Biology Reference
In-Depth Information
A. tumefaciens
, and in this section, we will discuss the basics of a common method.
We will provide guidelines for selecting appropriate transgenic plant lines that ex-
press GFP and mCherry fusions to tubulin and MAPs.
A. tumefaciens
causes crown gall disease in plants by transferring a piece of DNA
called the T-DNA (transfer DNA) from the tumor inducing, T
i
, plasmid into the
infected plant's genome (Lee & Gelvin, 2008;
Pitzschke & Hirt, 2010
). In nature,
the T-DNA encodes proteins that alter the physiology of the infected plant in order
to benefit the bacteria (Lee & Gelvin, 2008;
Pitzschke & Hirt, 2010
). The T-DNA
also encodes the majority of proteins required for virulence and horizontal (Lee &
Gelvin, 2008;
Pitzschke & Hirt, 2010
). The T-DNA is surrounded by short “border”
sequences that are recognized by the
A. tumefaciens
machinery that catalyzes the ge-
netic transfer (Lee & Gelvin, 2008). For routine use in the laboratory, the
A. tume-
faciens
T
i
plasmid has been split into two smaller pieces: the T-DNA binary vector
and the helper plasmid (
Komari et al., 2006
; Lee & Gelvin, 2008). The binary vector
contains the border sequences between which the fluorescent fusion sequence and
plant selectable marker cassette are placed. When choosing a selectable antibiotic
resistance gene, it is critical to pick a marker different than any the plant may be al-
ready expressing. Horizontal transfer of the T-DNA requires the proteins encoded by
the helper plasmid (
Hoekema, Hirsch, Hooykaas, & Schileroort, 1983
; Lee &Gelvin,
2008). Because the doubling time of
A. tumefaciens
is twice that of
E. coli
, it is more
efficient to perform the molecular cloning steps in
E. coli
. To facilitate this, the bi-
nary vector was engineered to contain an
E. coli
origin of replication, bacterial se-
lectable markers, and multiple cloning sites (
Komari et al., 2006
; Lee & Gelvin,
2008). Once the cassette is completed in
E. coli
, the binary vector is moved into
an
A. tumefaciens
strain containing a helper plasmid. Various binary vector systems
are publically available from the Biological Resource Center,
www.arabidopsis.org
(
Komari et al., 2006
).
15.1.1.1
Transgene construction
Building transgenes for plants is similar to building transgenes in other organisms. In
A. thaliana
, scientists do not have the ability to use homologous recombination to
control the site of transgene insertion into the genome. Because the expression of
the transgene can be affected by its position in the genome, it is especially important
in
A. thaliana
to follow best practices when building transgenic plants. The full ge-
nomic sequence including the native promoter, exons, introns, and UTRs should be
used for transgene construction. In practice, the promoter is usually defined as
2.5 kb of upstream sequence. The full genome sequence of
A. thaliana
is publically
available at
www.arabidopsis.org
. Whenever possible, the gene fusion should be
transformed into a null mutant background to verify that it is functional. The consti-
tutively active 35S cauliflower mosaic virus or ubiquitin promoters can be when pro-
tein overexpression is desirable. In plants, the most commonly used fluorescent
reporters currently are mCherry, green, yellow, and cyan fluorescent proteins
(GFP, YFP, and CFP). Originally, the
Aequorea victoria
GFP-coding sequence
needed to be altered for use in
A. thaliana
because plant cells recognized a cryptic
