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Plant Physiology and Biochemistry 46 (2008) 196e204
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Research article
Molecular characterization of a cDNA encoding DRE-binding
transcription factor from dehydration-treated
fibrous roots of sweetpotato
Yun-Hee Kim a,c, Kyoung-Sil Yang a, Sun-Hwa Ryu a,d, Kee-Yeun Kim a,e,
Wan-Keun Song a, Suk-Yoon Kwon b, Haeng-Soon Lee a,
Jae-Wook Bang c, Sang-Soo Kwak a,*
a
Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB),
Oun-dong 52, Yusong-gu, Daejeon 305-806, Republic of Korea
b
Plant Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea
c
Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea
d
Division of Wood Chemistry and Microbiology, Korea Forest Research Institute (KFRI), Seoul 130-758, Republic of Korea
e
Biotechnology Examination Team, Korean Intellectual Property Office, Daejeon 302-701, Republic of Korea
Received 28 April 2007
Available online 5 October 2007
Abstract
A new dehydration responsive element-binding (DREB) protein gene encoding for an AP2/EREBP-type transcription factor was isolated by
screening of the cDNA library for dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Its cDNA (referred to as swDREB1) fragment of 1206 bp was sequenced from, which a 257 amino acid residue protein was deduced with a predicted molecular weight of 28.17 kDa. A
search of the protein BLAST database revealed that this protein can be classified as a typical member of a DREB subfamily. RT-PCR and northern analyses revealed diverse expression patterns of the swDREB1 gene in various tissues of intact sweetpotato plant, and in leaves and fibrous
roots exposed to different stresses. The swDREB1 gene was highly expressed in stems and tuberous roots. In fibrous roots, its mRNA accumulation profiles clearly showed strong expression under various abiotic stress conditions such as dehydration, chilling, salt, methyl viologen (MV),
and cadmium (Cd) treatment, whereas it did not respond to abscisic acid (ABA) or copper (Cu) treatment. The above results indicate that
swDREB1 may be involved in the process of the plant response to diverse abiotic stresses through an ABA-independent pathway.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Abscisic acid; Dehydration; DREB; Fibrous root; Sweetpotato
1. Introduction
Drought stress acts as a key environmental factor that represents one of the principle limitations affecting plant species distribution and crop productivity [2,3]. Exposure to drought stress
triggers many common reactions in plants [31]. This leads to cellular dehydration, which causes osmotic changes and removal of
water from the cytoplasm into the extracellular space, resulting
* Corresponding author. Tel.: þ82 42 860 4432; fax: þ82 42 860 4608.
E-mail address: [email protected] (S.-S. Kwak).
0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2007.09.012
in decrease of the cytosolic and vacuolar volumes. Another consequence of this is the production of reactive oxygen species
(ROS), which, in turn, may negatively affect cellular structures
and metabolism. Therefore, drought triggers a wide variety of
plant responses, including the regulation of gene expression,
the accumulation of metabolites such as plant growth hormone
(i.e., abscisic acid (ABA)) or osmotic active compounds, and the
synthesis of specific proteins such as molecular chaperone and
antioxidant enzymes [1].
The study of dehydration-induced genes in Arabidopsis has
also revealed ABA-independent or ABA-dependent signal
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
transduction pathways [27]. In Arabidopsis, rd29A, rd29B,
cor78, and lti78 genes are differentially induced under conditions of dehydration, cold, salt, and exogenous ABA. Dehydration responsive element (DRE: TACCGACAT) functions in the
initial rapid response of rd29A to dehydration, salt, and low
temperature [32,33]. The DRE is an essential cis-acting element
for the regulation of rd29A induction in the ABA-independent
response to dehydration in Arabidopsis. DRE-related motifs
have been reported in promoters of several genes that are regulated by osmotic and low temperature stresses; these genes include kin1, cor6.6/kin2, and rd17/cor47 in Arabidopsis [11,34].
In a recent study, 16 genes containing DRE or DRE-related core
motifs (CCGAC) were identified in the promoters of dehydration-inducible Arabidopsis genes. This group of genes is a target
sequence of the DREB1 or DREB2 transcription factors [26].
AP2/EREBP DNA-binding proteins include the DREB or
CBF proteins that bind to DRE or C-repeats (CRT), respectively.
