Cloning of TaCIPK15 gene and interaction analysis with TaCBLs protein in wheat

【Objective】The purpose of the study was to clone the wheat serine / threonine protein kinases（CIPK） TaCIPK15 gene and analyze the interaction between the protein encoded by TaCIPK15 gene and wheat calcineurin B-like protein（TaCBLs），so as to provide reference for the in-depth study of the function of TaCIPK15 gene in the mechanism of response to adversity stress in wheat.【Method】The full length of coding region sequence（CDS）of wheat TaCIPK15 gene was amplified by PCR and analyzed by bioinformatics methods. The real-time fluorescence quantitative PCR（ qRTPCR）method was used to study its response to adversity，and the interaction between TaCIPK15 and TaCBLs was studied by yeast two-hybrid and bimolecular fluorescence complementation techniques.【Result】The CDS sequence of wheat TaCIPK15 gene obtained by cloning was 1302 bp in length，encoding 433 amino acid residues，which was 100% similar with the reference sequence XR_006454427.1 in the NCBI database. TaCIPK15 protein had a molecular weight of 48.48 kD，with a total number of atoms of 6896，a molecular formula of C₂₁₆₆H₃₄₈₇N₅₉₉O₆₂₈S₁₆，and theoretical isoelectric point（pI） of 9.38. TaCIPK15 protein had no transmembrane structure and signal peptide. TaCIPK15 protein contained conserved NAF structural domain and activation loop of CIPK family，and the amino acid sequence was most similar and the closest relative to that of barley HvCIPK15（XP_044946251）. The expression of TaCIPK15 gene was down-regulated under salt stress（NaCl）. The expression of TaCIPK15 gene was up-regulated in root under cold stress. The expression was up-regulated in root and leaf at 12 h after abscisic acid（ABA）treatment. TaCIPK15，TaCBL1 and TaCBL2 showed the ability to grow blue plaques on four-deficient media SD/-Trp/-Leu/-Ade/-His/X-α-gal/ABAin yeast two-hybrid test. In the bimolecular fluorescence complementation test，TaCIPK15 and TaCBL1 detected yellow fluorescence in the nucleus，and TaCIPK15 and TaCBL2 detected yellow fluorescence in the cytoplasm and cell membrane.【Conclusion】The TaCIPK15 gene belongs to CIPK gene family. TaCIPK15 interacts with TaCBL1 and TaCBL2 and has different interaction positions in cells. TaCIPK15 gene plays a role in Ca²⁺ signaling transduction in wheat in response to high salt stress，cold stress and ABA stress.