全 文 ：599Journal of Plant Physiology and Molecular Biology 2005, 31 (6): 599-606
Received 2004-12-02, Accepted 2005-10-08.
This work was supported by National Natural Science Foundation of
China (No. 30360008).
*E-mail: jianchunguoh@163.com; Tel: 86-898-66890635
Abbreviations AHK: Arabidopsis histidine kinase; AHP (AHP):
Arabidopsis histidine phosphate (gene); ARRs (ARR4 , ARR5):
Arabidopsis response regulators (gene); CKI (CKI1): Cytokinin in-
sensitive (gene); CRE1 (CRE1): Cytokinin receptor (gene); Hpt: His-
tidine-containing phosphoryl transfer protein.
Activation of Genes Presumed in Cytokinin Signal Pathway of Two-
component System in Response to the Increased Cytokinin Contents
in ipt-GUS Transgenic Arabidopsis
GUO Jian-Chun1,2*, HU Xin-Wen2, DUAN Rui-Jun2, FU Shao-Ping2
(1State Key Laboratory of Tropical Crops Biotechnology, 2Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical
Agricultural Sciences, Haikou 571101, China)
Abstract: The ipt-GUS activated transgenic
Arabidopsis had 20–25 fold higher cytokinin contents
than the wild type. Changes in cytokinin content in
vivo triggering gene expressions involved in signal
pathway of two-component system have been analyzed
on the day of 6 d, 12 d, 20 d and 30 d after seed culti-
vation on MS medium in light conditions. The results
showed that the two cytokinin receptors, His-kinase
CRE1 was more sensitive to the increased cytokinin
contents than CKI1. Arabidopsis response regulators,
ARR4 and ARR5, were induced by the increased cy-
tokinin contents a t different t ime after seed
germination. ARR4 responded to cytokinins at early
time of seed germination, especially on the 6 d when
seedlings were around true leaf initiation, while cyto-
kinins induced ARR5 activation after 6 d of seed cul-
tivation in light conditions, an obvious increase was
on the 20 d when seedlings were around inflorescent
shoot initiation. Hpt-type transmitter kinase AHP4 in-
creased its activation by cytokinin induction only be-
tween the 20 d and 30 d after seed cultivation in light
conditions, and an obvious increase was on the 20 d.
Key words: ipt-GUS transgenic Arabidopsis; cytokinins; gene
transcription; signal pathway of two-component system
Two-component system typically comprises a
sensor histidine kinase and a response regulator (Hoch
and Silhavy 1996). The phosphoryl group in two-com-
ponent system could be transferred directly from a sen-
sor histidine kinase to a response regulator, or indi-
rectly transferred through a histidine-containing phos-
phoryl transfer protein (Hpt) (Popas et al. 1996;
Appleby et al. 1997; Welch et al. 1993). Earlier stud-
ies suggested that cytokinins were involved in a two-
component system. An inspection of the Arabidopsis
databases revealed that Arabidopsis had at least 11
sensor His-kinases. Five of them (ETR1, ETR2, ERS1,
ERS2, and EIN4) have been demonstrated to be ethyl-
ene receptors (Chang et al.1993; Hua and Meyerowitz
1998; Hua et al. 1995; Sakai et al. 1998). CRE1
(identical with AHK4, WOL) and CKI1, CKI2 were
reported to be cytokinin-sensors (Kakimoto 1996;
Inoue et al. 2001; Suzuki et al. 2001). AtHK1 was pro-
posed to be a putative osmo-sensor (Urao et al. 1998).
The other two, AHK2 and AHK3 were recently re-
ported in Arabidopsis genome sequence databases to
be His-kinases. Kakimoto (1996) brought forward that
CKI1 was the first cytokinin receptor. But Inoue et al.
(2001) deduced that the CRE1 was more likely to be
the first cytokinin receptor. Natural downstream part-
ners of cytokinin receptor in cytokinin signal pathway
are Hpt-type transmitter proteins. There are at least five
genes coding for Hpt-type transmitter proteins in
Arabidopsis (AHP1–AHP5) (D’Agositino and Kieber
1999; Imamura et al. 1998, 1999). AHP1 is highly
expressed in roots, but is poorly expressed or not at all
in other tissues; and AHP2 and AHP3 have been de-
tected to express in all adult tissues (Miyata et al. 1998;
600 31卷 植物生理与分子生物学学报
Suzuki et al.1998). The expression of AHP5 has been
detected in both roots and leaves (Suzuki et al. 2000).