The Arabidopsis genome encodes 145 AP2/EREBP proteins
[24]. Among the AP2/EREBP proteins, the DREB subfamily
is reported to comprise 55 genes in Arabidopsis [24]. A major
transcriptional regulatory system is represented by DRE/C-repeat promoter sequences in stress-activated genes and
DREBs/CBF factors that control stress-related gene expression
[21,29]. A detailed analysis of expression showed that these
factors may be associated with various stress conditions. For
example, expressions of DREB1A/CBF3, DREB1B/CBF1, and
DREB1C/CBF2 are induced by low temperature. DREB1D/
CBF4, DREB2A, and DREB2B are induced by salt and dehydration. DREB1F, DREB2C, DREB2D, and DREB2F are slightly
induced by high salt treatment, whereas DREB2E is slightly
induced by ABA treatment. However, the expressions of
DREB1E, DREB2G, and DREB2H have not been detected
upon exposure to various stress conditions [7,21,22,24,28].
Sweetpotato (Ipomoea batatas) is known as a relatively
drought-resistant crop, and it is one of the most important root
crops in the world. However, sweetpotato has not been investigated in relation to the molecular basis of its drought stress tolerance, even though it is quite tolerant to unfavorable growth
conditions, including drought conditions. Moreover, no reports
have been published on DRE-binding transcription factor in
sweetpotato. The roots of plants are the primary sensors of
drought and salt stress [4,25]. Therefore, in this study we have
cloned a new DREB gene from the fibrous roots of sweetpotato
treated with dehydration. To analyze the roles of swDREB1 in
adaptation to stress in sweetpotato plants, its expression patterns
were examined in different tissues under various stresses.
2. Results
2.1. Isolation and sequence analysis of the swDREB1
To isolate the cDNA clone encoding DREB, a cDNA library was constructed from dehydration-treated fibrous roots
of sweetpotato and screened with a probe produced by PCR
using a DREB-conserved region. A positive clone containing
the largest insert was designated as an swDREB1, and was selected for further analysis.
197
Its cDNA is 1206 bp in length and encodes a deduced 257
amino acid residue protein with a predicted molecular mass of
28.17 kDa. Database searches revealed that the amino acid sequence of the SwDREB1 protein contains a conserved, 63
amino acid, DNA-binding domain that is present in a large
family of plant DNA-binding proteins. Its N-terminal includes
a basic residue, PKKRAGRKKFRETRHP, which might function as a nuclear localization signal (NLS). An acidic region in
its C-terminal (AP2/EREBP domain) was thought to be an activation domain for the transcription of DRE/CRT genes
(Fig. 1A). The swDREB1 protein also contains the DSAWRL
and LWSF motifs (Figs. 1 and 2). Alignment and comparison
of its amino acid sequence with those of other DREB genes
isolated from various plants showed that it shares 67% similarity with CaCBF1B and CaDREBLP1, and 61% similarity with
AtDREB1D/CBF4 (Fig. 2A). Phylogenic tree analysis of plant
DREB/CBF protein sequences indicated that the swDREB1
shares high homology with those in various plant species
such as tomato (LeCBF1), hot pepper (CaCBF1B and CaDREBLP1), cotton (GhDREB1A) (Fig. 2B). Among Arabidopsis DREBs, swDREB1 is more closely related with
AtDREB1/CBF than AtDREB2. Therefore, we suggest that
swDREB1 can be classified as a member of the DREB subfamily, and will likely play an important role in abiotic stressresponsive gene expression.
2.2. Genomic organization of swDREB1
To elucidate the genomic organization of swDREB1, the 30 UTR region was amplified from cDNAs, cloned to pGEM-T
Easy vector, sequenced, and used as probes. Southern blot
analysis of genomic DNA digested with EcoRI, EcoRV, and
HindIII is shown in Fig. 3. A single hybridizing band was observed, indicating that one copy of swDREB1 may exist in the
sweetpotato genome.
2.3. Differential expression of swDREB1
in intact sweetpotato tissues
The expression patterns of the swDREB1 gene were investigated in various whole plant tissues (L, leaf; S, stem; TR, tuberous root; FR, fibrous root; TPR, thick pigmented root) by
RT-PCR and northern blot analyses (Fig. 4). The RT-PCR results were well correlated with those of northern blot analysis
proving the validity of RT-PCR analysis. The results demonstrate that there is considerable variation in the levels of
swDREB1 expression in different sweetpotato tissues. The
swDREB1 gene was strongly expressed in stem, tuberous
root and fibrous root tissues, whereas it was weakly expressed
in leaf and thick pigmented root tissues.
