Cloning and functional analysis of ChaCBF1, a CBF/DREB1-like transcriptional factor from Corylus heterophylla × C. avellana.
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摘要: 为研究榛属植物的抗寒分子机理,以平欧杂交榛‘达维’为试材,采用同源克隆和RT-PCR技术克隆获得一个CBF/DREB1转录因子基因ChaCBF1,GenBank登录号为KT757373。该基因开放阅读框为666 bp,编码221个氨基酸,其相对分子质量为26.4 kDa,理论等电点为5.88。氨基酸序列比对结果显示ChaCBF1含有一个高度保守的AP2/ERF结构域,以及PKK/RPAGRxKFxETRHP和DSAWR等CBF蛋白特征序列。系统进化树分析发现ChaCBF1与垂枝桦BpCBF3蛋白的亲缘关系最近。通过基因枪轰击法使重组质粒p1302-ChaCBF1-GFP在洋葱表皮细胞瞬时表达,亚细胞定位结果显示ChaCBF1定位于洋葱表皮细胞的细胞核。通过实时荧光定量PCR检测了ChaCBF1基因在多种非生物胁迫下的表达模式,结果表明ChaCBF1基因的表达能够持续而强烈地响应低温信号,同时其表达也受干旱、高盐和ABA的诱导。通过农杆菌介导的花序侵染法将重组质粒p1301bar-ChaCBF1转化野生型拟南芥,对ChaCBF1基因的功能进行了研究。结果表明:过表达ChaCBF1的转基因拟南芥增强了对冷冻胁迫的耐受性,其成活率显著高于对照植株;转基因植株在正常和低温条件下能够显著上调冷响应基因RD29A和COR47的表达,并积累较高水平的游离脯氨酸和可溶性糖等渗透调节物质,从而提高其抗寒能力。研究结果表明,转录因子ChaCBF1基因在杂交榛冷响应途径中发挥重要作用。Abstract: To investigate the molecular mechanisms of cold-resistance of Corylus plants, a CBF/DREB1-like transcription factor gene, designated as ChaCBF1 with the GenBank accession number of KT757373, was cloned using homology-based cloning and reverse transcription-PCR (RT-PCR) from hybrid hazelnut ‘Dawei’ (C. heterophylla × C.avellana). The open reading frame of ChaCBF1 was 666 bp, encoding a protein of 221 amino acids, with a molecular mass of 26.4 kDa and a theoretical isoelectric point of 5.88. The amino acids sequence alignment showed that the ChaCBF1 protein contained a highly conserved AP2/ERF domain. In addition, ChaCBF1 also contained PKK/RPAGRxKFxETRHP and DSAWR motifs, which are signature sequences of CBF proteins. The recombinant plasmid p1302-ChaCBF1-GFP was transiently expressed into onion epidermal cells by particle bombardment method via a gene gun,and subcellular localization analysis revealed that ChaCBF1 protein mainly localized into the nucleus of onion epidermal cells. The expression patterns of ChaCBF1 under various abiotic stress conditions were determined using quantitative real-time PCR. The results showed that the expression of ChaCBF1 was strongly and constantly induced by low temperature, and also in response to drought, salt and ABA signals. To elucidate the function of ChaCBF1, the fusion construct p1301bar-ChaCBF1 was introduced into wild type Arabidopsis by Agrobacterium-mediated transformation using the floral-dip method. The results revealed that the transgenic Arabidopsis overexpressing ChaCBF1 enhanced tolerance to freezing treatment, and had significantly higher survival rates than wild-type plants. The expression of cold-regulated genes RD29A and COR47 were significantly upregulated and the contents of free proline and soluble sugars were also increased in transgenic plants under normal or low temperature conditions, and resulted in increased tolerance to cold. These results indicated that ChaCBF1 functions as a transcription factor and play crucial roles in cold-response pathway of hybrid hazelnut.
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