Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple

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1 Acta Botanica Sinica 2004, 46 (9): Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple CAO Qiu-Fen 1*, Masato WADA 2, MENG Yu-Ping 1, SUN Yi 1, CUI Gui-Mei 1 (1. Agricultural Biotechnology Research Center of Shanxi Province, Taiyuan , China; 2. Department of Apple Research, National Institute of Fruit Tree Science, National Agricultural Research Organization Shimokuriyagawa, Morioka, Iwate , 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 TF A 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 protein associated with the transition to flowering, suggesting that the ID1 gene product functions as a transcription regulator of floral development. The phenotype of id1 mutant and the expression analysis of ID1 suggested that the gene product act as a floral initiation signal like the florigen hypothesis. The shoot apex of higher plants contains an undifferentiated 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 responsible 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 different 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 Received 27 Oct Accepted 9 Mar Supported by the Funds from the Science and Technology Commission of Shanxi Province (021034) and Returning Overseas Student Affair Office of Shanxi Province ( ). * Author for correspondence. Tel +86 (0) Fax +86 (0) <caoqiufeng@yahoo.com.cn>.

2 ( 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, postgermination 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 EcoR, Not, and BamH adaptors for constructing the cdna library. The ligated cdnas were cloned into the EcoR restriction site of pbluescript SK + vectors (Stratagene, La Jolla, California, USA. The recombinant plasmids were transfected into Escherichia coli strain DH5α, 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 software (Software Development Co., Tokyo, Japan). The sequences 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 ' 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 ( 5 ' - GAAGATGGTTCCACAGTCAC-3') and 5RCS292 (5'- GCACTTGTCACATTTCCAGG-3') were designed according 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 polymerase (TaKaRa Biomedicals, Shiga, Japan). Primers C1 and 5RCS291 were used in the first amplification reaction to amplify fragments from 1 µg 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 annealing temperature of 54. The PCR products were subcloned into the EcoR restriction site of pbluescript SK + vectors, and transformed into E. coli strain DH5α. The clones with recombinant plasmids were detected, the plasmids were prepared and sequenced the same as described above. A 779 bp cdna, containing sequences of another zinc finger motif upstream of CS029, was obtained and designated CS 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 amplification 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 EcoR restriction site of the pbluescript 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 µg of total RNA was denatured at 65 for 10 min, then electrophoresed on 1.0% agarose formaldehyde-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 hybridization was following Kotoda et al 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 described above. cdna was synthesized in 20 µl reaction mixtures from 1 µg of total RNA using the RT-PCR HIGH Kit (Toyobo Co., Ltd. Osaka, Japan) including 5 reverse transcriptase buffer (4 µl), random primer (1 µl, dntps (2 µl, RNase inhibitor (1 µl and M-MLV reverse transcriptase (2 µl. The mixture was incubated at 30 for 10 min, 42 for 60 min, 99 for 5 min. PCR was performed using 1 µg of the reverse transcription product as a template, primers 5CS294 and 5RCS291, and the following amplification 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 µl of the PCR products were separated by electrophoresis in a 1.0% agarose gel and

3 CAO Qiu-Fen et al.: Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple 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.

4 Fig.1. (continued)

5 CAO Qiu-Fen et al.: Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple Fig.1. (continued) visualized by staining with 1 µg/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 fragment amplified between 5CS294 and 5RCS292 primers (Fig.1). Genomic DNA was digested by restriction enzyme EcoR, BamH or Hind, 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 chemiluminescence 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 reproductive phase was examined, we identified a novel zinc finger

6 gene with the 1.35 kb cdna fragment by random sequencing analysis and BLAST comparison with those in the GenBank. Based on the location of this motif, we hypothesized 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 fulllength 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 MdZF 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 bp long, and contained a 246 bp 5'-noncoding region, a 159 bp 3'-noncoding region, and an ORF encoding a 522-aminoacid 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, (LVVPS SSAAA GSGGR QQQQQ GEAAP).

7 CAO Qiu-Fen et al.: Molecular Cloning and Expression Analysis of a Zinc Finger Protein Gene in Apple C-end and N-end portions of the MdZF1 amino acid sequence were not homologous to other known plant proteins. However, the MdZF1 zinc finger region exhibited high homology 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 appropriately 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 floral 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 cotyledons 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 digested with rarely cutting enzymes (Hind, BamH, 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). EcoR ), and probed at high stringency with a DIG-labeled 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 Hind site. Figure 5 shows that there was a strong and a weak bands (7.9 and 4.3 kb, respectively) in EcoR lane, two strong bands and a weak band (3.8, 1.9 and 5.6 kb, respectively) in Hind lane and a weak and a strong bands (10.0 and 7.4 Fig.3. Northern blotting analysis in different tissues. Equal amount (10 µg) of total RNA isolated from flower organs, leaves, and shoot apexes were subjected by Northern analysis. The upper 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 cultured seedling; 2, stem from tissue cultured seedling; 3, leaves; 4, shoot apex; 5, sepals; 6, petals; 7, stamen; 8, pistils. Fig.5. DNA blotting analysis of apple genome. Genomic DNA (5 µg) from apples was digested with BamH (B), Hind (H) and EcoR (E), and hybridized with a probe prepared from a PCR-DIG CS029-4 (5CS294-5RCS291 primers).

8 kb, respectively) in BamH lane. The results indicated that the MdZF1was a single copy gene and may have another slightly homologous one. 3 Discussion Zinc finger proteins participate in numerous physiological activities in plants, including stress resistance and various 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 directly participate in activating the expression of salt-tolerance 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 monocotyledonous 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 homologous 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 suggest 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 homologous gene in Jonathan genome (Fig.5). We had already checked Fuji genome with the same probe, the resultant 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 expressed excess MDZF1 under 35S promoter. But the resultant transformants were never distinguished from wild type plants in flower formation or flowering time on photoperiods (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 investigation 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 respect 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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