Agriculture Reference
In-Depth Information
Fig. 10.3 Gene fusion is a
DNA construction in which
the coding sequence from
one gene (reporter) is
transcribed and/or
translated under the
direction of the controlling
sequence of another gene
(controller). (a) Gene
structure. (b) Gene fusion
consisting of two parts: the promoter or controller that senses the nutritional status
of the plant and directs the synthesis of a new product from the second component,
the responder. By replacing the original sequence of the responder gene with a new
and easily studied gene, called a reporter gene, it should be possible to obtain
valuable information about the activity of the promoter. Such a molecular manip-
ulation should provide information about the nutritional status of the plant by
simply measuring the activity of the reporter protein.
Plant biologists to study how a particular gene is controlled when measurement
of the gene product is too difficult have extensively used the gene fusion concept.
The elective tool used in this type of studies is the GUS gene fusion system, which
uses
uidA
from
E. coli
as a reporter gene. This gene encodes a
-glucuronidase able
β
to hydrolyse a wide range of
-D-glucuronide substrates producing coloured or
other compounds in amounts proportional to enzyme activity (Jefferson
1989
).
Assays for testing the activity of GUS in genetically modified plants are carried
out on plant material or plant extracts under laboratory conditions. A variety of
glucuronides is commercially available and can be used for fluorometric, spectro-
photometric, luminometric and histochemical GUS analyses, qualitative as well as
quantitative (Gallagher
1992
).
Unfortunately, reporter systems based on the activity of
β
-glucuronidase (GUS)
have severe intrinsic limitations that preclude their application under field condi-
tions since they require the enzyme (GUS) and its substrate be brought together to
produce the hydrolytic products necessary for the analysis. Likewise, luciferin-
luciferase imaging systems have been used in plants, but their application in the
field is hampered by the low level of light emission and the need for sensitive
photon-counting cameras to detect signal (de Ruijter et al.
2003
). Thus, new tools to
perform non-destructive analysis are essential to develop specific sentinel plants to
be directly used under field conditions.
In a pioneering paper, Jefferson (
1993
) listed some criteria useful to develop new
reporter systems suitable for agricultural molecular biology. Some of the key
criteria, such an in vivo reporter system are that it should be: (i) non-destructive;
(ii) non-disruptive to avoid physiological alteration of crop performance; (iii) useful
β
