non-plant-derived sequences are kept to a minimum. The T-DNA ofAgrobacteriumis
one source of foreign genetic material that could be eliminated using plant-derived
“P-DNA” sequences (Rommens et al. 2004). These are functional analogs of
Agrobacterium-derived T-DNAs, which have been shown to support the transfer of
DNA fromAgrobacteriumto plant cells. The transfer of DNA to plant cells is a relatively
rare event, and transformed cells are usually identified and regenerated with the aid of
selectable markers, such as antibiotic resistance genes, again often derived from bacteria.
Once these foreign selectable marker genes have served their purpose, they can be
removed, since they play no further role in the expression of the transgenic trait. One
method of removing such genes relies on the presence of an inducible recombination
system in the plant vector, which allows excision of a marker gene positioned between
recombination sites (Fig. 7.20).
Some marker genes, such as those conferring herbicide resistance, can be used to select
transformants and, at the same time, provide an economically important crop improvement
trait. In fact, about 75% of genetically modified crops are engineered for herbicide tolerance
(Castle et al. 2004). The two most commonly used herbicide resistance genes are derived
from the bacteriaStreptomyces hygroscopicusandBacillus licheniformis. Such bacterial
herbicide resistance genes could be replaced by plant-derived sequences. Several plant
genes that produce agronomically useful levels of herbicide resistance have now been
identified.
Plant genome sequence data and advances in plant molecular biology have provided the
means by which to identify and isolate plant sequences that have the potential for use in
Figure 7.20.XVE is a chimeric transcription factor. It contains three functional domains, a LexA
DNA binding domain (X), the VP16 activation domain (V), and the estrogen receptor binding
domain (E). The G10-90 promoter drives the constitutive and ubiquitous expression of XVE in trans-
formed plant cells. The XVE protein is then bound as a monomer in the cytosol of the cell by a cha-
perone protein HSP90, and the target gene is transcriptionally inactive. Application ofb-estradiol
causes a conformational change in E, which leads to the release of HSP90 and dimerization of the
receptor. On dimerization, the receptor is activated, allowing the protein to translocate to the
nucleus of the cell where it binds OLexAbinding sites of the promoter that is placed upstream of
the Cre recombinase. The VP16 activation domain activates RNA polymerase II, leading to the tran-
scription of the Cre recombinase gene. Cre recombinase allows recombination to occur between the
LoxPsites removing all intervening genes, including the selectable marker gene.
182 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION