Biomedical Engineering Reference
In-Depth Information
Nopaline T-DNA (23 kb) pTiC58
L border
R border
5
2
1 4 6a
6b
3
Fig. 12.10
Structure and transcription of
T-DNA. The T-regions of nopaline and
octopine Ti-plasmids have been aligned
to indicate the common DNA sequences.
The size and orientation of each transcript
(numbered) is indicated by arrows. Genetic
loci, defined by deletion and transposon
mutagenesis, are shown as follows:
nos
,
nopaline synthase;
ocs
, octopine synthase;
tms
, shooty tumour;
tmr
, rooty tumour.
tms
tmr tml nos
Octopine TL-DNA (13.6 kb) pTiAch5
L border
TR-DNA
R border
5
2
14 6a
6b
L border
R border
7
3
4
'
3'
2'
1'
0'
tms
tmr tml ocs
frs
mas ags
T-DNAs from a nopaline plasmid (pTiC58) and an
octopine plasmid (pTiAch5) are shown in Fig. 12.10
(Willmitzer
et al.
1982, 1983, Winter
et al.
1984).
Interestingly, nucleotide sequencing has revealed
that the T-DNA genes have promoter elements and
polyadenylation sites that are eukaryotic in nature
(De Greve
et al.
1982b, Depicker
et al.
1982, Bevan
et al.
1983a). This explains how genes from a bacterial
plasmid come to be expressed when transferred to
the plant nucleus. It is possible that the sequences
may have been captured from plants during the evo-
lution of the Ti plasmid. The ability of
Agrobacterium
to induce tumours in a wide variety of plants sug-
gested that T-DNA promoters, such as those of the
ocs
(octopine synthase) and
nos
(nopaline synthase)
genes, could be useful to drive transgene expres-
sion. These and other promoters used for transgene
expression in plants are discussed in Box 12.1.
resulting construct was called pGV3850 (Fig. 12.11).
Agrobacterium
carrying this plasmid transferred
the modified T-DNA to plant cells. As expected, no
tumour cells were produced, but the fact that trans-
fer had taken place was evident when the cells were
screened for nopaline production and found to be
positive. Callus tissue could be cultured from these
nopaline-positive cells if suitable phytohormones
were provided, and fertile adult plants were regener-
ated by hormone induction of plantlets.
The creation of disarmed T-DNA was an import-
ant step forward, but the absence of tumour forma-
tion made it necessary to use an alternative method
to identify transformed plant cells. In the experiment
described above, opine production was exploited
as a screenable phenotype, and the
ocs
and
nos
genes have been widely used as screenable markers
pBR322
Prototype disarmed Ti vectors
L border
We have seen that the Ti plasmid is a natural vector
for genetically engineering plant cells because it can
transfer its T-DNA from the bacterium to the plant
genome. However, wild-type Ti plasmids are not
suitable as general gene vectors because the T-DNA
contains oncogenes that cause disorganized growth
of the recipient plant cells. To be able to regener-
ate plants efficiently, we must use vectors in which
the T-DNA has been
disarmed
by making it non-
oncogenic. This is most effectively achieved simply by
deleting all of its oncogenes. For example, Zambryski
et al.
(1983) substituted pBR322 sequences for
almost all of the T-DNA of pTiC58, leaving only the
left and right border regions and the
nos
gene. The
Amp
R
R border
nos
pGV3850
(PTiC58 derivative)
vir
genes
Fig. 12.11
Structure of the Ti-plasmid pGV3850, in which
the T-DNA has been disarmed.
