关键词: 菘蓝, ItSnRK2.1基因, 克隆, 特性分析

Abstract: As crucial protein kinase in ABA pathway,SnRK2 regulates the expressions of stress responsive genes by regulating the activity of downstream related proteins,and which then significantly improves the resistance of plants to abiotic stresses such as high salinity and drought. The degenerate primers were designed according to the conserved sequences of UTR domains from SnRK2 cDNA sequences. And a full-length cDNA sequence of SnRK2,named as ItSnRK2.1,was cloned from Isatis tinctoria by RT-PCR method. The length of full-length cDNA（GenBank accession number：MF423122）was 1 305 bp containing a complete open reading frame of 1 071 bp and encoding 356 amino acids. The theoretical isoelectric point and putative molecular weight of the ItSnRK2.1 protein were 5.64 and 30.60 kD,respectively. The ItSnRK2.1 was a hydrophilic protein with 42 phosphorylation sites,contained two significant transmembrane domains and no signal peptide,and played a physiological role in the cytoplasm. The secondary structure of the ItSnRK2.1 protein contained 151 α-helixes,109 random coils,66 extended strands,and 30 β-turns. The ItSnRK2.1 protein contained an ATP binding site and a serine/threonine enzyme activity domain,and was at the same evolutionary branch with AtSnRK2.1,AtSnRK2.5 and StSnRK2.6. RT-PCR results showed that ItSnRK2.1 gene expressed mostly in roots,following in leaves,and marginally in stems of I. tinctoria seedlings. In conclusion,ItSnRK2.1 gene actively involved in the responses to high salt and drought stresses,but not sensitive to ABA stress,suggesting that the function of this gene was possibly associated with plant stress resistance,and independent of ABA regulatory pathway.

Key words: Isatis tinctoria, ItSnRK2.1 gene, cloning, characterization analysis