A new rice zinc-finger protein binds to the O2S box of the a-amylasegene promoter Rihe Peng 1 1, Quanhong Yao1, Aisheng Xiong1, Huiqin Fan1, Xian Li1, Youliang Peng2, Zong-Ming Cheng3 an
Trang 1A new rice zinc-finger protein binds to the O2S box of the a-amylase
gene promoter
Rihe Peng
1 1, Quanhong Yao1, Aisheng Xiong1, Huiqin Fan1, Xian Li1, Youliang Peng2, Zong-Ming Cheng3 and Yi Li4
1
Shanghai Key Laboratory of Agricultural Genetic and Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, China;2Department of Plant Pathology, Chinese Agricultural University, Beijing,
Sciences, University of Tennessee, Knoxville, USA;4Department of Plant Science, University of Connecticut, Storrs, USA
3A putative transcription factor, named RAMY, that binds
to the 20-bp O2S sequences of the regulatory region of the
Amy2 gene promoter has been identified using the yeast
one-hybrid system from a rice library The full length RAMY
cDNA clone encodes a 218-amino acid protein and is
homologous to the late embryogenesis-abundant protein
(LEA5) In vitro mutagenesis and electrophoretic mobility
shift assays confirmed that RAMY can bind with O2S
spe-cifically through an unusual zinc finger with a CXCX4CX2H
consensus sequence Low levels of RAMY mRNAs were
detected in rice leaves and roots by Northern blot
hybrid-ization The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization,
mRNA level occurs prior to that of the Amy2 mRNA level
in the GA-treated aleurone tissues These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice a-amylase gene
Keywords: rice zinc-induced protein; O2S box; yeast one-hybrid system
Cereal a-amylase genes have been one of the primary
systems for exploring the molecular mechanisms involved in
hormone-regulated gene expression in plants
ger-mination of cereal grains, the embryo releases GA to the
aleurone layer, where it induces the transcription of
a-amylase genes [1]
Functional analyses of a-amylase gene promoters using
transient expression assays with reporter genes have shown
both GA and abscisic acid (ABA) may interact with
transcriptional regulatory proteins or transcription factors
that bind to a short nuclear nucleotide sequence referred to
as the GA response element (GARE) [2–4] Lanahan et al
[5] has demonstrated that GARE mediates the hormonal
control of transcription in the promoter of the low-PI gene,
Amy32b At least three other distinct regulatory elements
have been found to be necessary for high-level a-amylase
gene expression regulated by GA A closely associated
group of elements is composed of an opaque-2-like protein
binding sequence (O2S), a sequence element with an
enriched pyrimidine nucleotide motif (the pyrimidine
box), the GARE, and box I (TATCCAT) [6] Using
quantitative transient expression assays, the most
import-ant elements have been found to be GARE and O2S; mutation or deletion of either GARE or O2S resulted in lower GA-induced transcription [4,5] Rogers and Rogers [4] found that both GARE and O2S functioned only when positioned in one orientation with respect to each other and with respect to the TATA box, and when the distance between them was relatively short In searching for factors that interact with the sequences and regulate a-amylase gene expression, a Myb protein, GAMyb (GA-responsive Myb protein), was isolated that may specifically bind to a portion of the GARE box [7] GAMyb is able to activate the expression of a-amylase and other GA-regulated genes [8] A zinc-finger protein has been identified by South-western screening with baits containing a GARE box and has been found to repress the expression of a-amylase and other genes [9] In Arabidopsis, at least three proteins, SPY, RGA and GAI, are thought to negatively regulate GA responses [10–12] However, although the O2S box is another important element for controlling the level of transcription in a-amylase gene, little is known about the regulatory proteins or transcription factors that bind to the O2S sequence Our interest in the mechanism of seed germination and development in rice prompted us to search for the O2S binding protein Using the yeast one-hybrid system, we screened the rice cDNA libraries using O2S-containing baits ATTGACTTGACCGTCATCGG from the low pI amy54 promoter [13] We have isolated
a cDNA clone, RAMY, which encodes a protein that contains a zinc-finger Our experimental data indicate that RAMY protein binds specifically to the O2S element We have also determined the importance of the amino acids within the binding domain of RAMY protein and analyzed the time course for the induction of RAMY and a-amylase mRNA by GA
Correspondence to Q Yao, Shanghai Key Laboratory of Agricultural
Genetic and Breeding, Agro-Biotechnology Research Center,
Shang-hai Academy of Agricultural Sciences, 2901 Beidi Road, China.
