Cloning and functional analysis of ChaCBF1, a CBF/DREB1-like transcriptional factor from Corylus heterophylla × C. avellana.

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.