Co-expression of AHP1, AHP2, AHP3 and AHP5 to-
gether with CRE1 suppressed the cytokinin-dependent
activation of cps::LacZin in Escherichia coli, but the
effectiveness of their signal titration was different
(AHP2/AHP5>AHP3>AHP1) (Suzuki et al. 2001).
Response regulators have been found in plants. In
Arabidopsis, members of ARR gene family are divided
into two groups, called type A and type B, which dif-
fer in their sequence and domain structure. Several
ARR genes in type A have been found to be respon-
sive to exogenous cytokinin treatment, such as, arr1
mutation resulted in reduced sensitivity to cytokinin
treatment in shoot regeneration assays in tissue culture;
over-expression of ARR1 increased the sensitivity to
cytokinin treatment in these assays; ARR1 mediated
the cytokinin induction of ARR6 transcription (Sakai
et al. 2001; Hutchison and Kieber 2002; Aoyama and
Oka 2003). ARR4 and ARR5 (also named IBC7 and
IBC6) displayed primary-response within 10 min after
exogenous cytokinin treatment, and reached a maxi-
mal induction at 30 to 40 min (ARR4) or 60 min
(ARR5), and then slowly declined. The steady-state
mRNA levels of ARR6, ARR7, ARR15, and ARR16
were elevated by exogenous cytokinin, in a pattern
similar to that of ARR5. Little increase or no change in
steady-state mRNA levels of ARR3, ARR8, ARR9 and
ARR17 were detected after exogenous cytokinin treat-
ment (Imamura et al. 1999; Brandstatter and Kieber
1998).
Although many researches have been done about
the role of cytokinins in inducing gene transcriptions
involved in cytokinin signal pathway of two-compo-
nent system, how the altered hormonal concentrations
in vivo trigger gene expressions is still not completely
understood. Questions remain whether the gene induc-
tion by exogenous cytokonin addition adequately
equals to the endogenous hormone increase. In this
work, how an increased endogenous cytokinin con-
tent affects the mRNA levels of selected genes involved
in cytokinin signal pathway of two-component system
has been investigated. The increased endogenous cy-
tokinin contents were obtained no-invasively by trans-
forming ipt-GUS genes to Arabidopsis that was acti-
vated by the pOp/LhG4 system through crossing the
reporter line harboring pOp-ipt-GUS with activator line
expressing LhG4 (Fig.1A).
1 Materials and Methods
1.1 Construction of plant binary vector pOp-ipt-
GUS and transformation
In plants, the presence of a short nucleotide re-
gion between a stop codon of the first open reading
frame and the initiation ATG codon of the second open
reading frame in transcription fusion gene is an essen-
tial prerequisite to achieve an efficient translation of
the second fusion partner. The coding sequence of
isopentenyl transferase (ipt, ID: 1224196) gene from
Agrobacterium tumefaciens fused with the coding se-
quence of GUS in transcription manner, which resulted
in ipt-GUS fusion. The resulting transcription fusion
genes were inserted downstream of pOp promoter in a
pVKH18 plant binary vector backbone, and then trans-
formed to Arabidopsis thaliana ecotype Columbia by
vacuum infiltration through A. tumefaciens GV3101.
The pOp promoter consists of a TATA box from
CaMV35S minimal promoter and two lac operators
upstream of the TATA box. The TATA box in pOp pro-
moter is silent for transcription activation and is acti-
vated by transcriptional factor LhG4, which is inde-
pendently expressed in another transgenic Arabidopsis
line called activator. The pOp-ipt-GUS fusion genes
were transformed to Arabidopsis called reporter. They
were silent firstly in transgenic reporter, and activated
by crossing the reporter line with the activator line (Fig.
1A) (Moore et al. 1998). After several generations, 20
independent single locus transgenic lines of pOp-ipt-
GUS were selected and designated as reporters.
