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Isolation and Expression Analysis of CBF4 from Vitis Amurensis Associated with Stress

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Isolation and Expression Analysis of CBF4 from Vitis Amurensis Associated with Stress Vol.4, No.5, 224-229 (2013) Agricultural Sciences http://dx.doi.org/10.4236/as.2013.45032 Isolation and expression analysis of CBF4 from Vitis amurensis associated with stress Chang Dong1,2, Meng Zhang1, Zhiying Yu1, Junpeng Ren1, Yang Qin2, Bailin Wang2, Lizhen Xiao2, Zhen Zhang1*, Jianmin Tao1* 1College of Horticulture, Nanjing Agricultural University, Nanjing, China 2Department of Horticulture, Heilongjiang Academy of Agricultural Science, Harbin, China; *Corresponding Authors: [email protected], [email protected], [email protected] Received 21 March 2013; revised 26 April 2013; accepted 15 May 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 response to stress. The cis-elements involved in stress response were analyzed to elucidate the molecular me- The C-repeat Binding Factor/Dehydration Re- chanisms of gene expression in response to stress. The sponsive Element Binding factor (CBF/DREB) C-repeat/Dehydration Responsive Element (CRT/DRE) responsive pathway is involved in plant respon- with the core CCGAC sequence is a cis-acting element in se to abiotic stress. In this study, we report the the promoter that regulates gene expression in response isolation of VaCBF4 (a complete cDNA) and its to cold and drought in Arabidopsis [3,4]. A similar motif promoter from Vitis amurensis through rapid was identified as the Low-Temperature Responsive Ele- amplification of cDNA ends and genome walking ment (LTRE) in cold-inducible genes [5]. techniques, respectively. The CBF4 transcript ac- At present, the most understood genetic system with cumulation of V. amurensis increased under an important role in cold acclimation is the Arabidopsis cold, salinity, and abscisic acid (ABA) and sali- CBF (CRT/DRE-binding transcription factors) cold re- cylic acid (SA) treatments, whereas that of Vitis sponse pathway [6]. Exposing Arabidopsis plants to low vinifera showed a different change. The tran- temperature or drought results in the rapid induction of a script levels of VaCBF4 in the roots, stems, lea- small family of transcriptional factors known as CBF1, ves, and petioles under cold, salinity, and ABA CBF2, CBF3(or as DREB1B, DREB1C, and DREB1A, and SA treatments were up-regulated in a time- respectively) [7], and CBF4 [8]. CBF/DREB-type pro- dependent manner. The presence of the cis-ele- teins belong to the AP2/ERF (APETALA 2/Ethylene Re- ments MBC, ABRE, and as-2-box in the VaCBF4 promoter further confirmed that this promoter is sponse Factors) DNA-binding protein family. Moreover, a component of the CBF transduction pathway, the constitutively over-expression of CBF genes induces which is involved in plant response to multis- the expression of downstream genes and improves the tress. freezing and salt tolerance of transgenic plants [9]. Despite the high conservation of CBF residues in Keywords: Vitis amurensis; CBF4; Stress; plants, some structural and regulatory differences were Transcript Accumulation observed in the CBF cold response pathway among plant species. For example, the CBF regulon of dwarf apple was found to be induced more than that of Arabidopsis 1. INTRODUCTION [10]. A previous work on blueberry showed that CBF Low temperature, high salinity, and drought are envi- genes from different species have dramatically different ronmental factors that limit the geographical distribution levels of induction under low temperature [11]. Although and affect the growth, development, and productivity of recent breakthroughs have increased the knowledge on plants. Plants respond and adapt to these stresses at the the molecular basis of the CBF cold pathway in herba- molecular, physiological, and biochemical levels. The ceous species, little is known in woody plants. To date, expression levels of various genes in plants are activated data on CBF genes from perennial plants include func- by these stresses [1,2]. The products of these genes tional analysis of CBF from cherry [12], eucalyptus [13], regulate the gene expression and signal transduction in blueberry [11], and apple [10]. Copyright © 2013 SciRes. OPEN ACCESS C. Dong et al. / Agricultural Sciences 4 (2013) 224-229 225 Grape is one of the most important fruit crops in the V, DraI, SacI, and StuI). After purification, the restric- world, and its production has been expanding as more tion fragments were ligated with Genome Walker adap- varieties and better quality wines become available. tors (Ad1 and Ad2, Table 1). The polymerase chain re- However, low temperature limits the geographical dis- action (PCR) products were amplified on each restriction tribution and affects the growth, development, and pro- fragment set using the primers C4-R1 and ada1 for the ductivity of this crop. Vitis amurensis is a freezing-tole- first step PCR and C4-R2 and ada 2 for the second step