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

Abstract

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

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.

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