全 文 ：Received 5 Aug. 2003 Accepted 10 Dec. 2003
Supported by the National Natural Science Foundation of China (30000112), Shanxi Provincial Natural Science Foundation, China (20001037)
and Science and Technology Development Foundation of Shanxi Educational Committee (2005).
* Author for correspondence. Tel: +86 (0)351 7016123; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 982-987
Expression of the Pathogenesis-related Gene Promoter—GUS
Reporter Fusion in Arabidopsis
ZHAO Shu-Qing*, LI Xin-Feng, GUO Jian-Bo
(Institute of Biotechnology, Shanxi University; Key Laboratory of Chemical Biology and Molecular Engineering,
The Chinese Ministry of Education, Taiyuan 030006, China)
Abstract: Pathogesis-related gene 1 (PR1) is a valid marker gene for systemic acquired resistance in
Arabidopsis thaliana L. Using PCR amplification, we cloned the upstream regulatory region of PR1 gene
from Arabidopsis Col-0. The fragment was then fused to the reporter gene encoding b-glucuronidase
(GUS) and introduced into Arabidopsis plant by Agrobacterium-mediated gene transfer. PCR analysis and
Southern blot hybridization of total DNA extracted from transgenic plants verified that the fusion gene had
been integrated into the Arabidopsis genome. GUS activity can be induced by chemical treatment in
transgenic Arabidopsis. The response was monitored by histochemical staining of GUS activity in situ, and
fluorimetric assay in tissue extracts. Transgenic plants containing PR1-GUS or other defense gene
promoter-reporter gene fusions may therefore provide specific assay systems for screening potential
activators of systemic acquired resistance.
Key words: Arabidopsis thaliana ; pathogenesis-related gene promoter; reporter gene; transformation
Systemic acquired resistance (SAR) is a plant defense
response usually triggered by incompatible pathogens
(Ryals et al., 1996). SAR is characterized by its broad-
spectrum disease resistance and activation of a set of patho-
genesis-related (PR) genes (Ward et al., 1991; Uknes et al.,
1992). From pathogen infection to establishment of SAR, a
number of signal transduction events occur in plants (Baker
et al., 1997; Smith, 2000). Recently, several components in
the signal transduction cascade to SAR have been recog-
nized in Arabidopsis. Salicylic acid (SA) is an important
signalling molecule in the establishment of SAR (Delaney
et al., 1994; Klessig and Malamy, 1994). The SA signal is
transduced through NPR1, a nuclear-localized protein that
interacts with transcription factors involving in regulating
SA-mediated PRs expression (Cao et al., 1994; Kinkema
et al., 2000). PR1 is considered a valid marker gene for SAR
in Arabidopsis thaliana (Uknes et al., 1992; Delaney et al.,
1994). Some chemicals that mimic natural signalling
compounds, such as 2, 6-dichloroisonicotinic acid (INA)
(Vernooij et al., 1995) and benzo(1, 2, 3)thiadiazole-7-
carbothioic acid S-methyl ester (BTH) (Friedrich et al., 1996;
Görlach et al., 1996; Lawton et al., 1996) can also activate
SAR and expression of SAR genes in the absence of patho-
gen infection (Ward et al., 1991; Uknes et al., 1992). Such
chemicals protect plants by induction of plant’s innate im-
munity rather than by direct toxic effects on the pathogens.
Thus, this kind of chemicals might have less health risks
and environmental impact than many of the conventional
agrochemicals currently used for plant protection. The
application of activators of SAR is a new way to fight plant
diseases. But first of all, it calls for developing a rapid
method for screening such novel agrochemicals.
SA is the only SAR activator derived from plants. Exog-
enous application of SA can activate SAR and expression
of SAR genes (Ward et al., 1991). In this paper, we report
the expression of the PR1 promoter-reporter gene fusion in
transgenic Arabidopsis plants by SA treatment. The find-
ing that the reporter gene activity can be induced in
transgenic plants by SA treatment suggests that the
transgenic plants containing PR1-GUS may provide con-
venient and specific assay system for screening compounds
which can activate plant’s natural resistance to disease.
1 Materials and Methods
1.1 Plant materials and growth conditions
Arabidopsis thaliana L. ecotype Columbia (Col-0) plants
were grown either in pots on vermiculite-mixed soil (rich
soil:vermiculite = 2:1, V/V) or on plates with MS medium
(Murashige and Skoog, 1962) as described by Zhao et al.
(2001). Plants on soil were kept in a growth room at 22 ℃
and 70% humidity with a 16 h photoperiod under fluores-
cent lamps at a light intensity of 100 to 120 µE·m-2·s-1,
while those on MS plates were kept in a growth chamber at
22 ℃ with continuous illumination (about 80 µE·m-2·s-1).
