Agriculture Reference
In-Depth Information
medium before bombardment, and expression of
a gene encoding green fl uorescent protein (GFP)
in scutellar cells after bombardment. Green fl uo-
rescent protein is encoded by the
gfp
gene from
jellyfi sh (
Aequorea victoria
). The protein fl uo-
resces green in living cells when blue or ultravio-
let (460-490 nm) light is shone on them (Color
Plate 32c) (Chalfi e et al., 1994; Pang et al., 1996;
Jordan 2000).
The transforming DNA consists of three com-
ponents needed for expression in wheat cells:
a promoter, a coding sequence, and a transcrip-
tion terminator (Fig. 18.1). Promoters are
DNA sequences that control gene expression,
usually found in front of coding sequences.
Although they are not themselves translated
into proteins, promoter sequences have informa-
tional content: they specify when and where a
gene will be transcribed. They can be thought
of as switches that are triggered in response to
intracellular and external environmental cues.
The coding sequence is transcribed into RNA
that is usually translated into protein. The tran-
scription terminator is placed after the coding
sequence and specifi es the end of the RNA
transcript and the site of polyA addition for
messenger RNAs.
A typical transformation experiment includes
at least two expression units, one incorporating a
gene intended only to allow selection of trans-
formed cells (selection gene), and one or two
experimental genes that are expected to affect the
trait being modifi ed or studied, referred to as the
gene(s) of interest. When the targeted trait is her-
bicide resistance, the gene of interest is also a
selection gene, so only one gene needs to be intro-
duced. For bombardment, the two expression
units are usually on separate plasmid DNAs (Fig.
18.1a). Joining is not necessary because the sepa-
rate plasmids are usually integrated together into
a
b
LB
RB
Integration
LB
RB
Or (more commonly)
Fig. 18.1
Structures of DNAs before (above arrows) and after (below arrows) biolistic (a) and
Agrobacterium
(b) mediated
transformation. The coding regions of selection genes and genes of interest are shown as open and fi lled rectangles, respec-
tively. Promoters and their direction of transcription are depicted by open or gray arrowheads. Noncoding regions that include
transcription terminators and located 3' to the coding regions are shown as striped rectangles. The promoters and 3' noncoding
regions may be the same or different for the selection gene and gene of interest. Bold dotted lines are plasmid backbone
sequences that include origins of replication for propagation in bacteria and antibiotic-resistance genes for bacterial transfor-
mation. Dotted lines are nongenic regions of T-DNA between the left (LB) and right borders (RB) for
Agrobacterium
transfer.
The simplest single-copy structures that can result from integration are shown below the vertical arrows. Solid lines depict
wheat genomic DNA of any length. For biolistic transformations, the selection gene and gene of interest can integrate at either
unlinked or linked locations. Drawings are not to scale.
