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Isolation and Expression Characterization of CBF2 in Vitis Amurensis with Stress

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Isolation and Expression Characterization of CBF2 in Vitis Amurensis with Stress Vol.4, No.9, 466-472 (2013) Agricultural Sciences http://dx.doi.org/10.4236/as.2013.49062 Isolation and expression characterization of CBF2 in vitis amurensis with stress Chang Dong1,2, Jianmin Tao1, Meng Zhang1, Yang Qin2, Zhiying Yu1, Bailin Wang2, Binhua Cai1, Zhen Zhang1* 1College of Horticulture, Nanjing Agricultural University, Nanjing, China; *Corresponding Author: [email protected], [email protected], [email protected] 2Department of Horticulture, Heilongjiang Academy of Agricultural Science, Harbin, China Received 5 June 2013; revised 5 July 2013; accepted 1 August 2013 Copyright © 2013 Chang Dong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT years, the C-repeat element-binding factor (CBF)/dehy- dration responsive element binding (DREB) cold re- The transcription factor VaCBF2, which interacts sponse pathway is the most recognized freezing tolerance with C-repeat/DRE and its promoter, was iso- pathway in plants [2]. All CBF proteins specifically bind lated from Vitis amurensis. The VaCBF2 amino to the C-repeat element (CRT)/dehydration responsive ele- acid sequence contained a conserved AP2 do- ment (DRE) in the promoter region of downstream genes main of 56 amino acids and a potential nuclear [3,4]. These proteins regulate their downstream gene localization sequence. The sequence of VaCBF2 expression and enhance the tolerance of plants to low showed a high level of homology with other temperature, drought, and high salinity. Thus, CBF regu- CBF2 family members. Phylogenetic analysis showed that the amino acid sequences may be lation has a fundamental function in cold acclimation [5]. CBF2 proteins with evolutionary relationship. Arabidopsis CBF1, CBF2, and CBF3 are major tran- Quantitative reverse-transcription polymerase scriptional factors affecting cold-inducible gene expres- chain reaction analysis indicated that the ex- sion [6,7]. CBF4 from Arabidopsis is weakly induced by pression of VaCBF2 gene in tissues (roots, stems, cold stress [8], whereas DREB2A is induced by drought leaves, and petioles) was induced by low tem- and salinity [9]. These transcript profiles suggest that perature, high salinity, and application of ab- CBFs have different expression levels in plants and that a scisic acid and salicylic acid in a time-depen- degree of cross-talk exists between these pathways [10]. dent manner but to different extents in the cold- Furthermore, CBFs widely exist in herbaceous plants hardy V. amurensis and the less cold-hardy Vitis that are responsive to cold [11]. Puhakainen et al. [12] vinifera. The presence of cis-elements such as demonstrated that the CBF pathway also exists in woody MYC and ABRE in VaCBF2 promoter further con- trees. Since then, CBF orthologs have been reported in firmed that this promoter was a component of various woody plants, including Vitaceae [13], poplar the CBF transduction pathway involved in plant [14], blueberry [15], peach [5], and apple [4]. Among response to multiple stresses. woody species, a positive correlation also exists between the cold tolerance and CBF transcript level of woody Keywords: Vitis amurensis; Stress; CBF2; species [4,5,14,16]. Therefore, the CBF cold pathway Expression appears to be conserved in woody plants. Vitis amurensis is one of the most widely used species for freeze-tolerant rootstock and winemaking in grape 1. INTRODUCTION cultivation. In this study, the VaCBF2 gene was isolated Abiotic stresses such as low temperature, drought, and from V. amurensis. We also compared CBF2 gene expres- salinity can adversely affect crop growth and develop- sion in the leaves of cold-tolerant wild V. amurensis and ment. Thus, improving stress tolerance in breeding is cold-sensitive Vitis vinifera “Manicure Finger” at different important. Plants respond and acclimate to environmen- times of cold treatment. Results confirmed the V. amu- tal stresses by activating network events from stress per- rensis transcript patterns in roots, stems, leaves, and peti- ception to effector gene expression [1]. Over the past oles at different times of low temperature, high salinity, Copyright © 2013 SciRes. OPEN ACCESS C. Dong et al. / Agricultural Sciences 4 (2013) 466-472 467 and abscisic acid (ABA) and salicylic acid (SA) treatments. qRT-PCR was performed in 20 mL volumes containing 10 mL of SYBR Premix Ex Taq mix (TaKaRa), 0.2 mM 2. MATERIAL AND TREATMENTS forward primer (5’-TGAGAACAAATGGGTGAGTG- Sixty-day-old in vitro samples of wild V. amurensis 3’), 0.2 mM reverse primer (5’-TGATGGAGGTTGCT- and V. vinifera “Manicure Finger” grown in Murashige GAAAA-3’), and 1 mL of diluted (1:10 v/v) cDNA. The and Skoog (MS) medium under a 16 