全 文 ：Received 27 Oct. 2003 Accepted 9 Mar. 2004
Supported by the Funds from the Science and Technology Commission of Shanxi Province (021034) and Returning Overseas Student Affair
Office of Shanxi Province (20011575).
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http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1091-1099
Molecular Cloning and Expression Analysis of a Zinc
Finger Protein Gene in Apple
CAO Qiu-Fen1*, Masato WADA2, MENG Yu-Ping1, SUN Yi1, CUI Gui-Mei1
(1. Agricultural Biotechnology Research Center of Shanxi Province, Taiyuan 030031, China;
2. Department of Apple Research, National Institute of Fruit Tree Science, National Agricultural Research Organization
Shimokuriyagawa, Morioka, Iwate 020-0123, Japan)
Abstract: A cDNA library was created from stem apex tissue from Jonathan apples (Malus domestica
Borkh.), harvested in June to August, during which the plant transitions from vegetative growth to
reproductive growth. From this library, we isolated an expressed sequence tag (EST) sequence containing
a zinc finger motif; using this sequence, a 779 bp cDNA fragment was obtained by using 5 RACE, and a final
full-length cDNA encoding an apple zinc finger protein (named MdZF1; GenBank accession number AB116545)
was obtained by further PCR. This zinc finger motif of MdZF1 has high homology with INDETERMINATE 1
(ID1) gene from maize which seemed to be involved in the transition to flowering. Northern blot and
RT-PCR analyses showed that the MdZF1 expressed in the root, stem, leaves, shoot apex and floral organs
of the apple, with expression levels higher in root, stem, leaves and floral shoot apex than that in floral
organs (sepals, petals, stamens and pistils). Genomic Southern analysis showed that there was a single
copy gene in apple genome.
Key words: Malus domestica zinc finger protein (MdZF1); cloning; expression
The zinc finger proteins belong to a family of regulatory
transcription factors that contain “finger-like” motifs; they
are found in most living organisms, from yeasts to plants
as well as mammals. The zinc finger motif can bind to DNA,
RNA and DNA-RNA hybrids, as well as to other zinc finger
proteins, and zinc finger-containing proteins normally have
two or more motifs and are responsible for controlling the
transcription and translation in an organism (Huang and
Liu, 1998). The classical zinc finger protein is the TFⅢA
type, also known as the Cys2/His2 or C2H2 type, which
has been extensively studied at a structural level (Sakamoto
et al., 2000). In plants, some Cys2/His2 zinc finger cDNAs
have been associated with stress resistance (Huang et al.,
2002; Wang and Yang, 2002) and growth (Colasanti et al.,
1998). Colasanti et al. (1998) cloned the INDETERMI-
NATE 1 (ID1) gene, which encoded a maize zinc finger pro-
tein associated with the transition to flowering, suggesting
that the ID1 gene product functions as a transcription regula-
tor of floral development. The phenotype of id1 mutant and
the expression analysis of ID1 suggested that the gene prod-
uct act as a floral initiation signal like the florigen hypothesis.
The shoot apex of higher plants contains an undifferen-
tiated cluster of cells, called the shoot apical meristem, which
maintains the propensity for vegetative growth and
development during the initial stages of plant growth, and
then switches to reproductive growth and development
when external and internal conditions trigger reproductive
signals. To investigate the molecular mechanisms respon-
sible for the transition of apple shoot apex from vegetative
to reproductive growth, we built a cDNA library from apple
shoot apex at the flower bud during differentiation stage
(June to August), and obtained a series of cDNA fragments.
By comparing the sequences from database in DDBJ/
EMBL/GenBank, we found several expressed sequence tag
（EST）sequences that contained a zinc finger motif. One
sequence showed high homology with maize ID1 gene ,
and we were able to assemble a full-length cDNA for this
zinc finger protein with 5′RACE and PCR techniques, which
we designated MdZF1 (Malus domestica ZINC FINGER
PROTEIN 1). Northern blot and RT-PCR methods were used
to confirm and analyze the expression of MdZF1 in differ-
ent apple tissues and floral organs. Southern blot was used
to analyze the copy number of MdZF1 in apple genomes.
1 Materials and Methods
1.1 Plant materials
Jonathan apple (Malus domestica Borkh.) tissues were
harvested between June and August. Tissue samples
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041092
(0.5-1.0 cm) were taken from the shoot apices every two
weeks, frozen in liquid nitrogen and stored at –80 ℃ for
construction of the cDNA library. Immature leaves, leaves
on the middle of newly-growing shoots, mature leaves, post-
germination cotyledons, shoot apices, sepals, petals,
stamen, pistils, leaves, roots, and stems from seedlings (one
month old) were harvested, frozen in liquid nitrogen and
stored at –80 ℃ for total RNA extraction, Northern blotting
and RT-PCR analysis.