2.4. Expression patterns of the swDREB1 to various
abiotic stresses in leaves and fibrous roots
In order to investigate the swDREB1 gene expression under
various abiotic stresses such as dehydration, ABA, cold, salt,
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
198
A
NLS
AP2/EREBP domain
N
Acidic region
C
DSAWRL
LWSF
B
NLS
AP2/EREBP
domain
Fig. 1. Sequence analysis of the swDREB1 full-length cDNA. (A) Schematic representation of the swDREB1 cDNA. Boxes represent an open reading frame including three motifs: the NLS (left black box), AP2/EREBP domain (DNA-binding domain, hatched box), and activation domain (acidic region, right gray box).
(B) Nucleotide and deduced amino acid sequences of swDREB1 cDNA. The AP2/EREBP domain is underlined, and the basic region in the N-terminal that might
act as an NLS is shown by thick underlining. The DSAWRL and LWSF motifs are indicated by a box.
MV, and heavy metal treatment, RT-PCR analysis was
conducted.
In the first step, sweetpotato plants grown for 50 days were
dehydrated at 25 C in a growth chamber. As shown in
Fig. 5A, the swDREB1 gene was differentially regulated by dehydration in leaves and fibrous roots. In leaves, the swDREB1
transcript was weakly expressed regardless of dehydration
time. Interestingly, its expression was rapidly increased in fibrous roots following dehydration treatment. The level of its
expression was transiently up-regulated at 1 h after exposure
to dehydration. At 24 h, its transcripts declined to an almost
undetectable level. When the plants were treated with
0.1 mM ABA, no difference in the transcript level was observed throughout the time course of the treatment
(Fig. 5B). The results of this experiment indicated that the expression of the swDREB1 gene is ABA-independent.
Sweetpotato plants were subjected to chilling at 4 C and
low temperature at 15 C for 16 h in order to examine the
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
A
NLS
199
AP2-EREBP domain
DSAWRL
LWSF
B
PaDREB1
GhDREB1A
SwDREB1
LeCBF1
CaCBF1B
CaDREBLP1
AtDREB1D / CBF4
TsDREB1
AtDREB1B / CBF1
AtDREB1C / CBF2
AtDREB1A / CBF3
AtDREB2A
AtDREB2B
Fig. 2. Multiple alignments (A) and phylogenetic trees (B) of the amino acid sequences of DREB/CBF proteins isolated from various plants. The signature
sequences ‘‘PKKRAGRKKFRETRHP’’, ‘‘DSAWRL’’, and ‘‘LWSF’’ appear in (A). The GenBank accession numbers are as follows: sweetpotato swDREB1
(EF433457); hot pepper CaCBF1 (AAQ88400), CaDREBLP1 (AAR88363); tomato LeCBF1 (AAK57551); Arabidopsis AtDREB1A/CBF3 (Q9M0L0),
AtDREB1C/CBF2 (Q9SYS6), AtDREB1B/CBF1 (P93835), AtDREB1D/CBF4 (Q9FJ93), AtDREB2A (O82132), AtDREB2B (O82133); Thellungiella salsuginea
TsDREB1 (AAS00621); sweet cherry PaDREB1 (BAC20184) and cotton GhDREB1A (AAP83936).
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
200
(kb)
EcoRI
EcoRV
HindIII
A
L
12
S
TR
FR
TPR
S
TR
FR
TPR
swDREB1
8
Tubulin
5
B
L
swDREB1
3
rRNA
2
1
5’-UTR
ORF
3’-UTR
Probe
(300 bp)
Fig. 3. Genomic southern blot analysis of swDREB1 from sweetpotato (Ipomoea batatas). Equal amounts (30 mg) of DNA were digested with the restriction enzymes, EcoRI, EcoRV, and HindIII, fractionated by electrophoresis,
transferred onto a Zeta-probe GT membrane, and probed with 32P-labeled
gene-specific DNA fragments.
effects of temperature stress on the levels of swDREB1
mRNAs. The swDREB1 gene responded differently to chilling
and low temperature conditions (Fig. 6A). Leaves and fibrous
roots from plants grown at 25 C served as controls. Under
15 C conditions, the swDREB1 gene was weakly induced in
leaves, whereas its expression was decreased in fibrous roots.