h light/8 h dark re- PCR regime consisted of denaturation at 94˚C for 4 min, gime at 25˚C were subjected to the following treatments followed by 40 cycles of 95˚C for 10 s, 60˚C for 20 s, for stress-responsive gene expression: cold stress by and 72˚C for 20 s. Melting curve analysis was performed transferring to 4˚C, salinity stress by supplying 200 mM from 80˚C to 95˚C at 0.5˚C intervals. VaCBF2 transcript NaCl, and hormonal treatments by directly supplying levels were normalized with grape malate dehydrogenase ABA (10 μM) or SA (5 μM) to plants at different times. gene as an internal control using the forward primer 5’-GCATCTGTGGTTCTTGCAGG-3’ and reverse pri- 2.1. Isolation of the VaCBF2 Gene mer 5’-CCCTTTGAGTCCACAAGCCAA-3’. The data of 0.5 h of V. vinifera in leaves at 4˚C were used as basis Sixty-day-old in vitro samples of wild V. amurensis for relative comparison. Relative expression was calcu- were grown in MS medium under a 16 h/8 h dark regime lated relative to a calibrator using the formula 2−ΔΔCt. Two at 25˚C. Total RNA was extracted from non-stressed or independent replicates were performed per experiment. cold-stressed plants in vitro using the Lagonigro’s me- thod [17]. First-strand cDNA was synthesized using Pri- 3. RESULTS mer Mix according to the instructions of ReverTra Ace RT Kit (TOYOBO, Japan). The complete coding se- 3.1. Identification of VaCBF2 Gene from V. quence was amplified using the gene-specific primers amurensis C2F (5’-TCATCAACATCCTCTCCTTCTCG-3’) and C2R The sequence of the V. amurensis cDNA clone includ- (5’-GCTAAAAGTGTGTATGGCAGTGA-3’). The prim- ed one open reading frame (ORF). Compared with the ers were designed based on the genome of Vitis GenBank database, this sequence resulted in an iden- (http://www.vitisgenome.it). Amplification was carried tical coding sequence to the CBF2 cDNA from V. v in if- out under the following conditions: 94˚C for 4 min; 36 era and V. r ip a r ia . This result clearly indicated that the cycles of 94˚C for 30 s, 60˚C for 45 s, and 72˚C for 60 s; gene in V. amurensis did not have introns interrupting and a final extension at 72˚C for 10 min. The polymerase its ORF. The coding regions encoded polypeptides of chain reaction (PCR) products were cloned and sequenc- 250 amino acids with a molecular mass of 27.55 kD and ed by pMD19-T vector. Sequence alignments were con- a theoretical isoelectric point of 10.23. The amino acid ducted using DNAMAN software (Version 5.2.2, Lynnon alignment of the sequence revealed that 97% and 96% Biosoft). of the residues were identical to the CBF2 of V. riparia 2.2. Promoter Isolation of VaCBF2 and V. vinifera, respectively. Moreover, the degrees of similarity to the CBF2 of Parthenocissus inserta and The isolation of the promoter sequence was carried out Eucalyptus grandis were high (94% and 73%, respec- using a Universal Genome Walker Kit (Clontech, USA) tively). Sequence alignment against various CBF pro- on 10 ng of V. amurensis genomic DNA digested by four teins suggested that this cDNA encoded a CBF-type pro- blunt-end-generating restriction enzymes (EcoRI, DraI, tein in V. a mure n s is (Figure 1). Therefore, the isolated SacI, and StuI). After purification, the restriction frag- cDNA was named VaCBF2. In other plants, basic resi- ments were ligated with Genome Walker adaptors. PCR dues similar to CBF2 were included in their N-terminal was amplified on each restriction fragment set using the regions. These residues potentially represented an un- primers C2-R1 (5’-GCTAAAAGTGTGTATGGCAG- clear localization signal (NLS) and putative APETALA TGA-3’) and C2-R2 (5’-CTTCACTCACCCATTTGT- 2/ethylene response factor (AP2/ERF) DNA-binding do- TCTCATT-3’) for first- and second-step PCRs, respec- main. VaCBF2 also contained acidic C-terminal frag- tively. Finally, the PCR products were cloned into pMD ments that can act as the transcriptional activation do- 19-T simple vector and sequenced. The sequences were main, but the LWSY signature was modified from LW- analyzed online by Plant CARE. SY to LWNHDFL in the grape protein. In addition, po- tential recognition motifs for CBF protein were de- 2.3. Expression Analysis of VaCBF2 by tected, such as DSAWR, Domain III, and Domain IV Real-Time Quantitative Reverse (Figure 1). These results indicated that VaCBF2 from V. Transcription (qRT)-PCR amurensis was a CBF2 or tholog of other plants but with RNA and cDNA were obtained as described above. unique features. Copyright © 2013 SciRes. OPEN ACCESS 468 C. Dong et al. / Agricultural Sciences 4 (2013) 466-472 Figure 1. Multiple alignment of VaCBF2 with the CBF2 proteins of Vitaceae, Parthenocissus, and Arabidopsis. The amino acid sequences are from NCBI [AtCBF2 (ABV27118), PtCBF2 (CBF94893), VrCBF (AAR28674), and VvCBF (AAR28677)]. The nuclear localization sig- nal, AP2, DSAWR, and LWNH domains are indicated by bars. 3.2.
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