Agriculture Reference
In-Depth Information
and thus the production of transgenic
protein in the plants, can be achieved.
3.
h e terminator sequence, which contains
the signal for the end of transcription and
for correct processing of the RNA.
4.
Localization signals. In case there is a
need for specii c targeting of the transgenic
protein towards particular organelles, the
nucleus, the endoplasmatic reticulum, the
vacuole or to the extracellular space, a signal
peptide and cleavage signals need to be
foreseen in the gene construct. In this case,
a translational fusion between the signal or
localization peptide and the coding sequence
is made.
5.
Translational fusion of tags. In particular
cases, signals for post-translational modii -
cation or peptide tags are added, especially
when the goal is to purify the recombinant
protein produced in the transgenic plant. In
such a case, it should be checked after clon-
ing whether the fusion between the coding
sequence and the tag are in frame.
and regeneration of transformed cells is
sui ciently high, one can avoid the use of
selectable markers by screening the plants
for the presence of transgenic DNA, and as
high throughput PCR screening is now
technically feasible, this is the preferred
approach, if possible (see Section 2.2.7).
For tissue explant co-cultivation, how-
ever, the incorporation and expression of a
transgene in a plant cell that subsequently
regenerates into a transgenic shoot is a rare
event. h erefore, a method is needed either
to kill the non-transformed cells, such that
only the transformed growing cells survive,
or a visible marker is needed to distinguish
the transformed from the non-transformed
cells.
Selectable marker genes result in a
selective advantage for the transformed
cells: either (i) because of the expression of
an enzyme that inactivates the selective
agent (detoxii cation); or (ii) because of the
expression of a resistant variant of the
endogenous enzyme that is the target of the
selection agent (tolerance). In the i rst case,
the selectable marker genes encode enzymes
that detoxify particular chemical products,
such as antibiotics or herbicides, providing
the transformed plant cell's resistance to
these chemicals. In the second case, the
selectable marker encodes an antibiotic- or
herbicide-tolerant target enzyme.
An example of the i rst group is the
antibiotic-resistance genes. Antibiotic-
resistance markers detoxify the antibiotics
by modifying them. For example,
neomycin
phos photransferase II
(NPTII) or
hygromycine
phosphotransferase
(HPT) specii cally phos-
phorylate neomycin/kanamycin/G418 and
hygromicin, respectively. Kanamycin and
hygromycin are taken up by the plant cells
and they bind on to a subunit of the
mitochondrial and chloroplast ribosomes,
thereby inhibiting translation and thus
blocking energy production and photo-
synthesis. h e phosphorylated kanamycin
and hygromicin compounds can no longer
bind to the ribosomes. h us, plant cells with
the NPTII or HPT enzymes are resistant to
kanamycin and hygromicin, respectively.
In this way, these antibiotic-resistance
selection markers are very often used to
In practice, transgene construction starts
with the on-paper design of the complete
gene sequence. Subsequently, the DNA
fragment is made in a test tube by cutting
and pasting dif erent gene elements
together; nowadays, the transgene encoding
DNA fragment is made synthetically by
polymerizing nucleotides in the desired
sequence.
Besides the transgene to be transferred to
the plant cell, a selectable marker gene is
linked to it most of the time, as expression
of this selectable marker allows identii cation
of the plant cells with the integrated
transgene construct. Indeed, only in some
systems is the transformation frequency
high enough, meaning higher than 1%, to
make simple PCR screening for transformed
cells with the transgene construct possible.
h is is, for instance, the case after protoplast
co-cultivation or after l oral dipping of
Arabidopsis l ower stalks with the relevant
A. tumefaciens
strain. h us, in case the
ei ciency
of
transformation/integration
