盐地碱蓬2个DREB1/CBF基因的克隆与表达调控分析

Cloning and Expression Analysis of Two DREB1/CBF Genes in Suaeda salsa L.

【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c DNA全长序列,分别命名为Ss CBF1和Ss CBF2。运用生物信息学软件对2个Ss CBF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Ss CBF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Ss CBF在低温、Na Cl、PEG以及ABA处理下的表达模式。【结果】Ss CBF1编码一个225个氨基酸的蛋白,预测分子量为25.4 k D,理论等电点为4.84。Ss CBF2编码一个由260个氨基酸组成的蛋白,预测分子量为28.6 k D,理论等电点为5.05。Ss CBF1和Ss CBF2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。Ss CBF1、Ss CBF2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Ss CBF1表达,而Ss CBF2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】Ss CBF1和Ss CBF2是盐地碱蓬的2个胁迫应答转录因子。在盐地碱蓬中,Ss CBF1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Ss CBF2则通过不依赖于ABA途径对低温胁迫产生响应。

【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 c DNA sequences of the two DREB1/CBF genes were isolated by the method of rapid-amplification of c DNA ends（RACE）,and they were named as Ss CBF1 and Ss CBF2,respectively.The structures and functions of the two proteins encoded by Ss CBFs were predicted by the bioinformatics software.In order to detect the subcellular localization of the two proteins,the coding sequences of the two Ss CBF 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 Ss CBF1 and Ss CBF2 to DRE/CRT cis-acting element and their transcriptional activities were investigated by using a yeast one-hybrid system.The expression of the two Ss CBF genes in response to low temperature,Na Cl,PEG and ABA were assessed by the real-time quantitative PCR.【Result】 Ss CBF1 encoded a peptide of 225 amino acid residues with a predicted molecular mass of 25.4 k D and a p I of 4.84.Ss CBF2 encoded a predicted protein of 260 amino acid residues with a predicted molecular mass of 28.6 k D and a calculated p I of 5.05.Ss CBF1 and Ss CBF2 both contained a typical AP2/ ERF domain and shared 53.5% and 45.4% identity at the levels of coding nucleotides and amino acids.However,the AP2/ ERF domains of the two Ss CBF genes were highly similar and shared 76.2% and 87.3% identity at the levels of nucleotides and amino acids,respectively.Ss CBF1 and Ss CBF2 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 Ss CBF1.However,the expression of Ss CBF2 could only be induced significantly by low temperature,not by drought,high salt and ABA.【Conclusion】 The Ss CBF1 and Ss CBF2 are two stress-responsive transcription factors of S.salsa.In S.salsa,Ss CBF1 is involved in the stress responses of cold,high-salt and drought through ABA-dependent pathways and Ss CBF2 is responsive to cold stress through ABA-independent pathway.