Fax: + 86 021 62209988, Tel.: + 86 021 62209988,
E-mail: pengrihe69@yahoo.com
Abbreviations: GST, glutathione S-transferase; O2S, opaque-2-like
protein binding sequence; GA, gibberellin; ABA, abscisic acid;
GARE, GA response element.
(Received 30 January 2004, revised 28 April 2004,
accepted 19 May 2004)
Trang 2trp–, ura–, leu–, his–) was used to rescue cDNA library
plasmids from yeast DNA preparations The bait plasmid
pLGD-265 UP1 is a URA-marked
plasmid carrying a lacZ reporter gene under the control of
the CYC1 minimal promoter [14]
The rice cDNA library was constructed in pPC86 vector,
which is a marked
7 yeast expression plasmid containing a
GAL4 activating domain under the control of the yeast
ADC1 promoter [15] The cDNA was derived from poly(A)
RNA isolated from 15-day-old rice (Oryza sativa L.,
genotype IR36) seedlings grown at 25C in a greenhouse
Yeast one-hybrid screen
A yeast one-hybrid screen was performed to isolate genes
encoding proteins that associated with the O2S box
ATTGACTTGACCGTCATCGG in the Amy2 gene
pro-moter [12] To construct bait plasmid, the cis-element
containing three copies of O2S was synthesized by PCR
using two primers: Amyb1: 5¢-ACCCTCGAGGTCGA
CGGTATCGATAAGCTTGATTGACTTGACCGTCA
TCGGATTGACTTGACCGTCATCG-3¢, Amyb2: 5¢-CA
GGATCCATCACGACAGTCAGTGCCGATGACGG
TCAAGTCAATCCGATG-3¢ (the PCR conditions were:
94C, 20 s; 58 C 30 s; 72 C, 30 s; 25 cycles) Following
PCR, the 110-bp fragment isolated from PAGE was
digested with restriction enzymes, and inserted into the
XhoI/BamHI sites of the vector pLGD
The lithium acetate protocol was used for yeast
transfor-mation [16] An overnight culture of yeast cells (0.5 mL) were
inoculated into 50 mL fresh YPD medium and grown for
4 h Yeast cells were centirfuged at 5000 g for 8 min, and the
pellets washed once with 20 mL sterile distilled water, and
then with 10 mL Tris/EDTA/LiAc (100 mMLiAc in Tris/
EDTA) Finally, the cell pellet was resuspended in 0.5 mL
Tris/EDTA/LiAc An aliquot of 50 lL yeast cells, 1 lg
plasmid DNA, 50 lg salmon carrier DNA and 300 lL Tris/
EDTA/LiAc containing 40% (w/v)
each tube, and the mixtures were incubated for 30 min at
30C with shaking (150 r.p.m.) After heat shock at 42 C
for 15 min, the collected yeast cells were resuspended in Tris/
EDTA buffer and plated on selective yeast media
The total DNA from each positive yeast clone was
isolated according to the method of Robzyk and Kassir [17];
DNA cloning was performed using to standard procedures
[18] The DNA extracted from yeast cells was electroporated
into E coli strain MC8 [19] The transformants were
selected on M9 minimal medium containing all amino acids
except tryptophan (M9-TRP), thus selecting
pPC86-con-taining colonies The plasmid DNA was re-introduced into
a yeast reporter strain to confirm the b-galactosidase
activity Two oligonucleotide primers GAL4 5¢-GGA
TGTTTAATACCACT-3¢ and TAD4 5¢-TTGATTG
GAGACTTGACC-3¢ derived from the DNA sequence
flanking the GAL4 activating domain and the ADC1
Southern–Northern blot analyses Extraction of plant DNA and Southern blotting analysis were performed based on the method of Bringloe [20] Total RNA was prepared from rice (O sativa L IR36); leaves and roots were treated with 10 lM GA, and control seedlings were treated with water by the standard method
