| CBLs which are recently discovered for a kind of plant specific calcineurin B-like proteins, and their target protein CIPKs (CBL-interacting protein kinases) were proved to play important roles in responding to stresses with signal transduction process in plants. Based on research results on model plants, CIPKs participated in responses of plants to abiotic stresses, such as high salinity, drought, low temperature, low potassium and plant hormones, etc. In this study, bioinformatics, semi-quantitative RT-PCR techniques were used to investigate the sequences and expression characteristic of the CBL-interacting protein kinases in maize (ZmCIPKs). Here, we screened and isolated ZmCIPK08, also its plant overexpression vector pBI221-ZmCIPK08 was constructed successfully, which laid the foundations for the further study on its function. The main results are as follows:1. Five candidate genes (GenBank accession No.EU974290, EU968441, AY104819, AY104530, AY104543, respectively) were analyzed bioinformatically. Sequence analysis results primarily suggested that these five genes belonged to the CIPK protein gene family. Phylogenetic analysis showed that, EU974290, EU968441, AY104819, AY104530, AY104543 in Zea mays had closer relationship with ZmCIPK16 (GenBank accession No.EU907939) in Zea mays, SbCIPK16 (GenBank accession No. FJ901202), SbCIPK21 (GenBank accession No. FJ901210), SbCIPK03 (GenBank accession No. FJ901215), SbCIPK08 (GenBank accession No.FJ901227) in Sorghum bicolor, respectively. The five candidate CIPK protein were speculated to have closer genetic relationship with the CIPKs in Zea mays and Sorghum bicolor. According to homology, EU974290, EU968441, AY104819, AY104530, AY104543 in Zea mays were temporarily named ZmCIPK17, ZmCIPK18, ZmCIPK21, ZmCIPK04, ZmCIPK08, respectively.2. Semi-quantitative RT-PCR results showed that, certain ZmCIPK21, ZmCIPK18, ZmCIPK04 and ZmCIPK08 expression level in leaves and roots of maize were detected under normal growth conditions, yet certain ZmCIPK17 expression level only in roots but not in leaves of maize were detected under the same conditions. The expression of ZmCIPK08 reached its highest level in leaves sooner than that of ZmCIPK04, yet the expression of ZmCIPK04 reached its highest level in roots sooner than that of ZmCIPK08 under high NaCl concentration, which indicated that the tissue expression patterns of ZmCIPK04 and ZmCIPK08 were different under osmotic stress. The expression levels of ZmCIPK21, ZmCIPK17, ZmCIPK04 and ZmCIPK08 were changed both in leaves and roots of different processing time, and the expression level of ZmCIPK18 was also affected both in leaves and roots of different processing time whether under high NaCl concentration or exogenous ABA, it is preliminarily speculated that these five genes are likely to exist in the ABA dependent stress response pathway, and this study provides a starting point to understand the regulatory mechanisms of ZmCIPKs.3. One full-length cDNA encoding a CIPKs homologue was isolated from maize (Zea mays) by RT-PCR and named as ZmCIPK08 (GenBank accession No. AY104543). The predicted ZmCIPK08 protein contained 451 amino acids with an estimated molecular mass of 50.87kDa and an isoelectric point of 6.15. The putative ZmCIPK08 protein structure contained a serine/threonine kinase domain in N-terminal region and a NAF domain in C-terminal region. ZmCIPK08 had higher amino acid sequence similarity with Sorghum bicolor CIPK08 (GenBank accession No.FJ901227), Vitis vinifera CIPK17 (GenBank accession No.FJ901245) and Arabidopsis thaliana CIPK8 (GenBank accession No.AF290193), which were 95%, 74% and 73%, respectively.4. The plant overexpression vector pBI221-ZmCIPK08 was constructed,and the DNA fragment in the coding region of ZmCIPK08 was in downstream of CaMV35S promoter of vector PBI221, restriction enzyme cutting confirmed that the gene had been correctly inserted in the vector. |
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