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A dicot plant has the following properties: (i) salt tolerant, and (ii) ready transformable with
Agrobacterium.
(a) Using a yeast system, design one strategy to clone the genes conferring salt tolerance.
(5 marks)
• Identify a yeast strain that is sensitive to salt (0.5) and determine the killing dosage
(0.5).
• Construct a cDNA library of the target plant (0.5) using a yeast expression vector
(0.5), preferably carrying an inducible promoter (0.5).
• Transform the yeast strain with the cDNA library (0.5).
• Screen/select on medium containing killing dose of salt (0.5); untransformed yeast
cells should be included as the negative control (0.5); the survived yeast cells
may contain cDNA clone of the salt tolerance gene (0.5).
• Verify the results by (i) growing the yeast with or without the inducer on medium
containing killing dose of salt (0.5) and (ii) re-transforming the original yeast
strain with plasmids containing putative clones and looking for a high survival
rate (e.g. 100%) of the transformed cells (0.5).
(b) After you have successfully cloned the putative genes in (a), suggest two tests to
verify that the cloned genes are responsible for salt tolerance in plants. (5 marks)
• Engineer the cDNA clone from (a) into a plant expression vector containing
appropriate selection markers (0.5) and a constitute promoter that can function
in plant (1);
• Drive the expression is either in sense (0.5) or antisense (0.5) orientation;
• Use co-integration / binary vector system (0.5) for Agrobacterium-mediated (0.5)
plant transformation;
• Transform the sense construct into a salt sensitive plant to see if the cDNA can confer
salt tolerance (1);
• Transform the antisense construct into the original salt tolerant plant to see if the salt
tolerance is reduced (1).
(c) You are asked to test if a target gene cloned in (a) is only expressed in a particular cell
type, explain what experimental approach you will employ. (5 marks)
OR
• Using the clone gene as the template, make labeled (0.5) antisense (1) RNA / single
stranded DNA probes (1) by in vitro transcription / biased PCR techniques
(0.5);
• Prepare sections from tissues of the target plant (0.5)
• Perform in situ hybridization (1) by hybridization the sections with probes (0.5)
described above;
• Detect the bound probes after washing steps (0.5).
(d) Two different genes (Gene X and Gene Y) were cloned in (a). Despite that either of
the Gene X or Gene Y can confer salt tolerance to certain extent, a strong synergistic
effect was observed when both Gene X and Gene Y were expressed in the same cell.
Design one experiment to test if the gene products of Gene X and Gene Y physically
interact with each other. (5 marks)
In the model plant Arabidopsis thaliana, random T-DNA insertional mutagenesis was
performed. Independent mutant lines were collected and propagated separately. Making use
of this tool, how can you study the functions of a cloned gene with known DNA sequence?
(5 marks)
• In the independently collected and propagated mutant lines, some may carry T-DNA
inserted in the gene of interest (0.5) and thus represent knock-out mutants /
loss-of-function mutants (0.5);
• These knock-out / loss-of-function mutants, the functions of the target gene can then
be studies by comparing to the wild type parent (1);
• To identify T-DNA insertional mutants of the target genes, PCR approach will be
used. One primer based on the sequence of the T-DNA (0.5) and one primer
based on the sequence of the cloned gene (0.5) will be used to perform PCR
reactions (0.5) on the genomic DNA prepared from randomly generated
mutant lines (0.5). Positive results indicate that the T-DNA and target gene
are in close proximity (0.5) suggesting that the T-DNA might have inserted in
the target gene. To facilitate screening, genomic DNA from pooled lines can
be used (0.5). Confirmation of successful insertions can be achieved by
sequencing the PCR products (0.5).
Question 3
• An EPO gene construct will be made so that the EPO is fused to a tag (0.5) such as
HIS-Tag (0.5); a cleavage site (0.5) should be introduced between the EPO
and His-Tag (0.5) for future separation of these two protein domains;
• The construct, driven by an appropriate promoter in a suitable vector (0.5); check the
host system) will be transformed into an animal cell (1; 0.5 if other eukaryotic
cell lines were used; no mark if bacteria were used) such as insect cells (0.5;
when baculovirus vector was used; using an animal cell line is to ensure that
animal specific complex sugar will be attached to the recombinant EPO
protein (0.5);
• After growing and harvesting the cells (0.5), the protein extract will be purified
through an affinity column (0.5).