ZHAO Shu-Qing et al.: Expression of the Pathogenesis-related Gene Promoter—GUS Reporter Fusion in Arabidopsis 983
All seeds were vernalized at 4 ℃ for 2 d before placement in
a growth environment.
1.2 Strains and plasmids
Agrobacterium tumefaciens strain EHA105, Escherichia
coli strain DH5a, pGEM-T and binary vector pBI101 (a gift
from Dr. HUANG Rong-Feng (Biotechnology Research
Institute, The Chinese Academy of Agricultural Sciences)
were used in this study.
1.3 Enzymes and reagents
Restriction endonuclease, Taq DNA polymerase, dNTP
and T4 DNA ligase were products of Promega (Madison,
USA). Four-MU was purchased from Sigma. X-Gluc and 4-
MUG were products of Bio Basic Inc. DIG DNA Labeling
and Detection Kit were from Roche (Mannheim, Germany).
HybondTMN+ was product of Amersham. Agarose gel
extraction kit was from Watson (Shanghai, China), and
other reagents used were products of A.R. grade. PCR
primers were synthesized by Sangon (Shanghai, China).
1.4 PCR amplification and cloning of PR1 gene promoter
fragment
Two primers, FPR and RPR, were designed and synthe-
sized according to the PR1 sequence in Arabidopsis
( a c c e s s i o n n u m b e r A F 0 9 6 2 9 4 ) . F P R : 5 -
GCCAAGCTTGATATACGA AGGCGGTAC-3 (containing
Hind Ⅲ site), RPR: 5-GCACCCGGGTTTTCTAAGTTGATA
ATGG-3 (containing SmaⅠ site). The Arabidopsis ecotype
Columbia genomic DNA was used as PCR template. PCR
was performed as follows: 94 ℃ 1 min, 60 ℃ 1 min, 72 ℃ 2
min, 35 cycles. The PCR product was ligated with pGEM-T
vector and transformed into DH5a of E. coli. White colo-
nies were selected on the LB plates containing
ampicillin+IPTG+X-Gal. The genomic DNA of Arabidopsis
was extracted from the leaves using the modified CTAB
method (He et al., 1999). Plasmid DNA preparation, restric-
tion enzymes digestion, ligation, transformation were car-
ried out essentially according to Sambrook et al. (1989).
1.5 Construction of plant expression vector
Nine hundred and ten bp PR-1 fragment from pGEM-T-
PR digested by HindⅢ and SmaⅠ was purified and in-
serted into the HindⅢ and SmaⅠ sites of pBI101, and
resulted in plant expression vector pBI101-PR. The recom-
binant plasmids were verified by plasmid DNA extraction,
PCR amplification and restriction endonuclease digestion.
1.6 Arabidopsis transformation
Plant expression vector pBI101-PR was transferred into
Agrobacterium tumefaciens EHA105 by freeze-thaw
method according to Höfgen and Willmitzer (1988).
Arabidopsis plants were transformed with the chimeric PR1-
GUS construct by floral dip method via Agrobacterium-
mediated transformation procedure (Clough and Bent, 1998).
Transformants were selected on MS medium containing 50
mg/L kanamycin, transferred to soil and allowed to self
pollinate. Plants homozygous for the PR1-GUS chimeric
gene were selected from T2 seeds and used for further
experiments.
1.7 PCR identification of transgenic plants
Two primers were designed and synthesized according
to the GUS gene sequence reported by Jefferson et al.
(1986). P1: 5-GCA ACTGGACAAGGCACT-3; P2: 5-AAT
AACGGTTCAGGCACA-3. PCR was conducted using
transgenic Arabidopsis genomic DNA as PCR template and
P1 and P2 as primers in the following conditions:
predenaturing at 94 ℃ for 3 min; 35 cycles of: 94 ℃ for 1
min, 52℃ for 30 s, 72 ℃ for 1 min; then keeping at 72 ℃.
The PCR positive seedlings were further confirmed by
Southern blotting analysis.
1.8 Southern blot of transgenic Arabidopsis plants
About 10 mg of genomic DNA of transgenic plants was
digested by HindⅢ and EcoRⅠ, separated on 0.8% agar-
ose gel electrophoresis, and transferred to a Hybond-N+
nylon membrane by Vacu Gene Pump. Southern blotting
analysis was performed according to the DIG DNA Label-
ing and Detection Kit instruction.
