盐地碱蓬2个DREB1/CBF基因的克隆与表达调控分析

doi:10.3864/j.issn.0578-1752.2016.12.017

摘要/Abstract

摘要： 【目的】从盐地碱蓬（Suaeda salsa L.）中克隆2个DREB1/CBF，分析其序列特征、编码蛋白的亚细胞定位和转录激活活性，以及在非生物胁迫下的表达模式，为进一步研究盐地碱蓬的抗逆机制提供依据。【方法】利用同源克隆法获得盐地碱蓬2个DREB1/CBF片段，采用RACE技术克隆获得cDNA全长序列，分别命名为SsCBF1和SsCBF2。运用生物信息学软件对2个SsCBF及其编码蛋白进行分析，并将它们分别与GFP融合构建植物表达载体，通过基因枪转化法导入洋葱表皮细胞进行瞬时表达，观察它们编码蛋白的亚细胞定位。利用酵母单杂交系统研究2个SsCBF与DRE/CRT顺式作用元件的结合特异性和转录激活活性。采用Real time-PCR研究2个SsCBF在低温、NaCl、PEG以及ABA处理下的表达模式。【结果】 SsCBF1编码一个225个氨基酸的蛋白，预测分子量为25.4 kD，理论等电点为4.84。SsCBF2编码一个由260个氨基酸组成的蛋白，预测分子量为28.6 kD，理论等电点为5.05。SsCBF1和SsCBF2均含有1个典型的AP2/ERF保守结构域，在核苷酸和氨基酸水平上分别具有53.5%和45.4%的同源性，而2个基因的AP2/ERF结构域在核苷酸和氨基酸水平上分别具有76.2%和87.3%的相似性。SsCBF1、SsCBF2归属于DREB亚组的A-1组，定位于细胞核内，均能与DRE/CRT顺式作用元件特异性结合，并激活下游报告基因的表达。低温、干旱、高盐和ABA能够诱导SsCBF1表达，而SsCBF2在低温处理下表达量上调，但对干旱、高盐和ABA处理不响应。【结论】SsCBF1和SsCBF2是盐地碱蓬的2个胁迫应答转录因子。在盐地碱蓬中，SsCBF1通过依赖ABA途径参与对高盐、干旱和低温等非生物胁迫的应激调控，而SsCBF2则通过不依赖于ABA途径对低温胁迫产生响应。

关键词: 盐地碱蓬, DREB1/CBF转录因子, 亚细胞定位, 酵母单杂交, 荧光定量PCR

Abstract:

【Objective】To provide basic information for understanding the resistant mechanism under abiotic stresses, two DREB1/CBF genes were cloned from Suaeda salsa L. At the same time, the sequence characteristics, subcellular localization and transcriptional activities of the two predicted DREB1/CBF proteins and their expression alteration in response to abiotic stress were analyzed. 【Method】The fragments of two DREB1/CBF genes were obtained by using the technique of homologous cloning. The full-length of cDNA sequences of the two DREB1/CBF genes were isolated by the method of rapid-amplification of cDNA ends (RACE), and they were named as SsCBF1 and SsCBF2, respectively. The structures and functions of the two proteins encoded by SsCBFswere predicted by the bioinformatics software. In order to detect the subcellular localization of the two proteins, the coding sequences of the two SsCBF genes were fused, respectively, downstream to the GFP sequence to obtain two expression vectors and they were separately transferred into onion epidermal cells by the biolistic method. The binding specificity of SsCBF1 and SsCBF2 to DRE/CRT cis-acting element and their transcriptional activities were investigated by using a yeast one-hybrid system. The expression of the two SsCBF genesin response to low temperature, NaCl，PEG and ABA were assessed by the real-time quantitative PCR.【Result】 SsCBF1 encoded a peptide of 225 amino acid residues with a predicted molecular mass of 25.4 kD and a pI of 4.84. SsCBF2 encoded a predicted protein of 260 amino acid residues with a predicted molecular mass of 28.6 kD and a calculated pI of 5.05. SsCBF1and SsCBF2both contained a typical AP2/ ERF domain andshared 53.5% and 45.4% identity at the levels of coding nucleotides and amino acids. However, the AP2/ ERF domains of the two SsCBF genes were highly similar and shared 76.2% and 87.3% identity at the levels of nucleotides and amino acids, respectively. SsCBF1 and SsCBF2 were classified into A-1 subgroup of the DREB subfamily and both localized to the nucleus. The two proteins were able to specifically bind to the DRE/CRT sequence and activate the expression of the down-stream HIS reporter gene in yeast. Low temperature, drought, high salt and ABA could induce the expression of SsCBF1. However, the expression of SsCBF2 could only be induced significantly by low temperature, not by drought, high salt and ABA. 【Conclusion】The SsCBF1 and SsCBF2 are two stress-responsive transcription factors of S. salsa. In S. salsa, SsCBF1 is involved in the stress responses of cold, high-salt and drought through ABA-dependent pathways and SsCBF2 is responsive to cold stress through ABA-independent pathway.