1.2 Isolation of the EST and full-length cDNA
Total RNA was extracted from shoot apex tissues of
Jonathan apples by a modified CTAB method as previously
described (Cheng et al., 1993), reverse transcribed to cDNA
with Oligo（dT）, and ligated with EcoRⅠ, NotⅠ, and
BamHⅠ adaptors for constructing the cDNA library. The
ligated cDNAs were cloned into the EcoRⅠ restriction site
of pBluescriptⅡ SK+ vectors (Stratagene, La Jolla,
California, USA). The recombinant plasmids were trans-
fected into Escherichia coli strain DH5a, and plated on
LB/ampiciline/IPTG/X-Gal. White clones were chosen for
amplification, followed by plasmid extraction. Forward and
reverse sequencing was carried out on a Hitachi SQ5500
sequencer (Hitachi, Tokyo, Japan) with a Thermo Sequenase
premixed cycle sequence kit (Amersham Biosciences, New
Jersey, USA), followed by analysis with DNA reading soft-
ware (Software Development Co., Tokyo, Japan). The se-
quences of the cloned cDNA fragments were compared
with those in the GenBank. A 1.35 kb EST, designated as
CS029, and was found containing a zinc finger motif.
1.3 5 rapid amplification of cDNA ends (5 RACE)
S p e c i f i c p r i m e r s 5 R C S 2 9 1 ( 5 -
GAAGATGGTTCCACAGTCAC-3) and 5RCS292 (5-
GCACTTGTCACATTTCCAGG-3) were designed accord-
ing to the sequence of the 1.35 kb EST fragment. For RACE,
PCR was performed using the above specific primers along
with cassette primers C1 and C2, and LA-Taq DNA poly-
merase (TaKaRa Biomedicals, Shiga, Japan). Primers C1 and
5RCS291 were used in the first amplification reaction to
amplify fragments from 1 mg template DNA from the cDNA
library. The PCR conditions were as follows: 30 cycles of 94
℃ for 30 s, 56 ℃ for 30 s, and 72 ℃ for 3 min. Primers C2 and
5RCS292 were used for RACE PCR as above, with an an-
nealing temperature of 54 ℃.
The PCR products were subcloned into the EcoRⅤ re-
striction site of pBluescriptⅡ SK+ vectors, and transformed
into E. coli strain DH5a. The clones with recombinant plas-
mids were detected, the plasmids were prepared and se-
quenced the same as described above. A 779 bp cDNA,
containing sequences of another zinc finger motif upstream
of CS029, was obtained and designated CS029-4.
1.4 Full-length cDNA amplification
Sense (5CS294(5-CTCTTCACCACAATTGGAGG-3）)
and antisense ( 3CS29(5-GTCACCGGAGATCATAACAT-
G-3）) primers were designed according to the sequences
of fragments CS029 and CS029-4 and used for PCR amplifi-
cation as follows: 95 ℃ for 10 min, followed by 35 cycles of
94 ℃ for 30 s, 54 ℃ for 30 s, and 72 ℃ for 3 min. The PCR
products were confirmed by electrophoresis, and subcloned
into the EcoRⅤ restriction site of the pBluescriptⅡ SK+
vector, a positive clone was picked and sequenced in both
forward and reverse directions. The sequence of the insert
was compared with those in the DNA database (DDBJ/
EMBL/GenBank).
1.5 Northern blotting analysis
Total RNA was extracted from various tissues of
Jonathan apple as described above. A 779 bp digoxigenin
(DIG)-labeled RNA probe was prepared with DNA fragment
CS029-4, using the DIG RNA Labeling Mix (Boehringer
Mannheim, Germany) according to manufacturer’s
instructions. Ten mg of total RNA was denatured at 65 ℃
for 10 min, then electrophoresed on 1.0% agarose formal-
dehyde-denaturing gels, stained with ethidium bromideand
transferred to a Hybond N+ (Amersham Biosciences, New
Jersey, USA). The membrane was hybridized overnight in
DIG EASY HYB (Roche Molecular Biochemicals, Germany)
solution containing 66 ng/mL of the probe. The hybridiza-
tion was following Kotoda et al.（2002）. CDP-Star
(Boehringer Mannheim, Germany) visualization of the bands
was performed according to the manufacturer’s
instructions, and bands were analyzed with an LAS-1000
plus Luminescent Image Analysis System (Fuji Film, Tokyo,
Japan).