In order to examine the time course of the GA-induced RAMYand Amy2 gene expression, steady state levels of their transcripts in the GA-treated aleurone tissues were determined with Northern blot hybridization De-embryo-nated rice half-seeds were de-husked, sterilized with 10% (v/v) commercial Clorox, and treated with 95% (v/v) ethanol for 30 s to remove the outer wax layer After rinsing with water, the rice de-embryonated half-seeds (20 g) were submerged in seed buffer (20 mM calcium chloride, 20 mM sodium succinate, pH 5.2) in cell culture dishes After 10 h of incubation in seed buffer, appropriate amounts of GA were added to a final concentration of
10 lM Isolated aleurone tissues were incubated with GA for 2, 4, 6, 8, 10, 12 or14 h
RNA was separated by gel electrophoresis and trans-ferred to a Hybond N+ nylon membrane (Amersham-Pharmacia) Hybridization was performed at 65C in 5· NaCl/Cit, 10% (w/v) dextran sulfate, 0.5% (w/v) SDS
and 0.1 mgÆmL )1 denatured salmon sperm DNA Filters were washed twice (each for 15 min) at 65C in 2· NaCl/ Cit, 0.1% SDS, and once in 0.1· NaCl/Cit, 0.1% SDS at
65C for 15 min
Production and analyses of GST–RAMY fusion protein The GST–RAMY fusion protein was generated by inserting RAMY gene open reading frame between the sites of BamHI and SacI in the vector pALEX [21] The primers used were RAMYZ (5¢-AGGATCCATGGCT CTCGCTCTCTCCACC-3¢) and RAMYF (5¢-AGA GCTCAGTGGTGGTGGTGGTGGTGCACTCGGGT ACGTGGTGAAAC-3¢) Mutated RAMY polypeptides were produced by in vitro mutagenesis using the follow-ing primers: C182SZ (5¢-CGTGTGGGCTGGATGCT CTCCT-3¢), C182SF (5¢-GAGGAGAGCATCCAGCCC AC-3¢), C184SZ (5¢-GTGGGCTGGATGCTCTGCTCG TCTG-3¢), C184SF (5¢-GCAGACGAGCAGAGCATC CAGC-3¢), C189SZ (5¢-CTGCTCGTGTGCTGGTTCTT CGTCCAC-3¢), C189SF (5¢-GAGGTGGACGAAGAAC CAGCACACG-3¢), H192AZ (5¢-TGCTGGTTCTTCGT GCACCTCTGCTGTAAC-3¢), and H192AF (5¢-GTGAG GCCCTGCTCGCTGTTACAGCAGAGGTGC-3¢) All mutant genes were cloned into a pUC18 vector and sequenced for confirmation The resultant mutant RAMY genes were then inserted into the BamHI/SacI sites of the vector pALEX
Trang 3The resulting GST–RAMY construct was used to
trans-form E coli BL21 Transtrans-formants were used to inoculate
50 mL cultures of LB/ampicillin and were grown overnight
at 37C and harvested by centrifugation Cell pellets were
resuspended in 9 mL phosphate buffer (pH 7.4), 20 mM
imidazole was added, and the bacteria were lysed by
sonication After centrifugation at 8000 g
4C, the GST–RAMY fusion protein was isolated using
HiTrap chelating columns according to the manufacturer’s
instructions (Amersham-Pharmacia) SDS/PAGE and
Coomassie blue staining were used to determine the purity
of the protein Protein concentrations were determined
using a protein assay kit (Bio-Rad) Mutant GST–RAMY
fusion protein
13 was expressed and purified in the same way as
for the GST–RAMY fusion protein
The mutant O2S box was synthesized by PCR using the
primers mAmyb1 (5¢-ACCCTCGAGATTGAGCTAGCC
GCTAGCTCAATCCGATG-3¢) The PCR conditions
were: 94C, 20 s; 58 C 30 s; 72 C, 30 s for 25 cycles
The core binding sequence CTTGA in the conserved O2S
domain was replaced by GCTAG A gel retardation assay
was carried out as described by Jensen [22] The XhoI and
