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VYACHESLAVOVA et al.
rbcL P SG Т P TG Т accD
rbcL
Chloroplast
DNA
accD
Homologous recombination
rbcL
Chloroplast DNA
P
SG
Т
P
TG
Т
accD
Fig. 3. A schematic diagram of plastid transformation. rbcL, gene of the large subunit of ribulose-bisphosphate-carboxylase/oxygenase;
accD, carboxytransferase β-subunit gene; P, promoter; T, terminator; SG, selective marker gene; TG, target gene.
ble. Nevertheless, this method has several serious
drawbacks: plastids do not maintain many posttranslational
modifications of proteins, such as glycosylation;
a number of heterologous proteins in plastids form
inclusion bodies that require repeated folding during
the purification process. Long-term investigations and
optimization of the technology for an individual
plant/construct is needed.
Transient Expression
Transient expression of foreign genes in plants is a
method based on temporary expression of a target
gene in specific plant organs [18]. At present, the following
main approaches are employed for transient
expression of genes in plants: Agrobacterium-mediated
transformation, expression with the use of plant viral
vectors [19], and magnifection.
Agrobacterium-mediated transient expression. As
mentioned above, the process of agrobacterial transformation
can be divided into four steps: attachment
of the bacterium to the plant cell wall, penetration of
T-DNA into the plant cell, integration of T-DNA into
the plant genome, and expression of genes from the
T-DNA region (Fig. 1). In contrast to stable transformation,
no integration of the transgene into the plant
genome occurs in this case, and its expression takes
place directly from the intact DNA of the agrobacterium
through the system of plant cell replication.
The proposed molecular mechanism of transient
expression of genes in plants is described in detail in
review [19]. It was shown, in particular, that after excision,
the T-DNA region exists in the bacterial cell as a
complex of single-stranded DNA (ssT-DNA) with the
bacterial proteins (T complex). The bacterial proteins
not only stabilize the structure of the T complex, they
are also involved in its transport to the host plant cell.
For double-stranded T-DNA to be formed, ssT-DNA
is released from the proteins of the mature T complex.
This process is assumed to involve both the agrobacterial
and plant proteins.
Transient expression with the use of viruses is based
on the knowledge of viral genomes. Viruses, including
plant viruses, have similar genome structures and carry
three main sequences: a sequence encoding the replicase
protein (involved in replication of viruses in the
host organism); a sequence encoding the transport
protein (involved in the spread of the virus in the host
organism), and a sequence encoding the viral envelope
protein (involved in the formation of viral particles).
Figure 4 presents a diagram showing the application of
viruses for transient expression of target genes and
sequences. In most cases, researchers use the following
approaches: substitution of a target sequence for
either the gene encoding the transport protein or the
gene encoding the viral envelope protein; insertion of
a target sequence between the sequences of the viral
genome; insertion of an epitope (an antigen used to
produce vaccines) as a part of the viral envelope protein
(Fig. 4). In the last case, the epitope will be delivered
on the surface of a viral particle.
Magnifection (Agrobacterium-mediated transfection)
is based on a combination of two methods of
transient expression (Icon Genetics). The process
includes vacuum infiltration of whole adult organisms
with dilute suspensions of agrobacteria carrying
T-DNAs encoding RNA replicons. In this case, the
agrobacteria mediate primary infection and movement
in cells, while the viral system mediates the
short-distance cell-to-cell movement, amplification,
and a high-level expression [20].
In general, transient expression of genes is characterized
by a number of advantages (table): a high efficiency
of gene expression and a high level of a target
protein; only several days are required after infiltration
with agrobacteria or viral particles to obtain a maximum
target protein yield (3–4 days for agrobacterial
transformation and 10 days for viral infection). Disadvantages
include the complexity of the agroinfiltration
RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012
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