Agriculture Reference
In-Depth Information
For example, in their experiments aimed at con-
trolling powdery mildew infection, Altpeter et al.
(2005) used a
GSTA1
gene promoter that is
pathogen-induced and primarily expressed in the
leaf epidermal cells. Transgenes aimed at provid-
ing drought resistance can use promoters induced
by stress, such as that of the barley (
Hordeum
vulgare
L.)
HVA1
gene (Hong et al., 1992).
Unfortunately, there are few promoter sequences
known in wheat or other cereals whose expres-
sion levels, inducibility, and tissue specifi city
are well-defi ned. Furthermore, an experimenter
may not know when and where a gene is best
expressed to have the desired impact. For these
reasons, and because high levels of expression are
usually desired, most wheat transformation
experiments to date have utilized one of the
three constitutive promoters described earlier or
the gene's own native promoter to express genes
of interest.
be transformed at a reasonable effi ciency. The
gene overexpression and suppression strategies
have been more often employed because these
experiments can be done with genotypes chosen
for ease of transformation.
Overexpression of a gene sequence can easily
be achieved by transformation to introduce more
copies of a native gene or to introduce construc-
tions consisting of the gene's coding region under
control of a nonnative (heterologous) but highly
expressed promoter. To suppress native gene
expression, early experiments utilized DNA con-
structions which were transcribed into antisense
RNAs that read in the direction opposite the
native mRNAs that were translated into protein.
Such transcripts would hybridize to their com-
plementary sense transcripts, forming double
stranded (ds)RNAs that could not be translated
into proteins (Fig. 18.2). Thus, antisense RNAs
decreased gene expression by disabling homolo-
gous coding messenger RNAs.
Some transformants containing constructs
designed to increase gene expression of native
genes were found instead to reduce the expres-
sion of those genes. This paradoxical phenome-
non, easily visualized in experiments to change
petunia fl ower color (Jorgensen 1995), was
called sense suppression or transgene-mediated
cosuppression (Matzke and Matzke 1995). It
could be explained by the unintended formation
of antisense RNA when newly integrated
sequences were transcribed from promoters
brought into proximity either by integration near
Applications for functional genomics
Many transformation experiments in wheat have
the goal of identifying the gene sequences and
molecular bases underlying trait expression.
Broad strategies for this kind of functional genom-
ics approach are increasing gene expression,
decreasing gene expression (suppression), and
complementation, whereby a functional gene is
added to a genotype that lacks it. The latter can
only be used in conjunction with transformation
when the genotype that lacks a gene function can
DNA
Transcription
sense
dsRNAs
antisense
Complementary RNAs
Hairpin RNA
Fig. 18.2
Arrangement of transgenes that can result in promoter read-through from adjacent wheat genomic DNA, or
adjacent transgenes that integrate in opposite orientations and lose one or both functional transcription terminators. Such
transgenes can be transcribed to form antisense (ending in open arrow) or hairpin RNAs. A promoter in wheat genomic
DNA is depicted as a solid horizontal arrowhead. The sense RNA transcribed from the gene-of-interest promoter ends in
a fi lled arrow. Complementary regions between strands that form double-stranded RNA are indicated by vertical lines.
Other symbols are defi ned in Fig. 18.1. Drawings are not to scale.