BamHI fragment containing three copies of the O2S box
was labelled using a random primer method with the
wild-type or mutant O2S motifs as competitors One nanogram
of labelled O2S DNA fragments, competitor DNA, 2.5 lg
poly(dI–dC) and 10 lg GST–RAMY binding protein were
mixed in 25 lL of DNA binding buffer [5 mM Hepes
pH 7.5, 2 mMMgCl2, 0.2 mMdithiothreitol, 1 mMCaCl2,
2% (w/v) glycerol
14 ] The mixture was incubated for 20 min
at room temperature and loaded onto a 6% polyacrylamide
gel After migration, the gel was fixed in 5% (w/v) glycerol,
5% (w/v) methanol, and 5% (w/v) acetic acid
DNA was then transferred to Whatman paper
autoradiographed
Results Screening for rice cDNA encoding the O2S binding protein
To isolate genes whose products bind to the O2S domain (ATTGACTTGACCGTCATCGG) in the Amy54 gene promoter, two plasmids were used in the yeast one-hybrid system The plasmid pPC86 contained a rice cDNA library to express GAL4–cDNA fusion proteins, and plasmid pLGD
17 -265UP1 was used as the bait with an insertion of three copies of the O2S domain at the 5¢ end
of the CYC1 mini-promoter region Following transfor-mation of the URA-marked plasmid pLGD-265 UP1 containing the O2S domain and the TRP-marked pPC86 plasmid carrying rice cDNA library
and selection on selective medium for 2 days, approxi-mately 5· 106 yeast transformants were overlaid onto SC-TRP-URA X-gal medium using nitrocellulose filters
In the first round of selection, 31 positive (blue) colonies were selected To verify the true positive clones after the first round selection, total yeast DNA was extracted and transformed into the E coli strain MC8 Following selection for the E coli strain MC8, 14 individual transformants were positive (blue) on medium containing X-gal
Nucleotide sequence and predicted amino acid sequence
of rice RAMY cDNA Using GAL4 and TAD4 primers, sequencing analyses of both strands of all 14 cDNA fragment
contained identical, overlapping sequences encoding the same protein The complete nucleotide sequence encoded a predicted protein of 218 amino acids (Fig 1)
A search in a number of random protein databases 22
revealed that there is no significant homology between
Fig 1.
32 The rice RAMY cDNA sequences
and the predicted product of its longest ORF
(GenBank accession no AY072712) The
putative DNA binding domain is underlined,
and the putative nuclear localization signal
is double-underlined.
RAMY 60 DGSSSSA AREVS WVPDPVTGHYRP SNFAGGRRRRPPRRPPRP 101 G.max LEAS 59 DTRDGSK AYSTD W PDPVTG Y YRP INHTPEIDPVELRHRLLR 100 N.tabacum LEA5 51 KWEESS -KKTTS WVPDPVTG Y YRP ESHAKEIDAAELRQMLLN 91 G.hirsutum LEA5 49 AMKESSSSETRAYSSA W PDPVTG Y YRP ENCGAEIDAAELREMLLN 94 V.radiata ARG2 52 KSGEEKVR- GGEKVS WVPDPVTG Y YRP EN-TNEIDVADMRATVLG 94 A.thaliana ARG21 53 KGVEES -TQKIS WVPDPKTG Y YRP ETGSNEIDAAELRAALLN 203 H.vulgare G3 59 REAEKA -AADSS WVPDPVTGHYRP ANRSSGADPADLRAAHLG 100
Fig 2 Alignment of the conserved domain of RAMY with some related proteins RAMY is compared with the LEA5 proteins from G max (accession no AAB38782), N tabacum (accession no AAC06242) and G hirsutum (accession no P46522), the ARG2 proteins from V radiata (accession no P32292) and A thaliana (accession no AAC19273), and with the G3 protein from H vulgare (accession no CAA55482).
