Agriculture Reference
In-Depth Information
expression proi ling, it is now possible to
identify and characterize the function of
genes and the resulting gene products in the
metabolic pathways of living organisms. h is
knowledge opens up enormous possibilities
for plant genetic engineering, allowing not
only the expression of a new protein but also
the ability to change complex metabolic
pathways in the plant. h is will allow the
scope of input traits to be broadened, but
especially it will open unlimited possibilities
to improve the quality of plant crops with
new or modii ed output traits.
used, either because it is derived from
a cDNA sequence and the regulatory
sequences are not available or because the
gene is derived from bacteria or another
organism using dif erent expression signals
than in plants, or because the expression
regulation of the original gene does not
result in the desired spatial and develop-
mental expression levels in the acceptor
plant. In this case, extra cloning work needs
to be done.
In short, a transgene requires the fol-
lowing necessary components:
1.
h e promoter, which is the on/of switch
that controls the gene transcription during
development in response to external biotic
and abiotic circumstances. h e promoter is
responsible for tissue- or organ-specii c
expression or for constitutive expression of
the coding sequence. An example of the
latter is the widely used 35S caulil ower
mosaic virus promoter. Genes under the
control of the 35S constitutive promoter are
highly transcribed throughout the whole life
cycle and in most of the tissues and organs
of the plant. h e promoter is, in most cases,
combined with the coding sequence by a
transcriptional fusion in the 5 untranslated
region.
2.
h e coding sequence of the gene of
interest. Often, this coding sequence is
adapted in order to achieve a better
expression in the transgenic plant. For
example, in the case of certain bacterial
genes, the adenine thymine (AT) content of
the coding sequence is much higher than in
plants and this results in aberrant premature
transcription termination. h is has been the
case for the gene encoding the
Bacillus
thuringiensis
-derived insecticidal protein
(BT). By substituting A-T nucleotide pairs
with G-C nucleotide pairs in the coding
sequence without signii cantly changing the
amino acid sequence, correct transcription
could be obtained. Another reason to change
the primary sequence is that the codon
usage can be very dif erent in the donor
organism than in the acceptor plant. h en,
the codon usage can be adapted by making
use of the redundancy of the genetic code.
In this way, enhanced translation ei ciency,
2.2.2 The cloning step: from gene
functional transgene construct
After the discovery of the gene coding for
the trait of interest and physically obtaining
the DNA for this gene, the next step is to
create a gene unit that will lead to the
functional expression of the trait of interest
in the acceptor plant. h is means that the
coding sequence for the trait needs to be
linked to the necessary regulatory elements
that are responsible for the transcription
and translation of the coding sequence,
i nally resulting in a functional protein. In
order to obtain a functional gene, dif erent
options are open, depending on the
relationship between donor and acceptor
organism and depending on the desired
spatial and developmental expression
pattern for the transgenic trait. Moreover,
when specii c demands on cell localization
for the transgenic protein need to be taken
into account, specii c targeting signals need
to be included in the construct.
h e easiest and most straightforward way
is when the intact gene is available and when
the regulatory elements are functional in
the acceptor plant and give rise to the
desired expression proi le. In this case, the
gene as such can be used for the gene
transfer: this means that the DNA sequence,
as it is obtained from the donor organism,
contains all the required information with
the coding sequence as well as regulatory
elements. However, in many cases, only the
coding sequence of the gene of interest is