1.6 RT-PCR analysis
Total RNA from various tissues was prepared as de-
scribed above. cDNA was synthesized in 20 mL reaction
mixtures from 1 mg of total RNA using the RT-PCR HIGH
Kit (Toyobo Co., Ltd. Osaka, Japan) including 5×reverse
transcriptase buffer (4 mL), random primer (1 mL）, dNTPs
(2 mL）, RNase inhibitor (1 mL） and M-MLV reverse tran-
scriptase (2 mL）. The mixture was incubated at 30 ℃ for 10
min, 42 ℃ for 60 min, 99 ℃ for 5 min. PCR was performed
using 1 mg of the reverse transcription product as a template,
primers 5CS294 and 5RCS291, and the following amplifica-
tion conditions: an initial denaturing at 95 ℃for 12 min,
followed by amplification cycles of 94 ℃ for 1 min, 56 ℃ for
1 min and 72 ℃ for 2 min. PCR was performed for 30, 35 and
40 cycles, respectively. Five mL of the PCR products were
separated by electrophoresis in a 1.0% agarose gel and
CAO Qiu-Fen et al.: Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple 1093
Fig.1. Nucleotide and deduced amino acid sequences of Malus demestica ZINC FINGER PROTEIN 1 (MdZF1) cDNA. The zinc finger
motifs #1 and #2 are underlined. Bold letters represent Cystein (C) and Histidine (H). Primers site for PCR reaction are shown by the
arrows. Initiation and stop codons for translation are marked by boxed letters, respectively.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041094
Fig.1. (continued)
CAO Qiu-Fen et al.: Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple 1095
visualized by staining with 1 mg/mL ethidium bromide.
1.7 Southern blotting analysis
Genomic DNA was extracted by the method reported
previously (Wada et al., 2002). The Southern analysis was
carried out using 779 bp DIG-labeled CS029-4 PCR frag-
ment amplified between 5CS294 and 5RCS292 primers
(Fig.1). Genomic DNA was digested by restriction enzyme
EcoRⅠ, BamHⅠ or HindⅢ, respectively and blotted onto
nylon membrane (Hybond N+). Hybridization was performed
in DIG EASY HYB solution with 20 ng/mL probe overnight
at 42 ℃, the membrane washing and detection by chemilu-
minescence were the same as that in Northern blotting
analysis.
2 Results
2.1 Cloning of MdZF1
Based on BLAST searching in the DDBJ/EMBL/
GenBank database, the EST sequence from apple shoot
apex during the transition stage of vegetative to reproduc-
tive phase was examined, we identified a novel zinc finger
Fig.1. (continued)
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041096
gene with the 1.35 kb cDNA fragment by random sequenc-
ing analysis and BLAST comparison with those in the
GenBank. Based on the location of this motif, we hypoth-
esized that the putative protein should have another zinc
finger motif upstream at the 5 end of the sequence.
Accordingly, we used 5 RACE to clone 779 bp fragment
upstream of the original EST and found another zinc finger
motif within it. Based on these sequences, we designed
additional primers to allow PCR amplification of the full-
length 2.0 kb cDNA fragment, the sequence of the insert
was compared with those in the DNA database (DDBJ/
EMBL/GenBank) and found to be homologues to those of
zinc finger motifs of Arabidopsis, maize, potato, tomato
and rice, which we designated MdZF1 .
2.2 Sequence and structural analysis of MdZF1
The nucleotide and deduced amino acid sequences of
MdZF1 are shown in Fig.1. The full-length cDNA was 1 974
bp long, and contained a 246 bp 5-noncoding region, a 159
bp 3-noncoding region, and an ORF encoding a 522-amino-
acid polypeptide that contained a typical Cys2/His2 zinc
finger motif (underlined in Fig.1). Database comparison
using the MdZF1 sequence as bait is shown in Fig.2. The
Fig.2. Alignments between Malus demestica ZINC FINGER PROTEIN 1 (MdZF1) and zinc finger proteins from other crops. These
Alignments show the comparison of sequences including zinc finger motif #1 and #2 from each plants. Identical amino acids among the
sequences are shown in shadows. AT, Arabidopsis thaliana; LE, Lycopersicon esculentum (tomato); MD, Malus domestica (apple); OS,
Oryza sativa (ssp. japonica (rice)); ST, Solanum tuberosum (potato); ZM, Zea mays (maize). *,the skipped sequence, 130-155
(LVVPS SSAAA GSGGR QQQQQ GEAAP).