1.9 Analysis of the GUS enzyme activity
Histochemical GUS assay was conducted according to
Stomp (1992). Transgenic Arabidopsis plants were sprayed
with 0.5 mmol/L SA or sprayed with water and the leaf was
sampled for GUS staining. Besides, transgenic plants and
wild-type Arabidopsis plants were grown on MS medium
with 0.5 mmol/L SA and the whole seedlings were sampled
for GUS staining.
Fluorimetric determination of GUS activity was per-
formed according to Jefferson (1987). Sixteen-day-old seed-
lings grown on MS medium and MS medium with 0.5
mmol/L SA were collected and frozen in liquid nitrogen.
Three replicates were taken for each treatment. GUS activ-
ity was measured at excitation wave of 365 nm and emis-
sion wave of 455 nm with flurospectrophotometer F-2500
（HITACHI, Japan）. Protein was determined by the
method of Bradford (1976) and the specific GUS activity
was determined by the rate of increase of fluorescence over
protein concentration (pmol·mg-1 protein·min-1).
2 Results
2.1 Amplification and cloning of PR1 gene promoter
fragment
The PR1 upstream region was amplified using genomic
DNA of Arabidopsis ecotype Columbia as template and
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004984
FPR, RPR as primers. Results demonstrated that a 910 bp
special fragment, identical to the predicted size, was ampli-
fied from Arabidopsis ecotype Columbia. The PCR prod-
uct was cloned into pGEM-T vector to generate pGEM-T-
PR. Digestion of plasmid pGEM-T-PR with HindⅢ and
SmaⅠ reobtained a fragment identical to the amplified
fragment, suggesting that the PCR product had been cloned.
2.2 Construction of the recombinant plasmid pBI101-PR
pBI101 was used as binary vector, plasmid pGEM-T-PR
was digested with Hind Ⅲ and SamⅠ to yield PR fragment.
The purified DNA fragments were subcloned into the
HindⅢ -SamⅠ sites of the pBI101 vector to generate the
plant expression vector pBI101-PR. PCR amplification
showed that a 910 bp fragment was amplified from the re-
combinant pBI101-PR. Digestion of the recombinant
pBI101-PR with restriction endonuclease Hind Ⅲ and Sam
Ⅰ reobtained a fragment identical to the amplified fragment,
suggesting that the PR1 fragment had been inserted into
pBI101.
2.3 Plant transformation and PCR identification
The binary vector was introduced to Agrobacterium
tumefaciens strain EHA105 and then transformed into
Arabidopsis plants by floral dip method. T1 seeds were
harvested and plated on MS medium containing 50 mg/L
Kan to select the transformants. More than 100 transgenic
plants were obtained through Kan resistance selection.
About 1/3 of these transgenic plants segregated three kana-
mycin-resistant: one kanamycin-sensitive in T2 population,
indicating that there is a single functional PR1-GUS
transgene in these lines. Plants homozygous for the
transgene were identified from the progeny of these lines
by resistance to kanamycin and were further verified by
PCR analysis and Southern blot. The partial fragment of
GUS was amplified using genomic DNA isolated from
transgenic plants as template and P1, P2 as primers. Result
showed that a 730 bp special fragment, identical to the pre-
dicted size, could be amplified from the DNA of transgenic
plants (Fig.1A). Seeds from PCR positive plants were har-
vested and used for Southern blot and analysis of GUS
expression.
2.4　Southern blotting analysis of transformed plants
Southern blot was used to further verify the presence of
GUS. According to the structure of pBI101-PR, There was
a HindⅢ site in the 5 end of foreign gene PR1, and there
was a EcoRⅠ site in the NOS terminator end of the binary
vector. Southern blot of genomic DNA of wild type and
homozygous transgene lines were digested with HindⅢ
and EcoRⅠ, and hybridized with the probe of DIG-labeled
GUS gene fragment. Transgenic plants showed a specific
band and the wild type had no such hybridization signal
(Fig.1B), suggesting that the GUS gene had integrated in
Arabidopsis genome.
2.5 Histochemical GUS assay
In order to investigate whether the PR1 promoter is able
to drive the GUS expression in transgenic plants, transgenic
plants were induced by 0.5 mmol/L SA and histochemical
staining was performed. The GUS activity could be de-
tected in the mature leaf of transgenic plants sprayed with
0.5 mmol/L SA, while there was no GUS activity observed
in transgenic plants sprayed with water (Fig.2A).
Thansgenic seedlings grown on MS medium with 0.5 mmol/L
SA showed a strong accumulation of the blue precipitate,
and the wild type seedlings grown on MS medium with 0.5
mmol/L SA showed no blue precipitate accumulation
(Fig.2B), suggesting that the 910 bp upstream region from
the PR1 startpoint could drive the GUS gene expression.