Key words: Suaeda salsa L., DREB1/CBF transcription factors, subcellular localization, yeast one-hybrid, quantitative real-time PCR

孙晓波，苏家乐，贾新平，梁丽建，肖政，邓衍明. 盐地碱蓬2个DREB1/CBF基因的克隆与表达调控分析[J]. 中国农业科学, 2016, 49(12): 2418-2429.

SUN Xiao-bo, SU Jia-le, JIA Xin-ping, LIANG Li-jian, XIAO Zheng, DENG Yan-ming. Cloning and Expression Analysis of Two DREB1/CBF Genes in Suaeda salsa L.[J]. Scientia Agricultura Sinica, 2016, 49(12): 2418-2429.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.12.017

https://www.chinaagrisci.com/CN/Y2016/V49/I12/2418

参考文献

[1] Song J, Fan H, Zhao Y Y, Jia Y H, Du X H, Wang B S. Effect of salinity on germination, seedling emergence, seedling growth and ion accumulation of a euhalophyte Suaeda salsa in an intertidal zone and on saline inland. Aquatic Botany, 2008,88(4): 331-337.

[2] 黄玮, 李志刚, 乔海龙, 李存桢, 刘小京. 早盐互作对盐地碱蓬生长及其渗透调节物质的影响. 中国农业生态学报, 2008, 16(1): 173-178.

Huang W, Li Z G, Qiao H L, Li C Z, Liu X J. Interactive effect of sodium chloride and drought on growth and osmotica of Suaeda salsa. Chinese Journal of Eco-Agriculture, 2008, 16(1): 173-178. (in Chinese)

[3] Song J. Root morphology is related to the phenotypic variation in water logging tolerance of two populations of Suaeda salsa under salinity. Plant and Soil,2009, 324: 231-240.

[4] Song J, Wang B. Using euhalophytes to understand salt tolerance and to develop saline agriculture: Suaeda salsa as a promising model. Annals of Botany, 2015, 115(3): 541-553.

[5] 邵秋玲, 谢小丁, 张方申, 崔宏伟, 曹子谊. 盐地碱蓬人工栽培与品系选育初报. 中国生态农业学报, 2004, 12(1): 47-49.

Shao Q L, Xie X D, Zhang F S, Cui H W, Cao Z Y. A preliminary study on the artificial cultivation and breeding selection of Suaeda salsa. Chinese Journal of Eco-Agriculture, 2004, 12(1): 47-49. (in Chinese)

[6] Guan B, Yu J, Wang X, Fu Y, Kan X, Lin Q X, Han G X, Lu Z H. Physiological responses of halophyte Suaeda salsa to water table and salt stresses in coastal wetland of Yellow River Delta. CLEAN-Soil, Air, Water, 2011, 39(12):1029-1035.

[7] Wang C Q, Liu T. Involvement of betacyanin in chilling-induced photoinhibition in leaves of Suaeda salsa. Photosynthetica, 2007, 45(45): 182-188.

[8] Wang Q, Zhou D, Wang M, Zhao Y, Chen Y, Yin M, Feng X. Chemical constituents of Suaeda salsa and their cytotoxic activity. Chemistry of Natural Compounds, 2014, 50(3): 531-533.

[9] Xu B, Zhang M, Xing C, Mothibe K J, Zhu C. Composition, characterisation and analysis of seed oil of Suaeda salsa L.. International Journal of Food Science & Technology, 2013, 48(4): 879-885.

[10] Zhao S Z, Yuan R, Sun H Z, Wang B S. Highly efficient Agrobacterium-based transformation system for callus cells of the C3 halophyte Suaeda salsa. Acta Physiologiae Plantarum, 2008, 30(5): 729-736.

[11] 赵术珍, 阮圆, 王宝山. 盐地碱蓬幼嫩花序的组织培养及植株再生. 植物学通报, 2006, 23(1): 52-55.

