Comparation Of Rice And Maize CIPK Gene Families And Functional Analysis Of Rice Zinc Finger Protein Gene ZFP252

Calcium is a universal second messenger in signaling pathways. Ca2+ signals are decoded by Ca2+-sensors in cell and in turn activate genes expression and physiological responses. The Ca2+-sensors calcineurin B-like proteins (CBLs) and their targets CBL-interacting protein kinases (CIPKs) constitute a distinctive calcium-signal network, and play key roles in regulation of stress responses in plant. Here, a CIPK gene family was identified in maize genome by bioinformatics, and compared with rice CIPK family in gene structures, evolution, and expression patterns. There are at least 43 members in maize CIPK family,13 more than that in rice, which might result by the expansion and duplication of maize genome. The gene structure of rice and maize CIPKs are similar, and both families’ members can be divided into intron-rich and intron-less groups. A high degree of amino-acid sequence homology exists between rice and maize CIPK proteins with a Ser/Thr kinase domain and a NAF motif necessary for interaction with CBLs. Phylogenetic analysis suggests that maize closely related with rice in evolution of CIPK genes. The digital expression reveals that most rice and maize CIPK genes ubiquitously express in various tissues, while maize CIPK5 may specifically express in pollen. Microarray data and semi-quantitative RT-PCR results show that 9 maize CIPK genes were induced by salt, drought, cold or heat stresses, and furthermore display similar expression patterns with the homologous rice CIPKs, suggesting that the homologous CIPK gene pairs in these two species may have similar functions. These results will better understand for the CIPK gene families and CBL-CIPK signal network in plant.Zinc finger proteins are key regulators in gene expression, development and stress responses. Rice gene ZFP252 encodes a C2H2 zinc finger protein which is a transcription factor involving in stress response, and significantly improved salt and drought tolerance in rice by over-expression. For understanding the function and molecular mechanism of ZFP252, the transcriptome levels between ZFP252-overexpressed lines and wild-type were assayed by microarray, and 187 differentially expressed genes, involving in multiple biological processes, were detected. Among them, one up-regulated gene encodes an E3 ubiquitin ligase OsRING-1, and three down-regulated genes encode TRAF-like protein, transcription factor Homeodomain and vesicle-related membrane protein VAMP724, respectively, were highly consistent in expression level among different lines over-expressing ZFP252. Moreover, ZFP252 and OsRING-1 were induced by salt and drought, whereas 3 other down-regulated genes were suppressed by salt. Therefore, it is likely that these 4 genes are regulated by ZFP252 in stress-responsive pathways, and the molecular mechanism of ZFP252 is proposed as follows:under salt or drought stress, ZFP252 activates the ubiquitin pathway via up-regulating OsRING-1 or down-regulating TRAF-like to improve the stress tolerance in plant; meanwhile, ZFP252 suppresses VAMP724 expression to prevent the vesicle-mediated H2O2 input to vacuoles and promote vacuoles to absorb Na+ and reduce cellular ion toxicity; ZFP252 might also activate the downstream stress-resistance pathway by suppression of Homeodomain.Since an E3 ubiquitin ligase, a key component in ubiquitination-mediated protein degradation, transcriptionally regulated by ZFP252, we analyzed the proteome level in ZFP252 over-expression rice by two-dimensional gel electrophoresis (2-DE), and found 33 differentially expressed protein spots by comparison with wide type. Twenty proteins were successfully identified by MALDI-TOF/TOF, and their mRNA levels were not significantly changed in transgenic line, suggesting that different protein level might cause by post-transcriptional regulation or protein metabolism. Among, most proteins are down-regulated protein involving in metabolisms of glucose, lipid and amino acid, or in photosynthesis, except for 4 up-regulated proteins, translation initiation factor 5A, cell wall component hydroxyproline-rich glycoprotein-like protein, and two reactive oxygen species (ROS) scavenger Glutathione S-transferase GSTF14 and NADPH quinone oxidoreductase 1. To decrease the metabolism level when in bad condition is a self-protection mechanism of plant. Therefore, it is likely that ZFP252 regulate the ubiquitination pathway to degrade proteins for decline the basic metabolism level in cell, and enhance the ability of self-protection in plant; meanwhile, ZFP252 may improve the synthesis of detoxification enzymes and cell wall components by post-transcription regulation, to clean the ROS induced by stresses and strengthen the cell wall, and increase stresses tolerance in plant.