1.2 Plant culture
Arabidopsis seedlings were grown in soil for
transformation. The seeds of wild type A. thaliana —
Columbia were sowed in a pot covered with nylon
mesh in a controlled environmental chamber under a
light intensity of 120–160 µmol m-2 s-1, 16 h light/8 h
dark photoperiod, 20–25℃, 50% relative humidity.
When the bolts were just emerging, the plants were
used for transformation.
Arabidopsis seedlings were grown on MS me-
601郭建春等: 在 ipt-GUS转基因拟南芥中双组分信号传导基因应答体内细胞分裂素的增加(英文)6期
dium for RNA isolation. The seeds from pOp- ipt-GUS
transgenic reporter, CaMV35S activator and their
crosses were sowed on the surface of MS medium with
sucrose 1% and kept at 4–8℃ for 2 d in dark, then
transferred to the light conditions with a light inten-
sity of 45–60 µmol m-2 s-1, 16 h light/8 h dark
photoperiod, at 22℃.
1.3 GUS histochemical assays
After 7 d, 14 d and 21 d the seeds were culti-
vated on MS medium in light conditions, the seedlings,
from reporters, activator and ipt-GUS activated
Arabidopsis, were incubated in solution of 0.3% form-
aldehyde (prepared with10 mmol/L MES and 0.3 mol/
L mannitol, pH 5.6) for 45 min at room temperature,
then followed three times’ washing with 50 mmol/L
NaH2PO4 (pH 7.0). The above seedlings were com-
pletely submerged in solution X-gluc for GUS stain-
ing 8–12 h at 37℃ [8 mL of 50 mmol/L sodium phos-
phate buffer (pH 7.2), 9 mg 5-bromo-4-chloro-3-
indolyl-D -glucuronide (X-gluc), 8 mL triton-X 100, 8
mL-mercaptoethanol]. The stained seedlings were
blanched with 70%–80% ethanol till the background
(Chlorophylls) disappeared.
1.4 Analyses of gene transcriptions by RT-PCR
After 6 d, 12 d, 20 d and 30 d the seeds were
cultivated on MS medium in light conditions, 50–100
mg of the seedlings at each culture time were harvested
for isolating total RNA with the TRIZOL reagent. Re-
verse transcription was performed immediately after
the isolation. The PCR reactions were carried out in a
volume of 50 mL, which included 2 mL of the reverse
transcription reaction solution, 3 mL (5 pm/mL) of gene
specific primers, 3 mL dNTPs (5 mmol/L), 5 mL
10×PCR buffer, and 31 mL ddH2O. The mixture was
preheated at 94℃ for 5 min before 3 U Taq DNA poly-
merase was added. PCR cycle conditions were 94℃
45 s, gene specific annealing temperature 45 s, 72℃
90 s. The PCR reactions were semi-saturated ones, and
cycles were decided by testing 20 cycles, 25 cycles,
30 cycles, 35 cycles, and 40 cycles of each gene. Ac-
tin primers 5 TTCCTCAATCTCATCTTCTTCC 3
and 5 GACCTGCCTCATCATACTCG 3 were used
to produce 1 100 bp fragments (annealing temperature,
54℃; 25 cycles ) . Two speci f ic pr imers 5
ATGGGAAGAGCAGAGTGTAGTTTGT 3 and 5
AATGGTTTCTCTTGCTTCCTCTGAA 3 were used
for CKI1 to produce 950 bp fragments (annealing
temperature, 60℃; 25 cycles). Two specific primers
5TCATCACAAGTCTCCGAAAC3 and 5 TC A
CATCCGCAGTCAT3 were designed for CRE1 to
produce 500 bp fragments (annealing temperature,
54℃; 26 cyc les ) . Two speci f ic p r imer s 5
TGCTGCAGTATGGCCAGAGACGGTGGTGTTTC3
and 5 GGCTGCAGTCAAAGCTTCCGTTTGTTT-
CCGT3 were designed for ARR4 to produce 512 bp
fragments (annealing temperature, 60℃; 28 cycles).
