Homework
1. Why is the Ti plasmid from A. tumefaciens well suited for developing a vector to transfer
foreign genes into plant chromosomal DNA?
The Ti plasmid carries the genes that allow the bacterium to infect the plant cells, and then insert
a region of their DNA called the T DNA into the chromosomes of the infected cells. This
property then makes it easier to manipulate this structure of T DNA to insert our gene of interest
into the plant chromosomes. The vir region in the Ti plasmid is essential for the mobility, and
separation of the T DNA region where the foreign gene has been inserted. The concept of binary
vectors where the vir genes are not present in the same plasmid with the T DNA region led to the
construction of a system for plant transformation.
2. How do (1) binary and (2) cointegrate Ti plasmid-based vector systems for plant
transformation differ from one another?
The difference between the binary vector system and the cointegrate vector system comes with
the generation of the final plasmid. In the binary system the plasmid has origins of replication
for both E. coli and A. tumefaciens. It also has selectable markers for both systems and has the
intact T-DNA with both left and right arms. The cointegrate vector system uses two plasmids
with homologous regions that will allow recombination to generate a final plasmid with all of the
necessary parts. Each of the two initial plasmids contains one origin of replication; one for A.
tumefaciens and one for E coli. When the plasmids recombine the final plasmid has both origins
of replication. The same is true for the left and right borders of the T-DNA. In addition, the
final T-DNA produced with the cointegrate system includes the homologous DNA sequence.
3. What are reporter genes and how are they used when plant cells are transformed?
Reporter genes are usually used to measure the expression level of a particular gene or to
determine the subcellular localization of a protein product. The reporter genes can be fused with
the target gene, thus creating a reporter construct without disturbing the original translational
frame of the protein. This forms a chimeric protein containing the target and the reporter protein.
When the gene-reporter construct is introduced into the cells, the chimeric protein is synthesized.
Depending on the localization of the chimeric protein, one can find out the production, and the
subcellular localization of the protein.
4. How are plants transformed by microprojectile bombardment?
A particle gun or gene gun is used to release DNA-coated gold or tungsten particles. Helium is
used to build pressure until a stopping disk bursts and lets the gases go. The gold or tungsten
particles penetrate the plant cells. This inserted DNA can then integrate into the plant DNA.
OR
The Ti plasmid mediated gene transfer doesn’t work for certain types of plants like rice, corn,
wheat etc which are monocots. The microprojectile bombardment initially started with gun
powder to shoot genes inside the plant chromosome. However, later it was modified, and use
helium which is pumped into a chamber. After it reaches a certain pressure, the disk ruptures and
moves the carrier plate/screen. On the screen are the gold-tungsten particles coated with the gene
of interest along with a selectable marker. The pressure of the gas propels the entire flying disk
downwards until it is stopped by the stopping screen which allows only the gold particles to pass,
and insert themselves into the cells of the plant.
5. Describe how you would isolate a root-specific plant promoter.
In order to isolate a root specific plant promoter, the promoter tagging vectors can be used. A
promoterless vector is inserted beside the right border of the Ti plasmid vector. If it is directed
towards the root, and the T-DNA gets inserted immediately downstream of a promoter region of
a functional gene in the root, transcription of the reporter gene occurs, which can be detected due
to the reporter protein, and hence the root promoter can then be identified and isolated.
6. How is foreign DNA targeted for integration into chloroplast DNA?
To integrate foreign DNA into chloroplasts should use microparticle bombardment. The DNA
pieces that are attached to the microparticles first have sequences added to flank the target DNA
piece that are homologous to the DNA in the chloroplast. It is important to remember that the
chloroplasts have their own genome but that it is prokaryotic in nature so it is easy to design
homologous regions that will be specific for the chloroplast. The homologous flanking regions
will allow recombination with the chloroplast genome after the particles are shot into the plant
cells and the target gene piece will be integrated into the genome. This is an important system
because there are many chloroplasts per plant cell so huge amounts of target protein can be
produced.
10. How would you downregulate the expression of a plant gene?
To downregulate the expression of a plant gene I would use cosuppression which is a technique
similar to RNA interference in which expression of an antisense version of the gene
downregulates expression. In addition, if this did not work well enough I could add the use of
ribozymes specific for whatever gene I wanted to downregulate. Ribozymes cleave mRNA in a
predictable fashion so they can be designed to cut the mRNA for the protein of interest.
11. What is the advantage of introducing foreign genes into chloroplast rather than nuclear
DNA?
The advantage of introducing foreign genes into chloroplasts is that there are many chloroplasts
per cell so the odds that transfection will occur are higher and the total amount of protein that can
be produced is much higher because the gene will be transcribed and translated much more
frequently.
15. Suggest a strategy that would facilitate the large-scale purification of soluble proteins,
such as antibody fragments, in plants.
One method of large scale protein purification involves the production of a fusion protein
between the water soluble target protein such as antibody fragments and an oleosin protein. The
oleosin molecules embed themselves within oil bodies in plant seeds so the fusion protein will be
attached to the oil body as well. Then all that must be done is separation of the oil bodies from
the rest of the plant material within the seed and then cleavage of the connection between the
soluble target protein and the oleosin protein molecule. The resulting extract will contain only
the soluble protein.
16. How do enhancer sequences facilitate plant gene expression?
Enhancer sequences enhance expression of the target gene in the plant cells because it helps to
recruit more RNA polymerase molecules to the gene of interest and therefore improves the yield
of mRNA that can be translated into protein product. The enhancer sequences can be added to
the gene sequence being integrated and the enhancer element itself will also be integrated into
the plant genome. The enhancer element will help to form the transcription complex, which will
result in higher production of mRNA (i.e., more transcription of the gene into mRNA).