Functional Analysis Of ZmCIPK24-2 From Maize

Salt stress exists in nature widely and affects the growth and development of crop plants,resulting in reduced crop yields.In the process of long-term adaptation,plants have evolved complex signal networks.With the development of genetics,genetic engineering and molecular biology,people have carried out in-depth research on the mechanism of salt tolerance in plant.Calcium is a central regulator of plant growth and development,and calcium ion(Ca²⁺)is a second messenger in plant cell.The Ca²⁺receptor calcineurin B-like protein（CBL）senses calcium signal and transmits it to the target protein CBL-interacting protein kinase（CIPK）.In this study,we identified an SOS2 homologous gene Zm CIPK24-2.The ORF of Zm CIPK24-2 is 1356 bp in length,encodes a ploypeptides of 452 amino acid rescides.Sequence analysis showed that Zm CIPK24-2 had a high sequence identity with SOS2and Zm CIPK24,containing the characteristic functional domain of CIPK gene family.Tissue-specific expression showed that Zm CIPK24-2 ubiquitously expressed in various tissue parts of maize,especially in pollen.Zm CIPK24-2 had different expression patterns in various tissues,suggesting that its site of action and functional mechanisms are variance.The expression of Zm CIPK24-2 under Na Cl stress was up-regulated.Overexpressed Zm CIPK24-2in the sos2 mutant,the survival rate and the root length of Zm CIPK24-2-overexpressed homozygous lines were significantly increased compared with sos2 mutant under high salt concentration.Subcellular localization experiments showed that Zm CIPK24-2 was localized in cytoplasm,cell membrane,and nuclear membrane.The evolution of SOS pathway is especially conserved in plants,and the CBL-CIPK complex involved in plants response to salt stress.In order to explore the regulation mechanism of Zm CIPK24-2 on salt tolerance,we contructed bait plasmid p DEST32-Zm CIPK24-2 to screening interacting proteins in maize Zm CBLs.Yeast two-hybrid experiments and the LUC complementation imaging assays proved that Zm CIPK24-2 interacted with Zm CBL1,Zm CBL4,Zm CBL8,and Zm CBL9respectively and could occur in plants,indicating that the activity and function of Zm CIPK24-2 protein may be potentially regulated by these Ca²⁺binding proteins Zm CBLs.Taken together,in this study,we identified a salt tolerance gene Zm CIPK24-2 in maize.These results suggest that Zm CIPK24-2 is involved in the regulation of plant growth and development,stress response and signal transmission,which provides a new experimental evidence for researching the function of CIPK family genes and analyzing CBL-CIPK signaling pathway in maize.