Specific primers 5 TGCGACAAGAGCTTTACA 3
and 5 ACACACCACCATTTTCATATC 3 were de-
signed for ARR5 to produce 650 bp fragments
(annealing temperature, 52℃; 25 cycles). Primers 5
GAGCGTCGGTCCCCACACTTCTATAC 3 and 5
AGCTCCAAGATGATGCAAACCCTAA 3 were
used for AHP4 to produce 220 bp fragments (annealing
temperature, 60℃; 25 cycles).
2 Results
2.1 ipt-GUS fused gene activation
The ipt-GUS genes were activated in the prog-
eny of pOp-ipt-GUS transgenic reporter crossed with
CaMV 35S LhG4 transgenic Arabidopsis activator
A47/1-C. As the LhG4 was promoted by 35S promoter,
the ipt-GUS fusion genes were constitutively
expressed.
The levels of cytokinins were analyzed by HPLC-
MS/MS in two single locus homozygous independent
lines of pOp-ipt-GUS transgenic Arabidopsis report-
ers (R1 and R2), their progeny of R1×A47/1-C and
R2×A47/1-C (IG-1 and IG-2), transgenic Arabidopsis
activator (A47/1-C) after the seeds were cultivated on
MS medium for12 d and 30 d in light conditions. The
cytokinin contents in the seedlings of IG-1 and IG-2
were 20–25 folds those of their respective inactive re-
porters (R1 and R2) when the seedlings were 12 d old,
and 15–20 folds those of their inactive reporters when
the seedlings were 30 d old (Fig.1B). Very weak GUS
staining was found at the shoot apical meristem of ac-
tivated ipt-GUS seedlings (Fig.1C). By RT-PCR
identification, ipt and GUS transcriptions were detected
602 31卷 植物生理与分子生物学学报
only in IG-1 and IG-2, but not in R1 and R2. The above
results showed that the pOp promoter was activated in
the crosses, and both ipt and GUS were correctly tran-
scribed and functional proteins were produced.
2.3 Sensitivity of cytokinin receptor CRE1 and
CKI1 to high endogenous cytokinin contents
The models of CKI1 transcription in the control
plants (no increased cytokinin content) of pOp-ipt-GUS
inactive transgenic reporter lines (R1 and R2), the ac-
tivator line A47/1-C, and wild type Columbia were
similar, which the transcription levels were higher on
the 6 d after seed cultivation in light conditions, fol-
lowed by a great decrease on the 12 d; and were unde-
tectable on the 30 d (Fig.3B1). In the seedlings with
activated ipt-GUS (IG-1, IG-2), where cytokinin level
was elevated, CKI1 transcript followed a similar pat-
tern except on the 12 d and 6 d when the CKI1 mRNA
level was slightly higher and lower than the controls
(Fig.3A1). The mRNA levels of CRE1in the control
plants were moderate on the 6 d, followed by a de-
cline on the 12 d and 20 d, then back up to the 6–d
level on the 30 d (Fig.3B2). In the ipt-GUS activated
seedlings, the mRNA levels of CRE1were higher than
those in the control plants on the 6 d, followed by a
large increase on the 12 d and 20 d (2–3 folds of ipt-
GUS inactive seedlings). On the 30 d, no significant
difference was found between the ipt-GUS activated
seedlings and inactive ones (Fig.3A2). These results
indicate that His-kinase CRE1 is more sensitive to cy-
tokinins than His-kinase CKI1.
2.4 Sensitivity of Hpt-type transmitter AHP4 to
high endogenous cytokinin contents
The transcriptions of AHP4 were low in all con-
trol plants at all stages (Fig.4B); and in the ipt-GUS
Fig.2 Expression levels of actin in all lines and time points
Columbia, wild type Arabidopsis. A47/1-C, CaMV35S-LhG4
transgenic activator Arabidopsis. R1 and R2, pOp-ipt-GUS
transgenic reporter Arabidopsis. IG-1and IG-2, progenies of
R1×A47/1-C and R2×A47/1-C. The data are the mean of the four
experimental replications. Error bar is SE.