2.6 Quantitative analysis of GUS expression in transgenic
plants
To accurately measure the level of GUS activity, a quan-
titative GUS assay was performed on transgenic plants and
the wild-type plants grown in the presence of SA, or in the
absence of SA. GUS activities in different transgenic lines
Fig.1. PCR and Southern blotting analysis of transgenic
Arabidopsis. A. PCR analysis of transgenic Arabidopsis. Lane M,
DL2000 DNA ladder marker; lanes 1-6, independent transgenic
lines. B. Southern blotting analysis of transgenic Arabidopsis.
Lane M, pBR328 DNA molecular weight marker, digoxigenin-
labeled; lane 1, wild type; lanes 2-5, independent transgenic lines.
ZHAO Shu-Qing et al.: Expression of the Pathogenesis-related Gene Promoter—GUS Reporter Fusion in Arabidopsis 985
showed significant difference, the highest activity appeared
in the transgenic line T28. As shown in Fig.3, without the
inducer, the background level of GUS activity was 3-fold
lower in the wild type than that in the transgenic plants
T28. Transgenic plants T28 grown in the presence of 0.5
mmol/L SA showed a 41-fold increase in GUS activity com-
pared to the uninduced plants, whereas in the SA-induced
wild-type plants, the increase in GUS activity was only 2-
fold. GUS staining was found consistent with GUS activity
measurement.
3 Discussion
The data show that the readily assayable enzymic marker
GUS can be induced in transgenic plants containing the
PR1-GUS gene fusion by treatment with SA. This pro-
vides the possibility for a novel assay to screen for chemi-
cals that induce a plant to turn up its own defense
mechanism, with positives then being tested by conven-
tional bioassays such as reduced disease indices as candi-
dates for agrochemicals that protect plants by enhance-
ment of natural resistance. Theoretically, as long as
transgenic plants containing PR1-GUS fusion treatment
with compounds which can activate SAR, the marker gene
PR1 of SAR must express, and the fused reporter gene
ought to express as well. We constructed the fusion gene
of PR1 promoter-GUS reporter，obtained the transgenic
Arabidopsis plant, and showed that PR1 promoter can drive
the GUS gene expression. Thus, this transgenic plant sys-
tem may provide convenient, sensitive and specific assay
systems for screening potential activators of natural resis-
tance to disease. A valid activator of SAR is to meet three
criteria: first, the compound and its metabolite have no di-
rect toxic effects on the pathogens; second, when the com-
pound activate SAR response, it can induce the same spec-
trum of disease resistance as do necrogenic pathogens;
and third, it can induce a suite of genes expression just as
pathogens induced SAR (Ryals et al., 1996). Therefore, the
activator of SAR may be useful as green pesticides for
plant protection.
Emerging evidence from analysis of plant defense re-
sponse has suggested the operation of several distinct
stress signal pathways for activation of the battery of dis-
ease resistance mechanisms. In parallel with SAR, induced
systemic resistance (ISR) and brassinosteroid-mediated
disease resistance (BDR) also existed in plants (Pietersa
et al., 1998; Nakashita et al., 2003). Various defensive mecha-
nisms are each through specific signaling pathway. The
understanding of plant defense responses network and the
mechanisms of controlling pathways will help develop novel
plant protection strategies for agronomic settings, and that
gene fusion assay of agrochemicals could be extended from
PR1 to other defense gene promoters responsive to differ-
ent stress signal pathways to identify distinct classes of
novel plant protective chemicals. Thus the gene fusion
approach may facilitate the discovery of new chemicals for
plant protection.
References:
Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar S P. 1997.
Fig.2. b-glucuronidase (GUS) staining of transgenic Arabidopsis
mature leaves (A) and whole seedlings (B). A. Left, GUS staining
of mature leaf of transgenic plant treated with H2O; right, GUS
staining of mature leaf of transgenic plant induced by SA. B. Left,
control (wild type); right, transgenic seedlings.
Fig.3. Effect of salicylic acid (SA) on GUS activity in PR1-GUS
transgenic plants.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004986
Signaling in plant-microbe interactions. Science, 276: 726-
733.
Bradford M M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem, 72: 248-
254.
Cao H, Bowling S A, Gordon A S, Dong X. 1994. Characteriza-
tion of an Arabidopsis mutant that is nonresponsive to induc-
ers of systemic acquired resistance. Plant Cell, 6: 1583-1592.
Clough S J, Bent A F. 1998. Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis
thaliana. Plant J, 16: 735-743.