Interestingly, the swDREB1 transcript was strongly induced at
4 C in leaves and fibrous roots, respectively, compared to that
observed under control conditions.
Similar to dehydration and chilling exposure, the transcript
of swDREB1 was induced by various chemical stress treatments
such as NaCl, methyl viologen (MV), and heavy metals. In
leaves, the transcript for swDREB1 was significantly induced
by 100 mM NaCl and 0.05 mM MV treatments, whereas its expression was slightly increased in fibrous roots (Fig. 6B).
To further elucidate the regulation of swDREB1, we analyzed the expression of swDREB1 in response to heavy metals
such as cadmium (Cd) and copper (Cu). Expression of
swDREB1 was induced in leaves and fibrous roots under
0.5 mM Cd-treated conditions, but did not respond to
0.5 mM Cu treatment (Fig. 6C). Overall, these results are consistent with the view that the swDREB1 gene is subject to control by dehydration, chilling, salt, and Cd treatment via an
ABA-independent pathway.
Fig. 4. Expression patterns of swDREB1 in various intact tissues of sweetpotato. Total RNAs were extracted from leaves (Ls), stems (Ss), tuberous roots
(TRs), fibrous roots (FRs) and thick pigmented roots (TPRs). (A) Reverse transcription of 1e2 mg of total RNA was carried out, and conventional PCR amplification reactions were performed using each gene-specific primer. cDNAs
were hybridized to tubulin gene-specific primers to assess relative amounts of
mRNA in each sample. Tubulin was used as a control for equal loading. Reaction products (20 ml) were analyzed by gel electrophoresis. (B) For northern
blot analysis, total RNA of 20 mg was denatured by heating at 70 C for
15 min in a denaturing buffer containing 50% formamide and 2.2 M formaldehyde, and blotted onto a Zeta-probe GT membrane. The blots were probed
with 32P-labeled gene-specific DNA fragment.
3. Discussion
The swDREB1 was isolated from dehydration-treated fibrous roots of sweetpotato, and was subsequently characterized in terms of abiotic stress. The deduced protein of the
swDREB1 gene has a conserved NLS, AP2/EREBP domain
and an acidic region, which suggests that the swDREB1 protein might function as a transcription factor in sweetpotato
plants (Figs. 1 and 2). Our results showed that only the Arabidopsis DREB1-type gene had the PKKRAGRKKFRETRHP
and DSAWRL amino acid sequences surrounding the AP2/
EREBP domain [23]. In the C-terminal region of swDREB1,
we also found the LWSF motif. In Arabidopsis, DREB1D/
CBF4 contains an LWSF motif. Among the AP2/EREBP proteins in the databases, swDREB1 is most closely related to the
hot pepper CaCBF1B and CaDREBLP1, and Arabidopsis AtDREB1D/CBF4, showing 61e67% sequence identity
(Fig. 2). These genes were reportedly expressed under various
stress conditions such as dehydration, cold, and high salinity
[7,8,17,24]. In hot pepper plants, expression of CaCBF1B
was found to be induced by cold, dehydration, and heat-shock
treatment, whereas CaDREBLB1 is induced by dehydration,
NaCl, and wounding treatment [8,17]. The transcript of
AtDREB1D/CBF4 is induced in Arabidopsis under high salinity
and dehydration conditions [7,24]. Therefore, swDREB1 was
classified as a member of the DREB subfamily, the members
of which are known to play important roles in abiotic stressresponsive gene expression.
