Functional Analysis Of ZmCIPK8 And Interacting Protein In Response To Stresses

CBL is a kind of calcineurin B-like proteins that is known to be unique in plants which is discovered in recent years.It plays an important role in the signal transduction process of plant response to stress by together with the target protein CIPK.Previous studies have shown that ZmCIPK8 plays an important role in the maize response to adversity stress.Analyzing the interaction between ZmCIPK8 and CBLs is significant for researching the molecular mechanism of ZmCIPK8 response to adversity stress.In this study,BiFC and plant transgenic technology were used to analysis the function of ZmCIPK8.Main conclusions are as follows:(1)ZmCIPK8 was fused with PS1300-GFP,then the fusion expression vector was transformed into Agrobaterium tumefaciens GV3101 strain.For transient expression in onion(Allium cepa)epidermal cells.For stable expression,Arabidopsis plants were transformed using the floral dipping method.The result showed ZmCIPK8 was located in cytoplasm.(2)We design specific primer and clone the promoter of ZmCIPK8,named as ZmCIPK8-Pro.The promoter of ZmCIPK8 was fused with PCAMBIA1391,named as PCAMBIA1391-ZmCIPK8-Pro.The construct was introduced into the Agrobaterium tumefaciens GV3101 strain,which was thereafter used to transform Arabidopsis thaliana by the floral dipping method.During early seedling development,expression of ZmCIPK8 was detected in cotyledon,hypocotyls and root except meristematic zone in root.GUS activity was induced by osmotic,ABA and drought.GUS activity was inhibited by salt and low potassium.(3)For generation of the BiFC vectors,the coding region of ZmCIPK8 was cloned into pUC-SPYNE,resulting ZmCIPK8-YNE;the coding regions of CBL1,CBL4 and CBL9 were cloned into pUC-SPYCE,resulting in CBL1-YCE,CBL4-YCE and CBL9-YCE.The fusion constructs were introduced into Arabidopsis mesophyll protoplasts prepared from rosette leaves by the polyethylene glycol-mediated transformation procedure.The BiFC results showd the ZmCIPK8 interacts with the ZmCBLl,4 and 9 in vivo.(4)Construction of plant expression vector of PCAMBIA1301-ZmCBL 1/9,the constructs were introduced into the Agrobaterium tumefaciens GV3101 strain,which was thereafter used to transform Arabidopsis thaliana by the floral dipping method.The results of seed germination experiments indicate that ZmCBLl in response to ABA during post-germination;ZmCBL9 can facilitates the germination of over ecpression strains.The results of stress experiments show that the roots of over expression strains is longer than the wild type under100?M ABA;the roots of over expression strains is less but longer than the wild type under 100mM D-mannitol;over expression strains are better than wild type under 375mM D-mannitol.The results suggested that ZmCBL9 facilitates the elongation of root uder osmosis condition.