Screening And Identification Of ZmCCaMK Interacting Proteins

As a second messenger in plant, Ca2+ plays an important role in plant growth, development and response to environment signals. Abiotic stresses, such as cold, drought, hot shot and salinity, alter the concerntration of Ca2+, starting Ca2+ signaling transduction in plant. Calcium/calmodulin dependent protein kinase (CCaMK) is an important Ca2+ sensor. CCaMK contains a kinase domain, a calmodulin binding domain and three EF-hand Calcium binding domains, and its autophosphorylation is dependent on Ca2+, its substrate phosphorylation activity is dependent on Ca2+ /CaM. CCaMK is a member of Ser/Thr protein kinase family. Many studies showed that CCaMK involved in plant growth and development, i.e. flower formation, light signaling, nodulation signaling, and response to stress.Our recent study showed that ZmCCaMK is involved in ABA-induced antioxidant defense and both hydrogen peroxide and drought can activate ZmCCaMK. Localization results showed that ZmCCaMK located in cytoplasm、nuclear and cell membrane. However, the detail mechanisms of ZmCCaMK in ABA-induced antioxidant defense still not clear. Therefore, in this study, the target proteins that interacte with ZmCCaMK were screened using yeast two-hybrid and the interaction was further confirmed by other methods. The results are as follows:1. First, pEXP32-ZmCCaMK, was constructed, and the toxicity of ZmCCaMK to yeast and self-activation of ZmCCaMK were detected. Then, using pEXP32-ZmCCaMK as bait plasmid, ZmCCaMK interacting proteins were screened by yeast two-hybrid in cDNA library of Zea Mays. Eventually, we screened two proteins,WRKY62 and NAM like protein(ZmZmNLP),which might interact with ZmCCaMK.2. To confirm the interaction between ZmCCaMK and ZmZmNLP, GST-pull down and co-immunoprecipitation were performed. Our results showed that ZmCCaMK interacts with ZmZmNLP in vivo and in vitro.3. The full-length of ZmZmNLP was cloned and constructed into the transient expression vector pXZP008, then transformed into maize mesophyll protoplasts. The localization of ZmCCaMK was detected by confocal. The results showed that ZmZmNLP is located in nucleus.