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
A
A
Dehydration
0
1
2
8
16
201
25°C
15°C
4°C
Control
NaCl
MV
Control
Cd
Cu
24 hrs
Leaves
Leaves
Tubulin
Tubulin
Fibrous roots
Fibrous roots
Tubulin
Tubulin
B
0
6
0.1 mM ABA
12
24
B
48
hrs
Leaves
Leaves
Tubulin
Tubulin
Fibrous roots
Fibrous roots
Tubulin
Tubulin
Fig. 5. Expression patterns of swDREB1 in leaves and fibrous roots of plants
grown in a growth chamber and subjected to dehydration and ABA treatment,
as detected by RT-PCR analysis. (A) Expression patterns of the swDREB1
gene in leaves and fibrous roots of sweetpotato by dehydration treatment. Total
RNA was extracted from leaves and fibrous root at 0, 1, 2, 4, 8, 16, and 24 h
after dehydration treatment. (B) Expression patterns of the swDREB1 gene in
leaves and fibrous roots of sweetpotato induced by ABA treatment. Total RNA
was extracted from leaves and fibrous root at 0, 12, 24, and 48 h after ABA
treatment. First-strand cDNA synthesis and PCR reaction were performed as
described in the instructions of the manufacturer. Tubulin was used as a control
for equal loading. Reaction products (20 ml) were analyzed by gel
electrophoresis.
C
Leaves
Tubulin
Fibrous roots
The responsiveness of the swDREB1 gene to dehydration
implies that its role in the transduction of abiotic stress signals
in sweetpotato is similar to that of DREB of Arabidopsis, and
occurs through an ABA-independent pathway (Fig. 5B). In
Arabidopsis and rice plants, the expressions of the DREB1type genes are specifically induced by low temperature stress,
whereas expressions of DREB2-type genes are induced by dehydration [5,21,24]. Structural features of swDREB1 resemble
the DREB1-type protein of Arabidopsis. The transcript of
swDREB1 was induced by both dehydration and cold. Moreover, its expression was induced by treatment with NaCl,
MV, or Cd (Figs. 5 and 6). These findings indicate that
swDREB1 is regulated by various abiotic stress signals, including dehydration and cold, in sweetpotato.
In a previous report, transgenic Arabidopsis plants overexpressing DREB1B/CBF1 cDNA under the control of the
Tubulin
Fig. 6. Expression of the swDREB1 gene in leaves and fibrous roots of plants
grown in a growth chamber subjected to various abiotic stress treatments, as
determined by RT-PCR analysis. (A) Expression of the swDREB1 gene in
leaves and fibrous roots of sweetpotato plants cultured in vitro and subjected
to low temperature (15 C) and chilling (4 C), as determined by RT-PCR
analysis. Plants were exposed to temperatures of 15 and 4 C for 16 h. Control
plants were incubated at 25 C. (B) Plants were treated with NaCl (100 mM)
and MV (0.05 mM) at 24 h. Control plants were kept in distilled water. (C)
Plants were treated with 0.5 mM Cd and Cu at 48 h. Control plants were
kept in distilled water. Total RNA was extracted from plant materials using
the CTAB method (Kim and Hamada [18]). After reverse transcription was
carried out as instructed by the manufacturer, PCR amplification reactions
were performed using each of the gene-specific primers. Tubulin was used
as a control for equal loading.
202
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
cauliflower mosaic virus (CaMV) 35S promoter were reported
to show strong freezing tolerance [12]. Moreover, overexpression of the DREB1A/CBF3 cDNA under the control of the
CaMV 35S promoter resulted in strong induction of target
stress-inducible genes, and the transgenic plants acquired
higher tolerance to drought, high salt, and freezing stresses
[6,14,21]. Recently, in transgenic rape, tomato, and tobacco
plants, an ectopically-expressed Arabidopsis DREB gene
showed enhanced resistance to water deficit, chilling, freezing,
and oxidative stresses [9,10,13,15]. These results indicate that
the DREB/CBF gene can be used to improve the multiple
stress tolerances of agriculturally important crops through
gene transfer. However, in the transgenic Arabidopsis, tomato,
and tobacco plants, overexpressions of DREB1A/CBF3 and
DREB1B/CBF1 caused severe growth retardation under normal growth conditions [9,10,14,15]. Use of the stress-inducible rd29A promoter instead of the constitutive 35S CaMV
promoter for the overexpression of DREB1A/CBF3 minimized
the negative effects on plant growth in transgenic Arabidopsis
and tobacco plants [14,15]. Rd29A promoter also showed functions in gene expression in response to stress in Arabidopsis
and tobacco plants [14,15,32,33].
