Isolation And Functional Analysis Of A Calcium-dependent Protein Kinase Gene ZmCPK4in Zea Mays

Calcium-dependent protein kinases (CDPKs) which are Ser/Thr protein kinases found in plants and some protozoa, play crucial roles in Ca2+-mediated signaling pathways. CDPKs participate in several important physiological and biochemical processes, including growth and development, transcriptional regulation, hormone response, cytoskeleton reorganization, plant stress signal transduction, and defense reaction. ABA as one of the five traditional plant hormone, plays an important role in plant adversity stress. Previous researches have shown that CDPKs widely involved in ABA signaling pathways. But the study of specific CDPK gene in ABA signal is still limited. In recent years, functions of CDPKs in antiviral defense reaction were also investigated, a growing number of studies confirm that CDPKs participate in signal-mediated disease resistance responses.We started with the above two aspects, explored the sequence characteristics, expression pattern of ZmCPK4and it’s physiological functions in transgenic Arabidopsis and tobacco. The main results are as follows:(1) We isolated ZmCPK4from maize leaves. The ZmCPK4encodes a protein of554amino acids with a predicted molecular mass of about61.2kDa. An alignment of the amino acid sequences of ZmCPK4and other CDPKs, showed that the ZmCPK4protein harbors all the four conserved domains. The phylogenetic tree showed that ZmCPK4belongs to subgroup I.(2) Ttransient expression of the ZmCPK4-GFP fusion protein in onion epidermal cells revealed that ZmCPK4was localized in the plasma membrane and nucleus membrane. Root tip cells of the transgenic Arabidopsis plants expressing the ZmCPK4-GFP fusion proteins revealed that ZmCPK4specifically localized in the plasma membrane.(3) ZmCPK4was shown to be expressed ubiquitously in all the tested organs/tissues, including primary root, pericarp, internode, adult leaf, silk, culm, seedling, endosperm, embryo and tassel by qRT-PCR. ZmCPK4exhibited a highest expression level in seedlings and embryo, whereas had a low expression in culm and endosperm. The transcript level of ZmCPK4could be induced by4℃, H2O2, NaCl, ABA, PEG, SA, MeJA and ETH.(4) ZmCPK4-overexpressing Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth, and ABA-induced stomatal closing and inhibition of stomatal opening, altered expression of a set of ABA responsive genes.(5) ZmCK4-overexpressing Arabidopsis lost less water and grown well, showed an enhanced resistance under drought stress.(6) ZmCPK4-overexpressing tobacco showed more severe symptoms of bleaching, decreased the antioxidant capacity under oxidative stress.(7) ZmCPK4-overexpressing tobacco exhibited enhanced sensitivity to PVX and TMV, more severe disease, more accumulation of pathogen and ROS, decreased activity of antioxidant enzymes POD and decreased antioxidant capacity.