Abstract: 【Objective】The objective of this study is to clone the stress resistance-related gene, analyze its sequence features, evolutionary relationships and expression characteristics, investigate its biological function during the stress tolerance of wheat, and to provide candidate gene and a theoretical foundation for clarifying molecular mechanism of stress resistance.【Method】Using an up-regulated EST obtained by cDNA chip as a probe to search the wheat EST databases, filter out the ESTs sequences with the homology of 97% of the probe, a full-length cDNA sequence was cloned from wheat by in silico cloning and reverse transcription PCR (RT-PCR) method. The conserved domains and sequence features of the gene were analyzed by bioinformatics’ methods. A phylogenetic tree was constructed using the MEGA 6.0 software, and then the cloned geneORF was inserted into the expression vector pMAL-c2X by EcoR I and HindⅢ digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.3 mmol·L^-1 IPTG for 1-5 h. The expression of the fusion protein was detected by SDS-PAGE. The expression profiles of the cloned genein various tissues and in response to cold, drought, heat and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR).【Result】The full-length cDNA sequence designated as TaC2DP1 from wheat is 1 356 bp in length, contains a 1 209 bp open reading frame (ORF), with 50 bp in the 5' UTR and 97 bp in the 3' UTR. TaC2DP1was predicted to encode a 402 amino acid protein with a molecular mass of 43.41 kD and isoelectric point of 4.30, it belongs to the acidic protein. BLAST analysis revealed that the protein contains a C2-domain and was predicted to be a Ca²⁺ binding domain. Multiple sequence alignment and phylogenetic tree analysis showed that TaC2DP1 had the closest evolutionary relationship with a C2-domain protein in Triticum urartu with unknown function, and shares 91% identity in amino acids. Protein structure prediction showed that TaC2DP1 had not transmembrane helix and disulfide bondand might be localized in cytoplasm. The prokaryotic expression vector of TaC2DP1, pMAL-c2X-TaC2DP1,was successfully constructed. The expression of fusion protein was obtained by inducing with IPTG and its relative molecular weight was 90 kD, which was consistent with the theoretical value. Real time quantitative PCR (qRT-PCR) analysis revealed that TaC2DP1 constitutively expressed in all tested roots, stems, leaves, immature ears, immature seeds, embryo and endosperm, the expression level of TaC2DP1 in immature ears was the highest, while its expression level was very low in seeds at 5 days after anthesis (DAA). The expression profiling revealed that TaC2DP1 was induced by plant hormone ABA and the expression of TaC2DP1 was steadily up-regulated in any of the time point under drought stress. TaC2DP1 was rapidly up-regulated within 0.5 h of cold and heat stress treatments and the expression level was 21 and 17 times than those of control, respectively. These results revealed that TaC2DP1 might be involved in stress resistance-related response of ABA signaling pathways in wheat leaves.【Conclusion】A full-length cDNA of TaC2DP1 was isolated and the typical a Ca²⁺ binding domain was found in the deduced proteins. The expression of TaC2DP1 was all up-regulated under drought, high and low temperature and ABA treatments, showing that TaC2DP1 was involved in the ABA dependent stress-responding gene network.It was deduced that TaC2DP1 might play an important regulation role under stress in wheat.

Key words: wheat (Triticum aestivum), C2 domain protein, gene cloning, expression analysis