(A. thaliana, Daucus carota, Zea mays, Capsicum annuum, and Lycopersicon
esculentum) were tested. Phytoene synthase genes were introduced into Z. mays
cell cultures, in order to determine the most active enzyme. The experiment
showed that psy enzyme of maize achieves the highest increment in b-carotene
expression. The second generation of golden rice was established; it used maize
phytoene synthase and was able to increase the b-carotene content 16–37 mg/g
(Paine et al. 2005).
15.4.3.4 Silencing by iRNA
The introduction of genes to increase the content of nutrients in plants involves the
overexpression of the enzymes that synthesize them. Unfortunately, this strategy has
been restricted by two main obstacles. In the first place, plants can respond to
increased nutrient levels activating degradation pathways and avoiding the accumulation of these metabolites recognized as foreign (Dixon 2005). On the other
hand, introducing gene copies can induce the phenomenon of cosuppression, where
excessive expression of these genes activate negative regulatory mechanisms
(Napoli et al. 1990). This tool has allowed the production of ‘‘Knock down’’
organisms with a decrease in gene expression. The silencing mechanism is activated
by double-stranded RNA (dsRNA), with the target mRNA complementary strands.
The dsRNA induction methods have been highly successful in reducing target gene
expression. Manipulating of specific tissues can be used for gene expression and
for gene silencing that degrades the desired product. The strategy could be used
constitutively, but it would cause side effects to the plant, the strategies are somewhat inclined to the regulation of specific promoters (Tang et al. 2007).
15.5 Transcription factors Manipulation
In plants, like in most of eukaryotes organisms, the genetic expression is mostly
handled at the transcription level. This regulation is mediated by a diverse group
of proteins known collectively as transcription factors (TFs). These molecules
recognize specific sequences of DNA in the promoters. Through protein–protein
interactions, the TFs mediate the construction of the basal machinery for transcription that results in the RNA polymerase II activation and the mRNA synthesis.
The TFs can be classified in families, based on the presence of conserved domains
for DNA binding. The regulation of multiple genes is carried out for the combinatory
action of transcription factors, like the union of TFs-binding protein–DNA or by TF
and a cis-regulatory element (Riechmann et al. 2000). The transcription factors are
considered a viable alternative in the manipulation of metabolic pathways in plants.
Because of its capacity to control multiple steps in a synthetic pathway, the
discovery of this master regulators was shown as a new strategy for metabolic
manipulation (Table 15.1).
436
L. A. Cruz-Ramírez et al.
esculentum) were tested. Phytoene synthase genes were introduced into Z. mays
cell cultures, in order to determine the most active enzyme. The experiment
showed that psy enzyme of maize achieves the highest increment in b-carotene
expression. The second generation of golden rice was established; it used maize
phytoene synthase and was able to increase the b-carotene content 16–37 mg/g
(Paine et al. 2005).
15.4.3.4 Silencing by iRNA
The introduction of genes to increase the content of nutrients in plants involves the
overexpression of the enzymes that synthesize them. Unfortunately, this strategy has
been restricted by two main obstacles. In the first place, plants can respond to
increased nutrient levels activating degradation pathways and avoiding the accumulation of these metabolites recognized as foreign (Dixon 2005). On the other
hand, introducing gene copies can induce the phenomenon of cosuppression, where
excessive expression of these genes activate negative regulatory mechanisms
(Napoli et al. 1990). This tool has allowed the production of ‘‘Knock down’’
organisms with a decrease in gene expression. The silencing mechanism is activated
by double-stranded RNA (dsRNA), with the target mRNA complementary strands.
The dsRNA induction methods have been highly successful in reducing target gene
expression. Manipulating of specific tissues can be used for gene expression and
for gene silencing that degrades the desired product. The strategy could be used
constitutively, but it would cause side effects to the plant, the strategies are somewhat inclined to the regulation of specific promoters (Tang et al. 2007).
15.5 Transcription factors Manipulation
In plants, like in most of eukaryotes organisms, the genetic expression is mostly
handled at the transcription level. This regulation is mediated by a diverse group
of proteins known collectively as transcription factors (TFs). These molecules
recognize specific sequences of DNA in the promoters. Through protein–protein
interactions, the TFs mediate the construction of the basal machinery for transcription that results in the RNA polymerase II activation and the mRNA synthesis.
The TFs can be classified in families, based on the presence of conserved domains
for DNA binding. The regulation of multiple genes is carried out for the combinatory
action of transcription factors, like the union of TFs-binding protein–DNA or by TF
and a cis-regulatory element (Riechmann et al. 2000). The transcription factors are
considered a viable alternative in the manipulation of metabolic pathways in plants.
Because of its capacity to control multiple steps in a synthetic pathway, the
discovery of this master regulators was shown as a new strategy for metabolic
manipulation (Table 15.1).
436
L. A. Cruz-Ramírez et al.