Trang 4RAMY with other proteins deposited in the databases.
However, the N-terminal half of RAMY is homologous
to the LEA5 proteins from Glycine max (GenBank
acces-sion no AAB38782), Nicotiana tabacum (accesacces-sion no
AAC06242) and Gossypium hirsutum (accession no
P46522), the ARG2 protein encoded by cDNAs isolated
from Vigna radiata (accession no P32292) [23] and
Arabid-opsis thaliana (accession no AAC19273), and the G3
protein encoded by Hordeum vulgare (accession no
CAA55482) [24] (Fig 2) The C-terminal half of RAMY
contained a motif with Cys and His residues similar to the
zinc finger C3H
23 (Fig 1, DNA binding domain)
Using the 720-bp full-length RAMY cDNA as a
probe for hybridization of rice genomic DNA that had
been digested separately with HindIII, XbaI, and BamHI,
we observed only one DNA band that hybridized with
the probe from each of the digested DNA samples
(Fig 3) Because there are no HindIII, XbaI, and BamHI
sites within the 720-bp cDNA, a single band observed in
the Southern blot hybridization experiment suggests a
single copy of the RAMY gene is present in the rice
genome
RAMY contains a novel zinc finger
Using the yeast one-hybrid system, the GAL4–RAMY
fusion protein exhibited a strong transcriptional activation
function in yeast cells The GAL4–RAMY fusion protein
24
bound to the cis-element in bait pLGD
induced transcription of the LacZ reporter gene
To examine whether RAMY protein binds to the O2S
sequence directly and specifically, we performed an
elec-trophoretic mobility shift assay experiment As shown in
Fig 4A, the presence of the purified GST–RAMY protein
resulted in a mobility shift of the 32P-labelled O2S DNA
Fig 3 Southern blot analysis of Oryza sativa genomic DNA Total
genomic DNA (10 lg per lane) was digested with the restriction
enzymes, HindIII (H), XbaI (X) and BamHI (B) The positive control
(CK) was pPC86 (RAMY) DNA digested with SalI Full-length
RAMY cDNA was used as the probe The molecular marker (in kb)
was kDNA digested with EcoRI and HindIII.
Fig 4 Characterization of the DNA binding affinity of RAMY recombinant protein to the Amy2/O2S sequences (A) The [a- 32 P]dATP labelled O2S probes were incubated in the presence or absence of GST
or GST–RAMY Lane 1, purified GST; lanes 2–5, GST–RAMY Binding is competed by the same length of fragment containing three copies of unlabelled Amy2/O2S sequence; lane 2, 50 · competitor DNA; lane 3, 10 · competitor DNA; lane 4, 5 · competitor DNA; lane 5, 1 · competitor DNA (B) Comparison of DNA binding pref-erences of RAMY protein to the mutants (M) and the wild-type O2S (W) Lane 1, Purified GST; lane 2, GST–RAMY; lanes 3–6, EMSA by preincubating 25-fold (25 ·) or 100 fold (100 ·) excess amounts of unlabelled DNA fragments F, free probe; S, shift probe.