Delaney T P, Uknes S, Vernooij B, Friedrich L, Weymann K,
Negrotto D, Gaffney T, Gut-Rella M, Kessmann H, Ward E,
Ryals J. 1994. A central role of salicylic acid in plant disease
resistance. Science, 266: 1247-1250.
Friedrich L, Lawton K, Ruess W, Masner P, Specker N, Gut-
Rella M, Meier B, Dincher S, Staub T, Uknes S, Métraux J P,
Kessmann H, Ryals J A. 1996. A benzothiadiazole derivative
induces systemic acquired resistance in tobacco. Plant J, 10:
61-70.
Görlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U,
Kogel K H, Oostendorp M, Staub T, Ward E, Kessmann H,
Ryals J. 1996. Benzothiadiazole, a novel class of inducers of
systemic acquired resistance, activates gene expression and
disease resistance in wheat. Plant Cell, 8: 629-643.
He Y K, Liu X F, Li J Y. 1999. Expression of Arabidopsis tryp-
tophan biosynthetic pathway genes: effect of the 5 coding
region of phosphoribosylanthranilate isomerase gene. Sci
China, 42: 274-280.
Höfgen R, Willmitzer L. 1988. Storage of competent cell for
Agrobacterium transformation. Nucleic Acids Res, 16: 9877.
Jefferson R A, Burgess S M, Hirsh D. 1986. Beta-Glucuronidase
from Escherichia coli as a gene-fusion marker. Proc Natl Acad
Sci USA, 83: 8447-8451
Jefferson R A. 1987. Assaying chemeric genes in plants: the GUS
gene fusion system. Plant Mol Biol Rep, 5: 387-405.
Kinkema M, Fan W, Dong X. 2000. Nuclear localization of NPR1
is required for activation of PR gene expression. Plant Cell, 12:
2339-2350.
Klessig D F, Malamy J. 1994. The salicylic acid signal in plants.
Plant Mol Biol, 26: 1439-1458.
Lawton K A, Friedrich L, Hunt M, Weymann K, Delaney T,
Kessmann H, Staub T, Ryals J. 1996. Benzothiadiazole in-
duces disease resistance in Arabidopsis by activation of the
systemic acquired resistance signal transduction pathway. Plant
J, 10: 71-82.
Murashige T, Skoog F. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant, 15:
493-497.
Nakashita H, Yasuda M, Nitta T, Asami T, Fujioka S, Arai Y,
Sekimata K, Takatsuto S, Yamaguchi I, Yoshida S. 2003.
Brassinosteroid functions in a broad range of disease resis-
tance in tobacco and rice. Plant J, 33: 887-898.
Pietersa C M J, van Wees S C M, van Pelt J A, Knoester M, Laan
R, Gerrits H, Weisbeek P J, van Loon L C. 1998. A novel
signaling pathway controlling induced systemic resistance
Arabidopsis. Plant Cell, 10: 1571-1580.
Ryals J A, Neuenschwander U H, Willits M G, Molina A, Steiner
H Y, Hunt M D. 1996. Systemic acquired resistance. Plant
Cell, 8: 1809-1819.
Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a
Laborstory Manual. 2nd ed. New York: Cold Spring Harbor
Laboratory Press.
Smith H B. 2000. Signal transduction in systemic acquired
resistance. Plant Cell, 12: 179-181.
Stomp A M. 1992. Histochemical localizayion of b-glucuronidase.
Sean R, Gallagher, GUS Protocols: using the GUS Gene as a
Reporter of Gene Expression. San Diego: Academic Press Inc.
103-114.
Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher
S, Chandler D, Slusarenko A, Ward E, Ryals J. 1992. Acquired
resistance in Arabidopsis. Plant Cell, 4: 645-656.
Vernooij B, Friedrich L, Ahl-Goy P, Staub T, Kessmann H, Ryals
J A. 1995. 2,6-dichloroisonicotinic acid-induced resistance to
pathogens does not require the accumulation of salicylic acid.
Mol Plant Microbe In, 8: 228-234.
Ward E R, Uknes S J, Williams S C, Dincher S S, Wiederhold D L,
Alexander D C, Ahl-Goy P, Metraux J P, Ryals J A. 1991.
Coordinate gene activity in response to agents that induce
systemic acquired resistance. Plant Cell, 3: 1085-1094.
Zhao S-Q（赵淑清）, Lin C（林春）, Yang X-H（杨晓贺）,
Wu W-H. 2001（武维华）. Isolation and genetic analysis
of Arabidopsis mutants with low-K+ tolerance. Acta Bot Sin
（植物学报）, 43: 105-107. (in English with Chinese
abstract)
(Managing editor: ZHAO Li-Hui)