Fig.1　Activation of ipt-GUS fusion genes by pOp/LhG4 system
A: The pOp/LhG4 expression system (Moore et al. 1998). B:
Cytokinin content of pOp-ipt-GUS activated Arabidopsis. C:
GUS histochemical assays. R1 and R2, inactivated reporter lines
of pOp-ipt-GUS. IG-1 and IG-2, activated lines from R1×A47/
1-C and R2×A47/1-C. A, activator Arabidopsis line CaMV35S-
LhG4 (A47/1-C). The legends are used in Fig.1B and C.
The seedlings with activated ipt-GUS showed a
number of visibly altered phenotypes, such as inhib-
ited root growth, longer and larger shoot, enlarged
shoot apical meristem, and serrated leaves.
2.2 Normalization of mRNA quality and quantity
by actin gene
Actin is highly conservative gene. In most cases,
actin specific primers spanning different exons were
used in RT-PCR reactions to check possible contami-
nation of genomic DNA in RT and the quality of the
RNA preparation. The amount of actin RT-PCR prod-
ucts was used to normalize the amount of RT-PCR of
other genes analyzed in the same lines and at the same
time. In general, the quantity of actin PCR product
was almost equal in all samples at all stages (Fig.2).
603郭建春等: 在 ipt-GUS转基因拟南芥中双组分信号传导基因应答体内细胞分裂素的增加(英文)6期
seedlings activated, the mRNA levels of AHP4 were
3–4 folds and about 2 folds those of in the control plants
on the 20 d and 30 d (Fig.4A).
2.5 Effect of high endogenous cytokinin contents
on the transcriptions of Arabidopsis response regu-
lator ARR4 and ARR5
In the control plants (R1, R2, A47/1-C,
Columbia), the transcriptions of ARR4 were only de-
termined on the 6 d and 12 d; and not any transcrip-
tion was measured on the 20 d and 30 d after seed
cultivation in light conditions (Fig.5B1). In the ipt-GUS
activated seedlings (IG-1, IG-2), the steady-state
mRNA level of ARR4 was significantly elevated on
the 6 d; slightly elevated mRNA level was detected on
the 12 d; and also not any transcription was determined
on the 20 d and 30 d (Fig.5A1). The transcription rates
of ARR5 were lower on the 12 d and 20 d than on the 6
d and 30 d in control plants (Fig.5B2); however, high
endogenous cytokinin contents enhanced the transcrip-
tion of ARR5 from 12 d to 30 d, an obvious enhance
was on the 20 d (Fig.5A2).
Fig.3 Transcription levels of CKI1 and CRE1 in response to high endogenous cytokinin contents
Fig.3A (A1, A2) shows the difference of the gene transcription levels between the ipt-GUS activated Arabidopsis. Fig.3B (B1, B2)
shows the gene transcription levels in control plants which have no cytokinin increase. Columbia, wild type; R, the average transcription
levels of gene in ipt-GUS inactive reporter line R1 and R2; A47/1-C, CaMV35SLhG4 activator. The data are the mean of the four
experimental replications. Error bar is SE. The legends of the symbols meet from Fig.3 to Fig.5.
Fig.4　Hpt-type transmitter AHP4 in response to high endogenous cytokinin contents
The legends see Fig.3.
604 31卷 植物生理与分子生物学学报
3 Discussion
Cytokinins have been reported to involve in a His-
kinase of two-component system. That cytokinin sig-
nal might employ a phosphorelay was the identifica-
tion of CKI1, a sensor histidine kinase homolog con-
sisting of a putative input domain, a histidine kinase
domain and a receiver domain. Over-expression of the
CKI1 gene induced typical cytokinin responses in ab-
sence of exogenous cytokinins, suggesting that CKI1
is a cytokinin receptor (Kakimoto 1996). Recently,
CRE1 was identified as a member of the same sensor
histidine kinase family from the fact that cre1 mutant
is cytokinin-insensitive. CRE1 has also been identi-
f ied a s a cyt ok in in r es pons ive sens or i n
Schizosaccharomyces pombe and E. coli (Inoue et al.
2001). Our results showed that His-kinase CRE1 was
more sensitive to cytokinins than His-kinase CKI1 (Fig.3).
His-kinases of cytokinin receptor CRE1 inter-
acted with certain AHPs through phosphorelay, the ef-
fectiveness of their signal titration was different
(AHP2/AHP5>AHP3>AHP1) (Suzuki et al. 2001).