Trang 5fragment on the gel, suggesting that the GST–RAMY
protein binds directly to the O2S sequence Because the
purified GST alone did not produce such a mobility shift,
the shift must be caused by RAMY protein Furthermore,
the unlabelled O2S DNA competed well with the 32
P-labelled O2S DNA but nonspecific DNA or mutant O2S
DNA did not (Fig 4B), demonstrating that the binding of
RAMY to the O2S is specific
We performed an in vitro mutagenesis experiment to
identify amino acid residues of RAMY protein that are
important for the DNA binding In the C-terminal region
of RAMY, there are three Cys and one His residues
Figure 5
26 shows that mutagenesis of Cys182 severely
reduced binding activity of RAMY to the O2S DNA
sequence Furthermore, mutagenesis of Cys184, Cys189 or
His192 abolished the binding of the protein to the O2S
sequence completely
RAMY mRNA accumulation in GA-treated tissues
To determine whether the expression of the RAMY gene is induced by GA, and the possible relationship between the expression of the RAMY gene and the Amy2 gene, we performed a Northern blot hybridization experiment As shown in Fig 6A, RAMY was expressed at low levels in leaves but almost not at all in roots
and leaves, expression of RAMY was significantly induced
by exogenous GA We also used aleurone tissues to study the time course of the GA-mediated induction of RAMY and Amy2 expression Figure 6B shows that RAMY mRNA was observed 4 h after the GA treatment, but an increase in Amy2 mRNA was seen only after 10 h of the
GA treatment The level of RAMY mRNA reached its maximum 10 h after the GA treatment and returned to its basal value within 14 h In contrast, Amy2 mRNA reached
a maximum at 14 h after the GA treatment (Fig 6B) These data suggest that RAMY could act as a regulatory or transcription factor for the expression of a-amylase genes Discussion
We have cloned a gene, RAMY, from a rice cDNA library using O2S-containing baits with the yeast one-hybrid method that encodes a zinc-finger protein
the O2S-box binding domain is present in the protein This conclusion is supported by the observations that the RAMY
Fig 5 DNA binding is mediated by the zinc finger domain (A) SDS/
PAGE showing the purity of the recombinant GST-fusion proteins
(lanes 2–6) The GST-fusion proteins were over-expressed in E coli
BL21 (DE3), extracted under nondenaturing conditions and purified
by affinity chromatography (B) Gel mobility assay with the purified
GST–RAMY fusion protein (lane 2) or GST-mutant RAMY fusion
protein (lanes 3–6), showing RAMY binding to DNA and the effects
of mutations within the RAMY DNA-binding domain on the ability
of the purified fusion protein GST–RAMY to bind a probe containing
three copies of the Amy2/O2S region.
Fig 6 RAMY transcript levels in a rice plant subjected to GA treat-ment (A) GA induced RAMY mRNA accumulation in leaves and roots L, leaves; L + GA, leaves treated with GA; R, roots; R + GA, roots treated with GA (B) Induction of RAMY protein and a-amylase (GenBank accession no AF411220) by GA Aleurone tissue was incubated with GA for 2, 4, 6, 8, 10, 12 or 14 h Total RNA samples were loaded (20 lg per lane), fractionated on a 1.2% formamide/ agarose gel, probed with a 32P-labelled RAMY probe 18S, RNA probed with a 32 P-labelled 18S rRNA probe a-amy, RNA probed with a 32 P-labelled a-amylase gene probe.
Trang 6to the O2S domain Several plant zinc-finger proteins are of
the cluster type with multiple repeated fingers separated by
sequences of different length The spacing between the
fingers is the main element to determine the specificity of
binding target sequence [25] However, we only detected one
zinc-finger domain in RAMY, perhaps because the different
structure of the zinc finger motif in RAMY influences the
binding domain A putative nuclear localization signal
[26,27] sequence is also found in RAMY (Arg90–Arg97),
suggesting that RAMY may be transported into the nucleus
through the nuclear pore complex using its own nuclear
localization signal
Database searches revealed RAMY was homologous
only to part of the LEA5 (late embryogenesis-abundant)
proteins LEA5 proteins display a hydrophobic N-terminal
half and a hydrophilic C-terminal half [28] This family of
proteins is further characterized by a highly conserved motif
of 12 amino acids with the consensus WAPDPVTGYYRP
