marker gene among progeny. Onlyconstitutivepromoters can ensure expression in all
tissues and at all stages of development (Figs. 9.1–9.4). The most frequently used consti-
tutive promoters were those associated with genes found in the T-DNA of the Ti plasmids,
in particular the nopaline synthase (nos) gene (Fraley et al. 1983; Bevan et al. 1983) or the
promoter of the cauliflower mosaic virus (CaMV), in particular that associated with the 35S
transcript. Although not of plant origin, both were among the best studied during the time
when transformation technologies were first being developed. The 35Spromoter is gener-
ally the stronger promoter of the two (Sanders et al. 1987) and provides an advantage in
selection efficiency, particularly in species where the selection procedure is not optimal.
These two promoters are generally effective over a very wide range of dicot species;
however, they were shown to not be very good in moncot species. For cereals, the rice
actin and maize ubiquitin 1 (Christensen and Quail 1996) promoters provide better alterna-
tives. Today, many other constitutive promoters have been isolated, but the 35Sand nos pro-
moters are still extensively used. Why is that? There are two reasons: (1) a large body of
knowledge on their performance and behaviors has accumulated since the mid-1980s,
and (2) they are widely available in most of the plant biotechnology laboratories world-
wide. If it works, and you have it, why use anything else? This question will be addressed
later in the chapter.
As we saw in the previous chapter, when constructing transformation vectors, a number
of factors must be considered in addition to the promoter selected to drive the expression of
marker genes. For example, the orientation of the promoter within the transferred DNA is
also extremely important. The 35Spromoter may interact with neighboring promoters in the
vector and plant sequences at the insertion site. It is known to radically alter the specificity
of tissue-specific promoters. This should not be particularly surprising as it often used in
activation-tagging experiments, in which it is randomly inserted into the genome to
elevate the expression of genes within its range of influence (Fig. 9.5). Field studies
Figure 9.5.Interactions occurring between marker genes and elements in the plant genome. Various
experimental strategies have been developed to probe and exploit the plant genome for functional
elements and genes. This includes the use of vectors in which reporter genes are introduced into trans-
genic plants without key regulatory elements such as promoters and enhancers. Activation of the
reporter is therefore dependent on the acquisition of the missing elements from the genome at the
site of insertion. These are calledenhancer traporpromoter trapexperiments. The frequency of trap-
ping such elements can be very high. The regulatory elements may be associated with expressed genes
(Px) or may lie dormant in the genome as cryptic elements (Pcryptic). An alternate strategy used to
activate genes of interest is by introducing strong constitutive enhancer elements alone. This is often
referred to asactivation taggingof genes. Interestingly, this strategy can be combined with selection
and/or screening techniques to recover genes within specific functional groups.
9.3. PROMOTERS 225