CAO Qiu-Fen et al.: Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple 1097
C-end and N-end portions of the MdZF1 amino acid se-
quence were not homologous to other known plant proteins.
However, the MdZF1 zinc finger region exhibited high ho-
mology with that of other zinc finger motif genes, including
genes found in Arabidopsis thaliana (AT) (88.05%
identity), rice (OS) (83.55% identity), tomato (LE) (85.43%
identity), potato (ST) (84.37% identity), and maize (ZM)
(80.50% identity) . Interestingly, when compared to MdZF1,
ID1 (ZM) contains an additional 25 amino acids between
the first and second zinc finger motif regions.
2.3 Expression of the MdZF1 gene
Total RNA extracted from various apple tissues or floral
organs was hybridized with CS029-4 as a probe (Fig.3). A
clear band was evident at 2.0 kb, indicating that an appro-
priately MdZF1-sized mRNA was expressed. MdZF1 was
expressed in apple roots, stems, leaves on the middle of
newly-growing shoot and shoot apices, but not in the flo-
ral organs, including the sepals, petals, stamen and pistils.
RT-PCR using MdZF1-specific primers (5CS294 and
5RCS291) produced a specific 864 bp band after 30 cycles
(Fig.4) in roots, stems, various leaf tissues including coty-
ledons and shoot apices but not in floral organs. After 35
cycles (Fig.4), MdZF1 expression was also identified in the
floral organs, but the expression level was lower compared
to that in the vegetative tissues. After 40 cycles (data not
shown), MdZF1 expression was identified in all the tested
tissues. This is consistent with the increased sensitivity of
RT-PCR, in that additional cycles were needed to identify
low level expression in floral organs that was not detected
by Northern blot.
2.4 Genomic analysis of MdZF1
Genomic DNA extracted from apple “Jonathan” was di-
gested with rarely cutting enzymes (HindⅢ, BamHⅠ,
EcoRⅠ ), and probed at high stringency with a DIG-la-
beled CS029-4 amplified PCR method. This CS029-4 probe
included zinc finger motif #1. The MdZF1 cDNA sequence
corresponding to CS029-4 probe had only one HindⅢ site.
Figure 5 shows that there was a strong and a weak bands
(7.9 and 4.3 kb, respectively) in EcoRⅠ lane, two strong
bands and a weak band (3.8, 1.9 and 5.6 kb, respectively) in
HindⅢ lane and a weak and a strong bands (10.0 and 7.4
Fig.3. Northern blotting analysis in different tissues. Equal
amount (10 mg) of total RNA isolated from flower organs, leaves,
and shoot apexes were subjected by Northern analysis. The up-
per picture shows extracted RNA patterns stained by ethidium
bromide and the right letters with arrows indicate each ribosomal
RNA. The lower picture shows Northern analysis hybridized
with CS29-4 RNA antisense DIG probe. 1, root from tissue cul-
tured seedling; 2, stem from tissue cultured seedling; 3, leaves; 4,
shoot apex; 5, sepals; 6, petals; 7, stamen; 8, pistils.
Fig.4. RT-PCR analysis of Malus demestica ZINC FINGER
PROTEIN 1 (MdZF1) in different tissues. The isolated RNA from
each tissue was synthesized to cDNA with reverse transcriptase,
and resultant cDNA was used as template for PCR reaction. The
primers were specific for MdZF1 zinc finger motifs (5CS294-
5RCS291). The PCR reaction cycles were different from top to
bottom (upper: 30, lower: 35 cycles). 1, root from tissue-cultured
seeding; 2, stem from tissue-cultured seeding; 3, leaves from
tissue-cultured seeding; 4, shoot apex; 5, sepals; 6, petals; 7,
stamen; 8, pistils; 9, post-germination cotyledons; 10, immature
leaves; 11, the middle of newly-growing shoots; 12, mature leaves;
M, marker (100 bp ladder).
Fig.5. DNA blotting analysis of apple genome. Genomic DNA
(5 mg) from apples was digested with BamHⅠ(B), HindⅢ(H)
and EcoRⅠ(E), and hybridized with a probe prepared from a
PCR-DIG CS029-4 (5CS294-5RCS291 primers).
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041098
kb, respectively) in BamHⅠ lane. The results indicated
that the MdZF1was a single copy gene and may have an-
other slightly homologous one.