Zhao S Z, Ruan Y, Wang B S. Tissue culture and plant regeneration from immature inflorescence explants of Suaeda salsa. Chinese Bulletin of Botany, 2006, 23(1): 52-55. (in Chinese)

[12] Qin F, Shinozaki K, Yamaguchi-Shinozaki K. Achievements and challenges in understanding plant abiotic stress responses and tolerance. Plant and Cell Physiology, 2011, 52(9): 1569-1582.

[13] Novillo F, Medina J, Rodriguez-Franco M, Neuhaus M, Salinas G J. Genetic analysis reveals a complex regulatory network modulating CBF gene expression and Arabidopsis response to abiotic stress. Journal of Experimental Botany, 2012, 63(1): 293-304.

[14] Agarwal P K, Agarwal P, Reddy M K, Sopory S K. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Reports, 2006, 25(12): 1263-1274.

[15] Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology, 2006, 57: 781-803.

[16] Lata C, Prasad M. Role of DREBs in regulation of abiotic stress responses in plants. Journal of Experimental Botany, 2011, 62(14): 4731-4748.

[17] Akhtar M, Jaiswal A, Taj G, Jaiswal J, Qureshi M, Singh N. DREB1/CBF transcription factors: Their structure, function and role in abiotic stress tolerance in plants. Journal of Genetics, 2012, 91(3): 385-395.

[18] Sakuma Y, Liu Q, Dubouzet J G, Abe H, Shinozaki K, Yamaguchi-Shinozaki K. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications, 2002, 290(3): 998-1009.

[19] Jaglo K R, Kleff S, Amundsen K L, Zhang X, Haake V, Zhang J Z, Deits T, Thomashow M F. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold response pathway are conserved in Brassica napus and other plant species. Plant Physiology, 2001, 127(3): 910-917.

[20] Choi D W, Rodriguez E M, Close T J. Barley Cbf3 gene identification, expression pattern, and map location. Plant Physiology, 2002, 129(4): 1781-1787.

[21] Dubouzet J G, Sakuma Y, Ito Y, Kasuga M, Dubouzet E G, Miura S, Seki M, Shinozaki K, Yamaguchi- Shinozaki K. OsDREB genes in rice, Oryza sativa L.,encode transcription activators that function in drought-, highsalt- and cold- responsive gene expression. The Plant Journal, 2003, 33(4): 751-763.

[22] Hang X, Fowler S G, Cheng H, Lou Y, Rhee S Y, Stockinger E J, Thomashow M F. Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. The Plant Jounal, 2004, 39(6): 905-919.

[23] Shen Y G, Zhang W K, Yan D Q, Du B X, Zhang J S, Liu Q, Chen S Y. Characterization of a DRE-binding transcription factor from a halophyte Atriplex hortensis. Theoretical and Applied Genetics, 2003, 107(1): 155-161.

[24] Xiong Y, Fei S Z. Functional and phylogenetic analysis of a DREB/CBF-like gene in perennial ryegrass (Lolium perenne L.). Planta, 2006, 224(4): 878-888.

[25] Chen M, Wang Q Y, Cheng X G, Xu Z S, Li L C, Ye X G, Xia L Q, Ma Y Z. GmDREB2, a soybean DRE-binding transcription factor, conferred drought and high-salt tolerance in transgenic plants. Biochemical and Biophysical Research Communications, 2007, 353(2): 299-305.

[26] Okamuro J K, Caster B, Villarroel R, Van Montagu M, Jofuku K D. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(13): 7076-7081.

[27] Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. 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. The Plant cell, 1998, 10(8): 1391-1406.

[28] 王平荣, 邓晓建, 高晓玲, 陈静, 万佳, 姜华, 徐正君. DREB转录因子研究进展. 遗传, 2006, 28(3): 369-374.

Wang P R, Deng X J, Gao X L, Chen J, Wan J, Jiang H, Xu Z J. Progress in the study on DREB transcription factor. Hereditas, 2006, 28(3): 369-374. (in Chinese)

[29] 陈国强, 王萍. 盐地碱蓬耐盐相关基因克隆研究进展. 生物技术通报, 2008, 5: 18-21.

Chen G Q, Wang P. Research on advancement of salt tolerant genes in Suaeda salsa. Biotechnology Bulletin, 2008, 5: 18-21. (in Chinese)

[30] Pang C H, Li K, Wang B. Overexpression of SsCHLAPXs confers protection against oxidative stress induced by high light in transgenic Arabidopsis thaliana. Physiologia Plantarum, 2011, 143(4): 355-366.

[31] 刘晓雪, 孙晓波, 王秀娥, 马鸿翔. 盐地碱蓬SsDREB基因的克隆与表达分析研究. 核农学报, 2011, 25(4): 684-691.