In our previous study, a strong oxidative stress-inducible
peroxidase (SWPA2) promoter from cultured cells of sweetpotato was isolated, and its function was characterized in transgenic tobacco plants and cultured cells that were subjected
to environmental stress [16]. Recently, in transgenic sweetpotato and potato plants, double overexpression of the antioxidant enzyme cDNA under the control of SWPA2 promoter
resulted in an acquired higher tolerance of transgenic plants
to MV-mediated oxidative stress, chilling, heating, and
drought stress [19,20,30]. These results indicate that the
SWPA2 promoter can be used to improve the multiple stress
tolerances of agriculturally important crops via strong defense
genes. Therefore, we expect that the overexpression of
swDREB1 under the control of SWPA2 promoter in transgenic
plants not only significantly enhances the tolerances of the
plant tissues to dehydration, cold, salt, and oxidative stress,
but also minimizes the negative effects on plant growth.
4. Materials and methods
4.1. Plant materials
Sweetpotato plants (I. batatas L. Lam. cv. White Star) were
grown in a growth chamber and harvested after 50 days to analyze expression of the swDREB1 gene in different tissues and
stress conditions.
4.2. Preparation of DREB probes
To prepare DREB probes, we obtained five cDNA sequences of AP2/EREBP DNA-binding proteins from an Arabidopsis database website (www.arabidopsis.org), and
subsequently designed primers for PCR. Accession numbers
of the sequences are as follows: AT4G25480: DREB1A/
CBF3, AT4G25490; DREB1B/CBF1, AT4G25470; and
DREB1C/CBF2, AT5G05410: DREB2A, AT3G11020:
DREB2B. The primers for three DREB1/CBF cDNAs were designed from the full-length ORF sequences, and the primers
for two DREB cDNAs were designed from the conserved
DRE-binding region sequences.
DREB1A/CBF3 Forward: 50 -ATGAACTCATTTTCTGCT-30 .
DREB1A/CBF3 Reverse: 50 -TTAATAACTCCATAACGA
TAC-30 .
DREB1B/CBF1 Forward: 50 -TTTGGCTCCGATTAC-30 .
DREB1B/CBF1 Reverse: 50 -TTAGTAACTCCAAAGCG-30 .
DREB1C/CBF2 Forward: 50 -ATGAACTCATTTTCTGCC-30 .
DREB1C/CBF2 Reverse: 50 -TTAATAGCTCCATAAG
GAC-30 .
DREB2A Forward: 50 -ATGGCAGTTTATGATCAG-30 .
DREB2A Reverse: 50 -TTAGTTCTCCAGATCCAA-30 .
DREB2B Forward: 50 -ATGGCTGTATATGAACAAA-30 .
DREB2B Reverse: 50 -TCAAATATCCAGAGAACTC-30 .
Polymerase chain reaction (PCR) was performed with approximately 10e50 ng of cDNA, a PCR premix kit containing
250 mM each of dNTPs, 1 unit of Taq DNA polymerase,
40 mM KCl, 1.5 mM MgCl2 in 10 mM TriseHCl (Bioneer,
Daejeon, Republic of Korea), and 10 pmol of each primer.
Amplification reactions consisted of 30 cycles of the following
regimen: 1 min at 94 C, 1 min at 53 C, and 1 min at 72 C.
Five different PCR products from 612 to 1008 bp in length
were cloned into pGEM-T Easy vectors (Promega, WI,
USA). The inserts were sequenced and found to be similar
to other published DREB sequences; the enzyme-cut inserts
were subsequently used as probes to screen the cDNA library.
4.3. Construction and screening of a cDNA library
For the construction of the cDNA library, total RNA was
isolated from fibrous roots of sweetpotato at 0, 0.5, 2, 6, and
10 h after dehydration, respectively. Poly(A)þ RNAs were
purified using the PolyATrac mRNA isolation system (Promega, Madison, WI), and the cDNA was synthesized from
poly(A)þ-enriched RNA using the SMARTÔ cDNA Library
Construction Kit according to the supplier instructions (Clontech, Japan). The ligated DNA was packaged with a lambda
packaging kit, and the library was amplified in Escherichia
coli strain XL-1-Blue (Clontech) prior to screening. The plaques were transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden), UV cross-linked,
and screened for hybridization with the radiolabeled probes.
Plaques with positive signals were purified by at least three
rounds of re-plating and screening. Phagemids were excised
from the phage by following the instructions of the manufacturer (Clontech).