RAMY can be grouped into the Lea5
presence of the canonical 12 amino acid sequence motif
(WAPDPVTGYYRP)
Most LEA5-like proteins are induced in embryos or
vegetative tissues by desiccation, ABA or high osmoticum
The soybean Lea5-like (D-73 like) cDNA accumulates in
desiccating seeds from 20 to 80 days after flowering
roots but not in leaves of drought-stressed plants [29,30]
The related gene (Di21) from Arabidopsis displays increased
transcript accumulation in roots and leaves after drought
induction, but is not detected in mature dry seeds of
nonstressed Arabidopsis plants [31,32] In cotton, the Lea5
transcripts are highly induced in mature leave of
water-stressed plants or in water-water-stressed detached leaves [28]
However, the evidence presented here indicates that RAMY
transcription is induced by GA The interaction between GA
and ABA may be important in controlling the a-amylase
gene expression
The effects of GA and ABA on a-amylase gene
transcription have been established from transient
expres-sion experiments using a-amylase promoter-reporter gene
constructs in aleurone protoplasts [33,34] Two physically
associated elements are essential: a GA response element
(GARE) regulated by GA and ABA, and an opaque-2
binding sequence (O2S) This is consistent with the
hypo-thesis that protein binding and interaction between two
separate binding sites are required for high-level
transcrip-tion and proper hormonal regulatranscrip-tion [5] The expression of
GAMybat the mRNA level is upregulated by GA [7] and
the increase in the GAMyb mRNA occurs before that of
Amy21 mRNA after GA treatment In addition, GAMyb
specifically binds to an Amy21 GARE, and transient
expression experiments have shown that GAMyb activates
transcription of a high-pI a-amylase promoter fused to a
reporter gene in the absence of GA These results suggest
that GAMyb is a GA-regulated transcription factor
required for transcriptional activation of the high-pI
established
Acknowledgements
We would like to thank Dr Qun Zhu for proving us the rice cDNA library This research was supported by China PR Committee of Science.
References
1 Jacobsen, J.V., Gubler, F & Chandler, P.M (1995) Gibberellin action in germinated cereal grains In Plant Hormones: Physiology, Biochemistry and Molecular Biology (Davies, P.J., ed.), pp 246–
271 Kluwer Academic Publishers, Dordrecht, The Netherlands.
2 Skriver, K., Olsen, F.L., Rogers, J.C & Mundy, J., (1991) cis-Acting DNA elements responsive to gibberellin and its antagonist abscisic acid Proc Natl Acad Sci USA 88, 7266–72770.
3 Gubler, F & Jacobsen, J.V (1992) Gibberellin-responsive ele-ments in the promoter of a barley high-pI a-amylase gene Plant Cell
31 4, 1435–1441.
4 Rogers, J.C & Rogers, S.W (1992) Definition and functional implications of gibberellin and abscisic acid cis-acting hormone response complex Plant Cell 4, 1443–1451.
5 Lanahan, M.B., Ho, T.-H.D., Rogers, W.S & Rogers, J.C (1992)
A gibberellin response complex in cereal a-amylase gene pro-moters Plant Cell 4, 203–211.
6 Huang, N., Sutliff, T.D., Litts, J.C & Rodriguez, R.L (1990) Classification and characterization of the rice a-amylase multi-gene family Plant Mol Biol 14, 655–668.
7 Gubler, F., Kalla, R., Roberts, J.K & Jacobsen, J.V (1995) Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high pI a-amylase gene promoter Plant Cell 7, 1879–1891.
8 Gubler, F., Raventos, D., Key, M., Watts, R., Mundy, J & Jacobsen, J.V (1999) Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone Plant J 17, 1–9.
9 Raventos, D., Skriver, K., Schlein, M., Karnahl, K & Rogers, S.W (1998) HRT, a novel zinc finger, transcriptional repressor from barley J Biol Chem 273, 23313–23320.
10 Jacobsen, S.E., Binkowski, K.A & Olszewski, N.E (1996) SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis Proc Natl Acad Sci USA 93, 9292–9296.
11 Peng, J., Carol, P., Richards, D.E., King, K.E., Cowling, R.J., Murphy, G.P & Harberd, N.P (1997) The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses Genes Dev 11, 3194–3205.
12 Silverstone, A.L., Ciampaglio, C.N & Sun, T (1998) The Arabi-dopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway Plant Cell 10, 155–169.
13 Huttly, A.K & Baulcombe, D.C (1989) A wheat a-Amy2 pro-moter is regulated by gibberellin in transformed oat aleurone protoplasts EMBO J 8, 1907–1913.
