Abstract:

The SikCDPK1 gene in the extreme habitat plant Saussurea involucrata can significantly improve the low temperature and drought tolerance of genetically modified tobacco. In order to further explore the expression characteristics and protein kinase activity of SikCDPK1 gene， real-time quantitative PCR was used to detect the expression level of SikCDPK1 gene in different tissues and organs of S. involucrata and the induced response mode of signal molecules CaCl₂， abscisic acid， gibberellin， salicylic acid and hydrogen peroxide， while Kinase-Glo? technology was used to detect the activity of SIKCDPK1 protein kinase， and the green fluorescent protein （GFP） fusion protein expression method was used to carry out the subcellular localization of SIKCDPK1 protein. The SikCDPK1 gene was found to be expressed in roots， stems， leaves， seeds， young stems and young leaves of S. involucrata and expression was induced by CaCl₂， abscisic acid， gibberellin， salicylic acid， hydrogen peroxide， but the response mode was different. Kinase activity analysis showed that SIKCDPK1 had kinase catalytic activity when Ca²⁺ was present， and SIKCDPK1 had almost no kinase activity after adding the Ca²⁺ chelating agent ethylene glycol tetraacetic acid. SIKCDPK1 kinase activity increased with increase in Ca²⁺ concentration， and SIKCDPK1 kinase activity reached 1/2 of the maximum activity when Ca²⁺ concentration （K_0.5） was 48.7 nm. When HisⅢ was used as the substrate， the Km value of SIKCDPK1 reached 43.8 μg·mL^-1， and SIkCDPK1 protein was localized to the nucleus. In summary， the expression of the SikCDPK1 gene had no obvious tissue specificity， and could respond to the induction of signaling molecules Ca²⁺， abscisic acid， gibberellin， salicylic acid， hydrogen peroxide. Ca²⁺ is required for SIKCDPK1 to exert protein kinase activity， and SIKCDPK1 mainly functions in the nucleus.

Key words: SikCDPK1 gene, expression, protein kinase activity