| The calcineurin B-like proteins (CBLs) are recently identified to be a kind of plant specific calcium sensors. CBL interacts with CIPK (CBL-interacting protein kinase). CBL and CIPK were proved to play important roles in responding to stresses and regulating certain developmental processes in plants. Based on studies on model plants Arabidopsis and rice, CIPKs perticipate in responsiveness of plants to abiotic stresses, such as high salinity, low potassium and drought. However, although researches on CIPKs have made much progress in Arabidopsis and rice in the recent years, only a few studies have been made and thus related information is limited in other plant species. Maize is one of the most important cereal crops in the world, its production is limited under abitotic stresses like drought and poor soil. Cloning CIPKs in maize not only shows theoretical meanings, but also benefits to practical application, providing potential candidate genes for improving stresses torlerance by genetic transformation in maize. Here, we isolated ZmCIPK31, a new CIPK in maize. Its biochemical property, expression profiles and fuctions have been analized. The main results were as follows.1. Cloning and sequence analysis of ZmCIPK31 geneThe full length of ZmCIPK31 cDNA was obtained by using RACE method, while the genomic DNA of ZmCIPK31 with the promoter region, 2189bp upstream of ATG initiation codon, was obtained by PCR method. The genomic DNA was composed of 14 exons and 13 introns in its coding region. And it contained a serine/threonine kinase domain in N-terminal region and a NAF domain in C-terminal region. A promoter motif search of this region showed that there were motifs related to light, stress, development, auxin and others.2. Protein kinase activity of ZmCIPK31The recombinant plasmids pET-ZmCIPK31 and pMAL-ZmCIPK31 were constructed for prokaryotic expression analysis. The results showed that the predicted target protein could be induced in the recombinant plasmid transformed E. coli BL21 strain cells. Protein soluble analysis proved the fusion protein His-tag-ZmCIPK31 was not soluble, while MBP-ZmCIPK31 was soluble. Thus, the purified and concentrated soluble MBP-ZmCIPK31 fusion protein was obtained for protein kinase assay. The prtein autophosphorylation assay showed that ZmCIPK31 possessed protein kinase properties and millimolar range of Mn2+ was effective cofactor.3. Expression analysis of ZmCIPK31 gene in maizeCertain ZmCIPK31 expression level in stem, root, embryo, filament, seed and leaf of maize were detected under normal growth conditions. And the relative expression level is the highest in filament. ZmCIPK31 was regulated by NaCl, cold and PEG, but not by ABA and dehydration.4. Subcellular localization of ZmCIPK31 and interation with ZmCBLsSubcellular localization analysis in onion epidermal cells and Arabidopsis mesophyll protoplasts showed that ZmCIPK31 expressed transiently in plasma membrane, cytoplasm and the nucleus. Yeast hybrid assay and BiFC assay showed that ZmCIPK31 interacted specifically with ZmCBL3 and ZmCBL4, and the NAF domain was necessary. ZmCIPK31-337C, however, exhibited more extensive affinities with ZmCBL2-1, 2-2, 5, 6 other than ZmCBL3 and ZmCBL4.5. Over-expression of ZmCIPK31 in ArabidopsisIn MS medium with or without 4°C treatment and in MS medium with 100 mM NaCl, the seeds germination rate and root length of all five transgenic lines were just like that of the control wild-type. However, in the medium with 125 mM or 150 mM NaCl, all transgenic lines showed significantly higher germination rates than the wild-type Arabidopsis. On the contrary, there was no significant difference of the root length between transgenic lines and wild type. |
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