ISSN 10214437, Russian Journal of Plant Physiology, 2012, Vol. 59, No. 3, pp. 413–418. © Pleiades Publishing, Ltd., 2012.
The promoter is an indispensable part of a func
tional gene; its sequence includes a variety of
ing elements, which are responsible for the promoter
binding with transcription factors , and this interac
tion then determines the level of both temporal and
spatial expression of the gene. Promoters have been
classified into those, which drive constitutive expres
sion; those, which allow the gene to be induced; and
those, which are either tissue or developmental stage
specific . Constitutive promoters are associated
with genes expressed throughout the life cycle and
throughout the plant, while inducible ones respond to
the presence of a particular physical or chemical cue.
The latter type has been exploited in certain genetic
engineering applications [3, 4]. The constitutive 35S
cauliflower mosaic virus (35S CaMV) promoter is
effective in many plant species and has been much
used for the investigation of transgenic function .
Despite promoter diversity, they share a number of
core elements, in particular the TATA CAAT boxes
. Once a promoter sequence has been determined,
its functionality can be assessed in a number of ways
[7, 8]. Several PCRbased methods have been devel
oped to isolate promoter sequences, and one of the
most effective of these is TAILPCR . Its advantage
lies in its simplicity, specificity, and efficiency. A more
recent method, termed SiteFindingPCR, was sug
gested by Tan et al. ; however, less effective TAIL
PCR provides a simpler means for designing the nec
essary primers. Here, we describe a modification of
The text was submitted by the authors in English.
TAILPCR, which combines the advantageous fea
tures of TAILPCR and SiteFindingPCR.
MATERIALS AND METHODS
Genomic DNA was extracted from the leaves of
chrysanthemum, using a conventional CTAB method
. The template DNA had an
ratio of ~1.8
and was diluted to 30–50 ng/mL. Three genes were
chosen from each of
Arabidopsis thaliana, Brachypo
dium distachyon, Oryza sativa, Physcomitrella patens,
Populus trichocarpa, Sorghum bicolor, Vitis vinifera
(Table 1). A 3kb stretch upstream of
the transcription start site of each gene was retrieved
from the ENSEMBL sequence database (http://
plants.ensembl.org/info/about/species.html) to ensure
that the promoter was included. A second 3kb
stretch lying 27–30 kb upstream of the transcription
start site of each gene was also retrieved to act as a
control. PLACE software (www.dna.affrc.go.jp/
PLACE/signalscan.html) was employed to identify
acting elements present. If the number of a
motif existed in single sequence was beyond 20 or
below 3, the motif was not involved in primer design in
the experimental group. Then the selected motifs were
compared in the experimental group to indentify what
were existed commonly, which means the motif pre
sented in > 10 of 24 genes. The conclusive chosen
motifs were targeted for PCR primer design (Fig. 1).
An adapter sequence plus 2–3 extra bases was added to
the 5' end of the primer targeting a motif sequence
(Fig. 1). Primer sequences were designed using primer
v. 5.0 software (Premier Biosoft, Palo Alto, United
States) (Table 2). A nested three step PCR strategy was
used to amplify the target promoter sequences. AD
primers were chosen as a positive control.
PCR Primers Targeting
Acting Promoter Elements in Plants
M. Zhao, Z. Liu, S. Chen, F. Chen, J. Jiang, and A. Song
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China;
fax: +862584395266; email: firstname.lastname@example.org
Received May 25, 2011
acting elements present in gene promoters are important for promoter function. Thermal
Asymmetric Interlaced PCR (TAILPCR) provides a simple means for isolating promoter sequences, but the
necessary primers can be troublesome to design. Here, we describe an approach, which targets
ments for TAILPCR. The method combines the advantages of TAILPCR and SiteFindingPCR. The new
method proved successful for isolating a number of plant promoter sequences.
plants, primer design,
acting elements, TAILPCR.
: AD—arbitrary degenerate; CE—
ment; TAILPCR—Thermal Asymmetric Interlaced PCR.