Abstract:Dehydration responsive element binding protein (DREB) belongs to the APETALA2/ethylene response factor (AP2/ERF) family in plants, which is a large group of plant-specific transcription factors. The DREBs widely participate in various abiotic stresses, such as drought, salt, heat, and cold. In order to explore the function of DREB in Malus zumi, a new gene named MzDREB2A (GenBank No. MH992511) was isolated and cloned using the method of rapid amplification of cDNA ends (RACE). The open reading frame of MzDREB2 was 1 197 bp without intron structure, encoding 398 amino acids and containing an AP2 domain with the molecular weight 43.9 kD and the isoelectric point 4.93. Bioinformatic analysis showed that MzDREB2A was a hydrophilic protein and had no transmembrane region, and the secondary structure contained 7.54% α-helix, 4.52% β-sheet, and 87.94% random coils. Homology analysis and phylogenetic analysis showed that MzDREB2A protein had the highest sequence similarity (about 99%) and closest genetic relationship to Malus sieversii. qRT-PCR analysis showed that MzDREB2A exhibited the highest expression level in roots followed by blades, and the lowest expression level in stems. The expression level of MzDREB2A sharply increased under NaCl or PEG-6000 treatment, and MzDREB2A expression reached the maximum when NaCl concentration was 150 mmol/L and PEG-6000 concentration was 20%, respectively. This study successfully cloned the MzDREB2A of AP2/ERF family in Malus zumi, which might provide a reference for further study on the function of MzDREB2A and the molecular mechanism of stress resistance in Malus zumi.
[1] 沈兵琪, 高晓宇, 王大玮, 等. 2017. 苹果DREB转录因子家族全基因组鉴定与分析[J]. 西北植物学报, 37(3): 460-469. (Shen B Q, Gao X Y, Wang D W, et al.2017. Genome-wide identification and analysis of the DREB transcriotion factor gene family in apple[J]. Acta Botanica Boreali-Occidentalia Sinica, 37(3): 460-469.) [2] 柴慈江, 孙世海, 王玉, 等. 2011. 珠美海棠叶片离体培养与植株再生[J]. 果树学报, 28(1): 124-128. (Chai C J, Sun S H, Wang Y, et al.2011. Plant regeneration from in vitro cultured leaves of Malus zumi[J]. Journal of Fruit Science, 28(1): 124-128) [3] Agarwal P K, Agarwal P, Reddy M K, et al.2006. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants[J]. Plant Cell Reports, 2006, 25(12): 1263-1274. [4] Arroyo-Herrera A, Figueroa-Yáñez L, Castaño E, et al.2016. A novel dreb2 -type gene from Carica papaya, confers tolerance under abiotic stress[J]. Plant Cell Tissue & Organ Culture, 125(1): 1-15. [5] Egawa C, Kobayashi F, Ishibashi M, et al.2006. Differential regulation of transcript accumulation and alternative splicing of a DREB2 homolog under abiotic stress conditions in common wheat[J]. Genes & Genetic Systems, 81(2): 77-91. [6] Elliott R C, Betzner A S, Huttner E, et al.1996. Aintegumenta, an APETALA2-Like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth[J]. Plant Cell, 8(2): 155-168. [7] Jiang C, Iu B, Singh J.1996. Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus[J]. Plant Molecular Biology, 30(3): 679-684. [8] Liu Q, Shinozaki K.1998. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis[J]. Plant Cell, 10(8): 1391-406. [9] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[J]. Methods, 25(4): 402-408. [10] Malhotra P K, Mittal A, Chopra R, et al.2016. Genetic augmentation of sugarcane with stress-inducible DREB1A, transcription factor using biolistic approach[J]. Sugar Tech, 19(5): 1-7. [11] Matsukura S, Mizoi J, Yoshida T, et al.2010. Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes[J]. Molecular Genetics & Genomics, 283(2): 185-196. [12] Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K.2012. AP2/ERF family transcription factors in plant abiotic stress responses[J]. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1819(2): 86-96. [13] Nakashima K, Shinwari Z K, Sakuma Y, et al.2000. Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression[J]. Plant Molecular Biology, 42(4): 657-665. [14] Nakashima K, Yamaguchi-Shinozaki K.2009. Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses[J]. Plant Physiology 149(1): 88-95. [15] Nordin K, Vahala T, Palva E T.1993. Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh[J]. Plant Molecular Biology, 21(4): 641-653. [16] Pagter M, Arora R.2013. Winter survival and deacclimation of perennials under warming climate: Physiological perspectives[J]. Physiologia Plantarum, 147(1): 75-87. [17] Sakuma Y, Liu Q, Dubouzet J G, et al.2002. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression[J]. Biochemical & Biophysical Research Communications, 290(3): 998-1009. [18] Sakuma Y, Maruyama K, Osakabe Y, et al.2006. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression[J]. Plant Cell, 18(5): 1292-1309. [19] Yamaguchi-Shinozaki K, Shinozaki K.1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress[J]. Plant Cell, 6(2): 251-264. [20] Zhao K, Shen X J, Yuan H Z, et al.2013. Isolation and characterization of dehydration-responsive element-binding factor 2C (MsDREB2C) from Malus sieversii Roem[J]. Plant & and Cell Physiology, 54(9): 1415-1430. [21] Zhao X J, Lei H J, Zhao K, et al.2012. Isolation and characterization of a dehydration responsive element binding factor MsDREBA5 in Malus sieversii Roem[J]. Scientia Horticulturae, 142(4): 212-220.