Cloning And Functional Analysis Of Water Stress Induced Gene ZmCIPK1 In Maize (Zea Mays.L) Seedlings

A new type of Ca²⁺ sensors unique to higher plants have been studied recently, they are the so-called CBLs for their similarity to B-subunit of yeast and animal calcineurin. Plant CBLs interact with and regulate the activity of a group of Ser/Thr protein kinases, referred to as CIPKs .The analysis of CBLs and CIPKs families revealed that they play an important role in the stress signal transduction pathway of higher plants.In our previous studies, we found a CIPK gene induced by water stress in maize, named ZmCIPK1 .To better understand the role of ZmCIPKl in stress-responsive pathway, we firstly cloned the ZmCIPK1 gene using RACE and RT-PCR, the full-long amplified cDNA fragment is 1652bp, including the 5'UTR (36bp) , the 3'UTR (200bp) and the coding region( 1416bp). ZmCIPK1 is predicted to encode a protein of 471 amino acids with an estimated molecular mass of 51 kDa, Database searches revealed that the deduced amino acid sequence of ZmCIPK1 has similarity with various Ser/Thr protein kinases. The putative kinase catalytic domain of ZmCIPK1 residues is in the N-terminal portion from 22 amino acids to 282 amino acids of the protein, and the tyrosine kinase catalytic domain is from 22 amino acids to 113 amino acids of the kinase catalytic domain of ZmCIPK1.The regulatory C-terminal domain of ZmCIPKl is partitioned into the two interaction domains,the NAF domain,which is responsible for CBL-CIPK interaction, and the adjacent PPI domain .The subcellular location presumed that the ZmCIPKl is localized to the plasma membrane .Southern blot result showed that two copys of ZmCIPK1 is present in maize genome. Northern blot analyses revealed that the expression of ZmCIPK1 is induced in maize seedings by drought, ABA and salt application, but not by cold. These observations may suggest that ZmCIPK1 regulates select pathway in response to drought, salt stress and ABA. Database searches revealed that ZmCIPK1 has the similar structure with SOS2, the SOS2/ZmCIPKl complementation analysis suggested that ZmCIPK1 plays an important role in the germinating seeds of Arabidopsis under salt stress. Transgenic Arabidopsis plants with double-stranded ZmCIPK1 RNA were generated mediated by Agrobacterium. Transgenic plants showed a significant delay in germination on mannitol-containing medium and a lower survival rate on sodium chloride-containing medium but no difference in adult plants . We also tested whether ZmCIPKl -overexpressing plants were altered in stress response during seeding germination and adult plants, but no difference was detected between wild-type and ZmCIPK1 -overexpressing plants .These results indicated that ZmCIPKl take part in the response to drought and salt during plants seeding. The ZmCIPKl coding region was amplified by PCR,cloned in frame into the vector pET-30a(+) ,and transformed into Escherichia coli BL21 (DE3 ) cell .The ZmCIPK1-fusion protein were highly incubated with 1.0mM isopropyl 8-D-thiogalactoside .Soluble analysis shows the induced protein is insoluble.In summary, plants have evolved complex molecular mechanisms by which they tolerate and adapt to adverse growth conditions for survival. A single stimulate can product different signal pathways, there is much overlap in these pathways. Because CIPKs are a large family of CBL-associated kinases, additional studies on the interaction of CIPKs and CBLs family may identify the molecular mechanisms of stress responses and other stimulus-response coupling processes in plants.