3 Discussion
Zinc finger proteins participate in numerous physiologi-
cal activities in plants, including stress resistance and vari-
ous developmental processes (Sakamoto et al., 2000 ) . The
Arabidopsis thaliana STZ (salt tolerance zinc finger protein)
gene, which encodes a zinc finger protein, was expressed
in a salt-sensitive yeast strain and led to increased salt
tolerance, indicating that the zinc finger protein might di-
rectly participate in activating the expression of salt-toler-
ance genes (Lippuner et al., 1996). Similarly, typical C2H2
zinc finger proteins, as well as salt-tolerance-related genes
have been isolated from cotton and rice (Huang et al., 2002;
Wang et al., 2002). Some other zinc finger proteins play
important regulatory roles in plant growth.
We obtained a 2 kb cDNA fragment MdZF1, from a cDNA
library of apple stem apex tissue. It had two typical zinc
finger motifs. The zinc finger structural regions and the
spacer region between them had above 80% homology with
dicotyledonous Arabidopsis and potato, and monocotyle-
donous rice and maize, implying that they were evolved
from one ancestral gene. The maize ID1 and ID1-like gene
“P1” genes reported by Colasanti (1998) shared highly ho-
mologous zinc finger motifs with MdZF1 gene identified in
this study; these motifs were of the C2-H2 and C2-HC
patterns. The addition of 25 amino acids between the two
zinc finger regions in ID1, compared with those in other
plants, indicated that distinctive regions have been evolved
in the homologous genes in each species. ID1 mRNA was
expressed in immature leaves, but not in the shoot apex and
floral organs. ID1-deficient maize is unable to flower
normally; the mutant maintains a prolonged vegetative
growth period, and may produce floral inflorescences with
vegetative growth characteristics. These observations sug-
gest that ID1 products play as a transcription regulator for
the floral initiation or transport of various substances to
the shoot apex. The P1 gene, which is similar in structure to
ID1, was expressed in both mature and immature leaves
and shoot apex. The MdZF1 gene identified in this study
has zinc finger motifs similar to those encoded by the ID1,
P1 and AT genes from maize and Arabidopsis thaliana, as
well as the PCP1 gene from Solanum tuberosum (Kuhn
and Frommer, 1995); however, MdZF1 was expressed in
roots, stems, cotyledons, young leaves, mature leaves,
shoot apex, and various leaf tissues (leaves on middle of
newly growing shoot and leaves from seedlings). In
contrast, the ID1 and P1 genes were expressed in leaves,
shoot apices and stems, but not in roots. MdZF1 was not
detected in floral organs by Northern blotting analysis, but
after the RT-PCR cycles were increased, MdZF1 expression
was detected in them. Thus, the result implied that MdZF1
is expressed at very low levels in floral organs, and it mainly
functions in roots, stems, leaves and stem apex.
The genomic Southern analysis with CS029-4 probe,
which contained the zinc finger motif #1, showed MdZF1
was a single copy gene and might be another slight ho-
mologous gene in Jonathan genome (Fig.5). We had al-
ready checked “Fuji” genome with the same probe, the re-
sultant patterns were the same (data not shown). If the zinc
finger motif binds the specific DNA sites, the MdZF1 and
ID1 showed a similar regulatory manner because their zinc
finger parts showed high homology (Fig.2). In Zea mays,
the id1 mutant had apparent phenotypic effects on flower
transition. It means clearly that the ID1 gene plays a key
role in flower development. Then the MdZF1 from apple
was also expected the similar function for flowering. We
have made MdZF1-transformed Arabidopsis, which ex-
pressed excess MDZF1 under 35S promoter. But the result-
ant transformants were never distinguished from wild type
plants in flower formation or flowering time on photoperi-
ods (data not shown). It indicated that apple MdZF1 could
not be involved in flowering events of Arabidopsis. For
further research, the complementation experiment of Z. mays
id1 mutant with apple MdZF1 will be needed for investiga-
tion of the physiological role.
Based on the expression patterns of MdZF1 and the
analysis of ID1 and P1 functions, we propose that MdZF1
may be an apple growth-related regulatory gene. With re-
spect to the study of regulatory genes involved in apple
flowering, we have already isolated two homologues of
FLORICAULA/LEAFY, AFL1 and AFL2, from apple tree
flower buds (Wada et al., 2002). Kotoda et al. (2002) also
obtained the AP1-homogenous gene MdMADS5 from
apple. The function of MdZF1 and its relationship with
these flowering regulatory genes are needed to investigate
in future work.
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