Liu X X, Sun X B, Wang X E, Ma H X. Cloning and expression analysis of SsDREB gene from Suaeda salsa L.. Journal of Nuclear Agricultural Sciences, 2011, 25(4): 684-691. (in Chinese)

[32] Sun X B, Ma H X, Jia X P, Chen Y, Ye X Q. Molecular cloning and characterization of two novel DREB genes encoding dehydration-responsive element binding proteins in halophyte Suaeda salsa. Genes & Genomics, 2015, 37: 199-212.

[33] Chen S, Songkumarn P, Liu J L, Wang G L. A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiology, 2009, 150(3): 1111-1121.

[34] 孙晓波, 贾新平, 邓衍明, 梁立建. 盐地碱蓬DREB同源基因SsDREB的功能研究. 植物遗传资源学报, 2015, 16(3): 624-632.

Sun X B, Jia X P, Deng Y M, Liang L J. Functional study of a DREB homologous gene SsDREB from Suaeda salsa L.. Journal of Plant Genetic Resources, 2015, 16(3): 624-632. (in Chinese)

[35] Cao Z F, Li J, Chen F, Li Y Q, Zhou H M, Liu Q. Effect of two conserved amino acid residues on DREB1A function. Biochemistry, 2001, 66(6): 623-627.

[36] Wang Z L, An X M, Li B, Ren Y Y, Jiang X B, Bo W H, Zhang Z Y. Identification and characterization of CBF/DREB1- related genes in Populus. Forestry Studies in China, 2008, 10(3): 143-148.

[37] Gilmour S J, Zarka D G, Stockinger E J, Salazar M P, Houghton J M, Thomashow M F. Low temperature regulation of Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. The Plant Journal, 1998, 16(4): 433-442.

[38] Wang Y M, He C F. Isolation and characterization of cold-induced DREB gene from Aloe vera L.. Plant Molecular Biology Reporter, 2007, 25(3): 121-132.

[39] Liu L, Cao X L, Bai R, Yao N, Li L B, He C F. Isolation and characterization of the cold-induced Phyllostachys edulis AP2/ERF family transcription factor, peDREB1. Plant Molecular Biology Reporter, 2011, 30(3): 679-689.

[40] Gao F, Chen J M, Xiong A S, Peng R H, Liu J G, Cai B, Yao Q H. Isolation and characterization of a novel AP2/EREBP-type transcription factor OsAP211 in Oryza sativa. Biologia Plantarum, 2009, 53(4): 643-649.

[41] Jiang F, Wang F, Wu Z, Li Y, Shi G, Hu J, Hou X. Components of the Arabidopsis CBF cold-response pathway are conserved in non-heading chinese cabbage. Plant Molecular Biology Reporter, 2011, 29(3): 525-532.

[42] Yang W, Liu X D, Chi X J, Wu C A, Li Y, Song L L, Liu X M, Wang Y F, Wang F W, Zhang C, Liu Y, Zong J M, Li H Y. Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA- independent pathways. Planta, 2011, 233(2): 219-229.

[43] Wang Q, Guan Y, Wu Y, Chen H, Chen F, Chu C. Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice. Plant Molecular Biology, 2008, 67(6): 589-602.

[44] Wang Q J, Xu K Y, Tong Z G, Wang S H, Gao Z H, Zhang J Y, Zong C W, Qiao Y S, Zhang Z. Characterization of a new dehydration responsive element binding factor in central arctic cowberry. Plant Cell Tissue and Organ Culture, 2010, 101(2): 211-219.

[45] Navarro M, Marque G, Ayax C, Keller G, Borges J P, Marque C, Teulieres C. Complementary regulation of four eucalyptus CBF genes under various cold conditions. Journal of Experimental Botany, 2009, 60(9): 2713-2724.

[46] Peng Y L, Wang Y S, Cheng H, Sun C C, Wu P, Wang L Y, Fei J. Characterization and expression analysis of three CBF/DREB1 transcriptional factor genes from mangrove Avicennia marina. Aquatic Toxicology, 2013(140/141): 68-76.

[47] Nakashima K, Yamaguchi-Shinozaki K. Abiotic stress adaptation in plants, physiological, molecular and genomic foundation. Springer Netherlands, 2010: 199-216.

[48] Verslues P E, Zhu J K. Before and beyond ABA, upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress. Biochemical Society Transactions, 2005, 33(Pt 2): 375-379.

[49] Hirayama T, Shinozaki K. Perception and transduction of abscisic acid signals: keys to the function of the versatile plant hormone ABA. Trends in Plant Science, 2007, 12(8): 343-351.