The expression of AHPs was identified in different
organs or tissues (Miyata et al. 1998; Suzuki et al.1998,
2000). In our current study, the increased cytokinins
elevated AHP4 transcription level only at a certain stage
around 20 d (Fig.4). The above results suggest whether
the individual AHPs may transmit phosphoryl group
from AHK at different stages with different
effectiveness. In wild type Arabidopsis, seedlings ini-
tiate inflorescence around 20 d, whether AHP4 trans-
mits phosphoryl group from the cytokinin receptor to
regulate the inflorescent shoot initiation, further evi-
dences need to be accumulated.
Arabidopsis response regulators (ARR4, ARR5)
have been found to be specifically sensitive to exog-
enous cytokinins (Imamura et al. 1999; Brandstatter
et al. 1998). In ipt-GUS activated Arabidopsis, ARR4
and ARR5 responded to cytokinins at different time.
The enhanced transcription of ARR4 was only checked
on the 6 d; and ARR5 enhanced its transcription from
12 d to 30 d (Fig.5).
Even though cytokinins have been presumed in
signal transduction of two-component system about
twenty years, the genes, which respond as cytokinin
receptors, the phosphoryl group transmitters and the
response regulators still have not been definitively
identified. The information involved in the present re-
search is helpful to understand the cytokinin signal
transduction pathway in two-component system.
Fig. 5　Arabidopsis response regulators in response to high endogenous cytokinin contents
The legends see Fig.3.
605郭建春等: 在 ipt-GUS转基因拟南芥中双组分信号传导基因应答体内细胞分裂素的增加(英文)6期
References
Appleby JL, Parkinson JS, Bourret RB (1997). Signal
transduction via the multi-step phosphorelay: not
necessarily a road less traveled. Cell, 85: 717–723
Aoyama T, Oka A (2003). Cytokinin signal transduction in plant
cells. J Plant Res, 116: 221–231
Brandstatter I, Kieber JJ (1998). Two genes with similarity to
bacterial response regulators are rapidly and specifically
induced by cytokinin in Arabidopsis. Plant Cell, 10: 1009–
1020
Chang C, Kwok SF, Bleeker AB, Meyerowitz EM (1993).
Arabidopsis ethylene-response gene ETR1: similarity of
product to two-component regulators. Science, 262: 539–
544
D’Agositino IB, Kieber JJ (1999). Molecular mechanism of
cytokinin action. Curr Opin Plant Biol, 2: 359–364
Hoch JA, Silhavy TJ (1996). Two-component signal
transduction. Cell, 86: 865–875
Hua J, Chang C, Sun Q, Meryerowitz EM (1995). Ethylene
insensitivity conferred by Arabidopsis ERS gene. Science,
269: 1712–171
Hua J , Meyerowitz EM (1998). Ethylene responses are
negatively regulated by a receptor gene family in
Arabidopsis thaliana. Cell, 94: 261–271
Hutchison CE, Kieber JJ (2002). Cytokinin signaling in
Arabidopsis. Plant Cell, 14: 47–59
Imamura A, Hanaki N, Umeda H, Nakamura A, Suzuki T,
Ueguchi C, Mizuno T (1998). Response regulators
implicated in His-to-Asp phosphotransfer signaling in
Arabidopsis. Proc Natl Acad Sci USA, 95: 2691–2696
Imamura A, Hanaki N, Nakamura A, Suzuki T, Taniguchi M,
Kiba T, Ueguchi C, Sugiyama T, Mizuno T (1999).
Compilation and characterization of Arabidopsis thaliana
response regulators implicated in His-Asp phosphorelay
signal transduction. Plant Cell Physiol, 40: 733–742
Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato
T, Tabata S, Shinozaki K, Kakimoto T (2001).
Identification of CRE1 as a cytokinin receptor from
Arabidopsis. Nature, 409: 1060–1063
Kakimoto T (1996). CKI1, a histidine kinase homolog
implicated in cytokinin signal transduction. Science, 274:
982–985
Miyata S, Urao T, Yamaguchi-Shinozaki K, Shinozaki K (1998).
