Cloning And Functional Analysis Of Foxtail Millet SiCBL4 And SiCIPK24 Genes

Foxtail millet(Setaria italica(L.)Beauv.)is one of the crops widely planted in arid and semi-arid regions of China.However,the yield and quality of millet were severely restricted by various abiotic stresses,such as salt and drought.CBL proteins(calcineurin B-like protein)were identified as a unique type of calcium sensor in plants.CBLs activate the downstream calcium signal transduction pathways through interaction with CIPKs(CBL Interacting Protein Kinase).The CBL-CIPK signaling pathway plays an important roles in response to various abiotic stresses.In this study,we cloned SiCBL4 and SiCIPK24 genes from millet and analyzed their expression profile under different abiotic stresses,such as salt,drought,oxidative stress,high temperature,cold and ABA.The overexpression vector of SiCBL4 and SiCIPK24 were constructed and transformed into Arabidopsis thaliana for determine its functions.The promoter of SiCBL4 gene was also used to control the expression of a luciferase reporter gene for investigate its activity in protoplasts.qRT-PCR results showed that both SiCBL4 and SiCIPK24 were induced by salt,ABA or Paraquat.Phylogenetic relationship and sequence analysis indicated that SiCBL4 and SiCIPK24 display a highly degree of conservation with the plant CBL and CIPK family.Remarkably,SiCBL4 and SiCIPK24 have a highly amino acid sequence identity to their homologous genes of SOS pathway in Arabidopsis,maize and rice.SiCBL4 was mainly expressed in young leaves,while SiCIPK24 was expressed in roots,shoots,leaves and inflorescences.The subcellular localization in millet protoplast showed that SiCBL4 was mainly located in plasma membrane while SiCIPK24 in the cytoplasm.Bimolecular fluorescence complementation(BiFC)and yeast two-hybrid assay(Y2H)results indicated that SiCBL4 acted with SiCIPK24,and the SiCBL4/SiCIPK24 complex was located in the plasma membrane.Complementation test of Arabidopsis mutants(sos3,sos2-1)and overexpression of SiCBL4 and SiCIPK24 in wild-type Arabidopsis,showed that SiCBL4 and SiCIPK24 have the similar function of AtCBL4(sos3)and AtCIPK24(sos2-1).In addition,we found that mutation of the N-myristoylation site of the SiCBL4 protein will influence the subcellular localization and the signal transduction under salt stress using site mutation method.Futhermore,a WRKY transcription factor SiWRKY17 was found to negatively regulate the SiCBL4 expression.This study would enrich CIPK members in plant kingdom and provides important information for further elucidating the function and mechanisms of the CBL/CIPK network system responsive to stresses in foxtail millet.