rant wild grape species native to Northern China. This PCR (Table 1). The PCR products were cloned and se- species can survive below 40˚C [14]. V. a mure n s is is one quenced by the pMD19-T vector. of the most widely used species for rootstock and wine- making in grape cultivation. However, little is known 2.2. Sequence Analysis about the freezing tolerance of V. amurensis at the mo- Homology searches were performed using BLAST lecular level. with default parameters at NCBI In this study, the VaCBF4 gene was isolated from V. (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequ- amurensis. We also compared VaCBF4 gene expression ence alignment of CBFs was performed using Crustal in the leaves of cold-tolerant and cold-sensitive grapes 1.83, and shading was carried out using DNAMAN with that in tissues of V. amurensis at different times of software (Version 5.2.2, Lynnon Biosoft). The CBF pro- low temperature, high salinity, and abscisic acid (ABA) tein sequences necessary for constructing a phylogenetic and salicylic acid (SA) treatments, and then confirmed its tree were retrieved from the GenBank database and de- transcript patterns under stress. picted using Crustal 1.83. Phylogenetic analysis was based on the neighbor-joining method using Mega 4.1 2. MATERIAL AND TREATMENTS software with 1000 bootstrap replicates and substitution Sixty-day-old in vitro samples of wild V. amurensis model of Poisson correction. Promoter sequence analysis and Vitis vinifera’ Manicure Finger’ grown in Murashige was performed using PLACE (http://www.dna.affrc.go.jp) and Skoog (MS) medium under a 16 h light/8 h dark re- and Plant CARE. gime at 25˚C were subjected to the following treatments for stress-responsive gene expression: cold stress by 2.3. Real-time Reverse Transcription transferring to 4˚C, salinity stress by supplying 200 mM Quantitative-PCR (RT-qPCR) NaCl, and hormonal treatments by supplying ABA (10 Total RNA from different treatments and tissues were μM) or SA (5 μM) directly to the plants at different digested with RNase-free DNAase I (TaKaRa, Japan) to times. remove genomic DNA contamination. cDNA fragments were synthesized as aforementioned. Transcript level was 2.1. Isolation of VaCBF4 determined on an ABI PRISM 7300 (Applied Biosystems, Total RNA from non-stressed or cold-stressed wild Foster City, CA, USA). For each reaction, 20 ng of samples in vitro was extracted using the CTAB method cDNA was used with SYBR Premix Ex Taq mix (Ta- (Lin et al., 2008) and converted to cDNA using Primer KaRa, Japan), including primers C4rt1 and C4rt2 for the Mix according to the instructions of Rever Tra Ace RT CBF4 gene. All experiments were run in triplicates with Kit (Toyobo, Japan). The partial coding sequence of the different synthesizedc DNA fragments. A positive control CBF4 genes was amplified using gene-specific primers, was provided through a parallel analysis using the grape C4-F and C4-R (Table 1). The primers were designed malate dehydrogenase gene as an internal reference [15], based on the genome of Vitis (http://www.vitisgenome.it). and was calculated relative to a calibrator using the for- −△△Ct The 3’ regions of the corresponding cDNA fragments mula 2 . were obtained by 3’ rapid amplification of cDNA ends (RACE) using the cDNA amplification kit (Clontech, 3. RESULTS CA, USA) with the gene-specific primers 16326 and 3.1. Identification of the VaCBF4 Gene from C4-rF2 for the first step and 16324 and C4-rF3 for the V. amurensis second step (Table 1). Amplification was carried out under the following conditions: 94˚C for 4 min; 36 cy- We constructed a cDNA admixture template from the cles of 94˚C for 30 s, 60˚C for 45 s, 64˚C for 60 s, and cold-treated and non-treated in vitro leaves to identify 72˚C for 60 s; and a final extension at 72˚C for 10 min. the gene related to low temperature. A CBF4-like partial The isolation of the promoter sequence was carried out sequence was found, suggesting that the corresponding using a Universal Genome Walker kit (UGWK, Clontech, gene was probably induced by low temperature. Given USA) on 10 ng of V. amurensis genomic DNA digested that this sequence contained the intact 5’ end of the Open by four blunt-end-generating restriction enzymes (EcoR Reading Frame (ORF) of a putative CBF4 gene, we can Copyright © 2013 SciRes. OPEN ACCESS 226 C. Dong et al. / Agricultural Sciences 4 (2013) 224-229 Table 1. Primers used in this study. Primer Sequence (5’ – 3’) C4-F CGACACTTCAGTCTTCACCGTT C4-R ACTGAACTCCCACCTACCCTCT C4-rF2 GGAGGAAGAAGTTTCGGGAGAC 16326 GGTGGTAGAGCTCGCAGGACTGCAGCTGACTG C4-rF3 CAAGATGAGAGGAGGGAAGAGAGTGA 16324 AGAGCTCGCAGGACTGCAGCTGACTGACTAC Ad1 GATTTACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT Ad2 PO4-ACCAGCCC-NH2 C4-R1 CCCACCTACCCTCTGTTACTTT ada1 GATTTACGACTCACTATAGGGC C4-R2 TCACTCTCTTCCCTCCTCTCATCTTG ada2 ACTATAGGGCACGCGTGGT C4rt1 GACACTTCAGCCTTCACCG C4rt2 CCTCGCATACCCACTTCC VaCBF4 MNTTSPPYSDPHPLVCNWDSLNLPDSDGGSEELMLASTHPKKRAGRKKFRETRHP 55 obtain the rest of the sequence by 3’ RACE assay.
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