Char acter izat ion of genes fo r two-compon en t
phosphorelay mediators with a single HPt domain in
Arabidopsis thaliana. FEBS Lett, 437: 11–14
Moore I, Gälweiler L, Grosskopf D, Schell J, Palme K (1998).
A transcription activation system for regulated gene
expression in transgenic plant. Proc Natl Acad Sci USA,
95: 376–381
Popas F, Wurgler-Murphy M, Maeda T, Witten EA, Thai TC,
Saito H (1996). Yeast hog1 MAP kinase cascade is
regulated by a multistep phosphorelay mechanism in the
SLN-YPD1-SSK1 “two-component”osmo-sensor.
Cell, 86: 845–848
Sakai H, Hua J, Chen QG, Medrano LJ, Bleecker AB, Meyerowitz
EM (1998). ETR2 is an ETR1-like gene involved in
ethylene signaling in Arabidopsis. Proc Natl Acad Sci
USA, 95: 559–567
Sakai H, Honma T, Aoyama T, Sato S, Kato T, Tabata S, Oka
A ( 20 0 1 ) . AR R 1 , a t r an scr ip t ion f ac t o r f o r
genesimmediately responsive to cytokinins. Science, 249:
1591–1592
Suzuki T, Imamura A, Ueguchi C, Mizuno T (1998). Histidine-
containing phosphotransfer (Hpt) signal transducers
implicated in His-to-Asp phosphorelay in Arabidopsis.
Plant Cell Physiol, 39: 1258–1268
Suzuki T, Sakurai K, Imamura A, Nakamura A, Ueguchi C,
Mizuno T (2000). Compilation and characterization of
histidine-containing phosphotransmitters implicated in
His-to-Asp phosphorelay in plants: AHP signal transducers
of Arabidopsis thaliana. Biosci Biotechnol Biochem, 64:
2486–2489
Suzuki T, Miwa K, Ishikawa K, Yamada H, Aiba H, Mizuno T
(2001). The Arabidopsis sensor his-kinase can respond to
cytokinins. Plant Cell Physiol, 42(2): 107–113
Urao T, Yakubov B, Yamaguchi-Shinozaki K, Schinozaki K
(1998). Stress-responsive expression of genes for two-
component response regulator-like proteins in Arabidopsis
thaliana. FEBS Lett, 427: 175–178
Welch MK, Oosawa K, Aizawa SI, Eisenbach M (1993).
Phosphorylation-dependent binding of a signal molecule
to the flagellar switch of bacteria. Proc Natl Acad Sci
USA, 90: 8787–8793
606 31卷 植物生理与分子生物学学报
在ipt-GUS转基因拟南芥中双组分信号传导基因应答体内细胞分裂素
的增加
郭建春 1,2*，胡新文 2，段瑞军 2，符少萍 2
(中国热带农业科学院 1热带作物生物技术国家重点实验室，2热带生物技术研究所，海口 571101)
摘要：ipt-GUS转录融合基因在拟南芥植物中表
达，其体内细胞分裂素的含量可达到野生型的
20~30倍。从拟南芥种子萌发后的 6、12、20和
30 d四个时间分析了植物体内细胞分裂素含量的提
高对其双组分信号传导系统中基因的影响。研究
发现：细胞分裂素受体基因 CRE1比 CKI1基因更
容易被增加的植物细胞分裂素诱导表达。拟南芥
植物细胞分裂素反应调节基因ARR4和ARR5在植
物发育的不同时期应答植物体内增加的植物细胞分
裂素，ARR4的应答反应比 ARR5早，种子萌发
后的第 6天幼苗真叶形成初期，ARR4基因被明显
诱导；而ARR5的应答反应在幼苗真叶形成后的几
个时间段均能检测到，并且在种子萌发后的第 20
天，花枝形成开始时特别明显。在双组分信号传
导途径中，从受体到反应调节基因传导磷酸基团
的传导基因AHP4在幼苗发育的后期种子萌发后的
第 20和 30天，应答植物体内增加的植物细胞分
裂素，并且在花枝形成初期比较明显。
关键词：ip t -GU S 转基因拟南芥；细胞分裂素；基因转
录；双组分信号传导系统
中图分类号：Q7 4
国家自然科学基金项目(No.30360008)资助。
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