14 Guarente, L (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast Methods Enzymol 101, 181–191.
Trang 715 Chevray, E.M & Nathans, D (1992) Protein interaction
clon-ing in yeast: identification of mammalian proteins that react with
the leucine zipper of Jun Proc Natl Acad Sci USA 89, 5789–
5793.
16 Gietz, D., Jean, A.S & Woods, R.A (1992) Improved method for
high efficiency transformation of intact yeast cells Nucleic Acids
Res 20, 1425.
17 Robzyk, K & Kassir, Y (1992) A simple and highly efficient
procedure for rescuing autonomous plasmids from yeast Nucleic
Acids Res 20, 3790.
18 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular
Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor
Laboratory Press, New York, USA.
19 Dower, S.J., Miller, J.F & Ragsdale, C.W (1988) High efficiency
transformation of E coli by high voltage electroporation Nucleic
Acids Res 16, 6127–6145.
20 Bringloe, D.H., Dyer, T.A & Gray, J.C (1995) Developmental,
circadian and light regulation of wheat ferredoxin gene expression.
Plant Mol Biol 27, 293–306.
21 Panagiotidis, C.A & Silverstein, S.J (1995) pALEX, a dual-tag
prokaryotic expression vector for the purification of full-length
proteins Gene 164, 45–47.
22 Jensen, E.O., Marcker, K.A., Schell, J & de Bruijin, F.J (1988)
Interaction of a nodule specific, trans-acting factor with distinct
DNA elements in soybean leghaemoglobin Ibc35¢ upstream
region EMBO J 7, 1265–1271.
23 Yamamoto, K.T., Mori, H & Imadeki, H (1992) Novel mRNA
sequences inducted by indole-3-acetic acid in sections of
elongat-ing hypocotyls of mung bean (Vigna radiata) Plant Cell Physiol.
33, 13–20.
24 Speulman, E & Salamini, F (1995)£GA3-regulated cDNAs
from Hordeum vulgare leaves Plant Mol Biol 28, 915–926.
25 Meshi, T & Iwabuchi, M (1995) Plant transcription factors Plant Cell Physiol 36, 1405–1420.
26 Park, K.J & Kanehisa, M (1998) NLS (nuclear localization sig-nal) prediction ICR Annual Report 5, 52–53.
27 Robbins, J., Dilworth, S.M., Laskey, R.A & Dingwall, C (1991) Two interdependent basic domains in nucleoplasmin nuclear geting sequence: identification of a class of bipartite nuclear tar-geting sequence Cell 64, 615–623.
28 G
33 alau, G.A., Wang, H.Y.-C & Hughes, D.W (1993) Cotton Lea5 and Lea14 encode atypical late embryogenesis-abundant proteins Plant Physiol 101, 695–696.
29 Yamamoto, K.T (1994) Further characterization of auxin-regu-lated mRNAs in hypocotyl sections of mung bean Vigna radiata (L.) Wilczek: sequence homology to genes for fatty-acid desaturases and atypical late-embryogenesis-abundant pro-tein, and the mode of expression of the mRNAs Planta 192, 359–364.
30 Burns, W.C., Maitra, N & Cushman, J.C (1996) Isolation and characterization of a cDNA encoding a LEA5-like protein from soybean (U66316) (PGR96-103) Plant Physiol 112, 1398.
31 Gosti, F., Bertauche, Vartanian, N & Giraudat, J (1995) Abscisic acid-dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana Mol Gen Genet 246, 10–18.
32 Xu, D., Duan, X., Wang, B., Hong, B., Ho, T.-H.D & Wu, R (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice Plant Physiol 110, 249–257.
33 Bethke, P.C., Schuurink, R & Jones, R.L (1997) Hormonal sig-naling in cereal aleurone J Exp Bot 48, 1337–1356.
34 Lovegrove, A & Hooley, R (2000) Gibberellin and abscisic acid signaling in aleurone Trends Plant Sci 5, 102–110.