4.4. Southern blot analysis
Genomic DNA of sweetpotato was extracted from leaves
according to the method described by Kim and Hamada [18],
digested with EcoRI, EcoRV, and HindIII (Roche, Manheim,
Y.-H. Kim et al. / Plant Physiology and Biochemistry 46 (2008) 196e204
Germany), electrophoresed on 0.8% agarose gel, and blotted
onto Zeta-probe GT membrane (Bio-Rad, CA, USA). The blots
were hybridized to a 32P-labeled probe from the 30 -untranslated
sequence specific to their own cDNAs. Hybridization was carried out in 0.5 M sodium phosphate (pH 7.2), 7% SDS, and
1 mM EDTA at 65 C.
4.5. Northern blot analysis
For northern blot analysis, total RNA of sweetpotato was
extracted from leaves according to the method described by
Kim and Hamada [18]. The RNA was denatured by heating
at 70 C for 15 min in a denaturation buffer containing 50%
formamide, and 2.2 M formaldehyde was separated on agarose
gel and blotted onto a Zeta-probe GT membrane. Hybridization was performed as a southern blot analysis.
4.6. RT-PCR analysis
Quantitative RT-PCR was employed to measure the transcript levels of the swDREB1 gene. First-strand cDNA was
then synthesized using MMLV Reverse Transcriptase (Clontech) from 1 mg of total RNA in a 20 ml reaction volume. PCR
amplification reactions were initially incubated at 94 C for
5 min followed by 26e28 cycles at 94 C for 30 s, 57 C for
30 s, and 72 C for 45 s. Reaction products (20 ml) were analyzed by gel electrophoresis. Gene-specific primers used for
PCR reactions were as follows: the swDREB1 primer set (50 CTATTCGCCCTATTCCTAT-30 , 50 -CAAATTCAAGCCTTGG
TATC-30 ) used to amplify a 114 bp product from cDNA coding
for swDREB1. RT-PCR for amplifying transcripts of the
swDREB1 gene was performed at least three times to confirm
the accuracy of the results. The total synthesized cDNA was
also used to amplify the tubulin gene as an internal standard using tubulin gene-specific primers (50 -CAACTACCAGCCAC
CAACTGT-30 , 50 -CAAGATCCTCACGAGCTTCAC-30 ).
4.7. Stress treatment
For stress treatments, we used sweetpotato plants that had
been grown in the growth chamber at 25 C for 50 days. For
dehydration treatment, sweetpotato leaves and fibrous roots
were collected after 0 (untreated control), 1, 2, 4, 8, 16, and
24 h after treatment. For abscisic acid (ABA) treatment, sweetpotato plants were incubated at 25 C for 48 h in a glass bottle
containing 400 ml of 0.1 mM ABA. Sterile water was used as
a control for ABA treatment. For temperature stress treatment,
plants were exposed to conditions at 15 C (low temperature),
4 C (chilling), and 25 C (control) for 16 h, respectively. For
NaCl (100 mM) and methyl viologen (MV, 0.05 mM) treatments, sweetpotato plants were incubated in a glass bottle containing 400 ml of each chemical solution at 25 C for 24 h. For
heavy metal treatments, sweetpotato plants were incubated in
0.5 mM of CdSO4 or CuSO4 at 25 C for 48 h. Sterile water
was used as a control for chemical stress treatment. All treated
plant materials were immediately frozen in liquid nitrogen and
stored at 70 C until further use.
203
4.8. Analysis of DNA and protein sequences
Sequence identities were identified using the BLAST program of the NCBI web-server, and multiple sequence alignment
was obtained with Clustal X and the GeneDoc program. To predict the isoelectric point (pI ), molecular weight, and signal peptides of deduced proteins, ExPasy (http://www.expasy.org/
tools), PSORT (http://psort.ims.u-tokyo.ac.jp), and SoftBerry
(http://www.softberry.com) programs were used.
Acknowledgments
This work was supported by grants from the BioGreen21
Program (20070301034015), Rural Development Administration, Korea, from the Environmental Biotechnology National
Core Research Center, Korea Science & Engineering Foundation (KOSEF), from the Korea Foundation for International
Cooperation of Science & Technology (KICOS), and from
the KRIBB Initiative Program. We are grateful to Dr. Cha
Young Kim for his valuable comments to the manuscript.
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