Cloning And Molecular Mechenism Analysis Of Stress Tolerance Genes ZmCIPK12and ZmCIPK21from Maize

CBL-interacting protein kinases (CIPKs) which contain Ser/Thr kinase domain are unique protein kinase in higher plants. They especially interact with CBLs (calcineurin B-like proteins). Recent studies reveal that CBL-CIPK network plays an important role in stresses signaling transductions pathway.Maize CIPKs is induced by diversity abiotic stresses. In this study, the expression pattern of ZmCIPK12showed that ZmCIPK12was up-regulated under dehydration, salt and exogenous ABA treatments. The ORF of ZmCIPKl2is1512bp in length, encodes a polypeptides of503amino acid rescides. Overexpression of ZmCIPK12could enhance the salt and osmotic tolerance in transgenic plants. The yeast two hybrid assay showed that ZmCIPK12can interact with CBL4and CBL8, and BiFC results indicated they were cell membrane localization after interaction. Four GRF (GRF1, GRF2, GRF3, GRF4) and a ZmPPase4protein were found after ZmCIPK12used as bait for screening maize cDNA library. In addition, pull-down assay showed that ZmCIPK12could interact with ZmPPase4in vitro, and ZmPPase4was phosphorylated by ZmCIPK12in vitro in kinase assay. Expression of maize ZmPPase4in Escherichia coli showed that the growth rate of transformed ZmPPase4Escherichia coli decreased obviously than the empty vector under pH4.0in LB liquid medium. In contrast, the growth rate of the transformed ZmPPase4Escherichia coli was higher than empty vector under pH9.0in LB. These results suggested that ZmCIPK12might play as an important role in osmotic stresses. We supposed that ZmCIPK12interact with CBL4or CBL8for signaling transduction, and then phosphorylated the ZmPPase4to regulate the osmotic potential by driving the H+gradient.Salt stress represents an increasing threat to crop growth and yield in saline soil. In this study, we identified a maize calcineurin B-like protein-interacting protein kinase (CIPK), ZmCIPK21, which was primarily localized in the cytoplasm and the nucleus of cells and displayed enhanced expression under salt stress. Over-expression of ZmCIPK21in wild-type Arabidopsis plants increased their tolerance to salt, as supported by the longer root lengths and improved growth. The downstream stress-response genes, including dehydration-responsive element-binding (DREB) genes were also activated in transgenic plants over-expressing ZmCIPK21. In addition, introduction of the transgenic ZmCIPK21gene into the Arabidopsis mutant cipkl-2rescued the salt-sensitive phenotype under high salt stress. Measurement of Na+and K+content in transgenic plants showed that over-expression of ZmCIPK21decreased accumulation of Na+and allowed retention of relatively high levels of K+, thereby enhancing plant tolerance to salt conditions.