The Functional And Biochemical Study Of Plant Protein Kinase CIPK And CK1A

Phosphorylation,which is catalysed by protein kinase,is one of the most important posttranslational modifications in plant.It is a key mechanism for the regulationof almost all signal transduction pathways.Several protein kinases have been determined in many plant species,and they were involved in plant development,plant stress resistant,and many signal transduction.CIPK and CK1 are two protein kinase families which are widely studied in plants.CIPK plays an important role in the process of stress response in the CBL-CIPK system.CK1 was reported playing key role in regulating flowering time,root development,and microtubule regulation.On the basis of the earlier work of our lab and collaborate,we chosed the plant CIPK and CK1 as our research object in this dissertation.The two kinase gene families were analysed by bioinformatictools;real-time quantitative PCR was used to analysethe gene expression patterns;the over-expression mutants of CIPK and loss of function mutants of CK1 were used for functional research in stress signal transduction and devolopment regulation.The main results are as follows:?1?The DNA and protein sequences of the CIPKs were analyzed by bioinformatic methods.Gene structure analysis showed that SbCIPKs could be divided into two groups,the multiple introns subgroup and intron-free subgroup.And it can be finely divided in two independent branches in the phylogenetic analysis.The genomic evolution analysis showed that the segmental duplication was both found in the multiple introns subgroup and intron-free subgroup,while the tandem duplication was only found in the intron-free subgroup.SbCIPKs contain two conserved functional domains: the N-terminal kinase domain and the C-terminal NAF domain.The kinase domain is the catalytic domain with highly conservation.The NAF domain is the domain about the CBL interaction,with a relatively low conservation.It showed that the promoter region of the SbCIPKs contain multiple stress signal responsive element in Plant CARE analysis.The RT-PCR also showed that the expression level of SbCIPK wasinduced under alkali stress,indicating that the SbCIPKs might play a role in the salt stress resistance.?2?Our earlier work showed that At CIPK14 play key roles in salt stress response,so the highly homologous SbCIPK4 was chosed in this study.The wild type plant suffered more growth inhibition than SbCIPK4 overexpression mutants under salt stress condition.In the mean time,stress related genes level were highly induced in SbCIPK4 overexpression transgenic plants.These results indicated that SbCIPK4 may increase plant resistance to salt stress by upregulating the stress related gene expression.The subcellular localization of GFP:SbCIPK4 showed that SbCIPK was located in the nucleus.?3?The SbCIPK4 can interact with SbCBL5 proteinsin the yeast two hybrid screening.The SbCIPK4 interacts with the N-terminal domain of SbCBL5.Pull-down assay also verified the interaction between SbCIPK4 and SbCBL5 in vitro.Under salt stress,it showed that both SbCBLs and SBCIPKs were induced by Na₂CO₃ inthe co-expression analysis.?4?The CK1A promoter contains a large number of light response element and stress response elements according to the plant CARE analysis.CK1A was highly expressed in severalgrowth stages and organs,especially in the flowers and seedsmicroarrayand Q-PCRassay.ck1a mutant showed a late flowering phenotype under long day conditions,with downregulated expression of the flowering genes FT,SOC1,and CO,especially the FT gene.The prokaryotic expression and purification system of CK1A kinase domain was successfully established,and thepurified CK1A recombinant protein showed auto-phosphorylated activity in vitro.In summary,we investigated and elucidated the function and mechanism of CIPK in stress response,and the biological function of CK1A in regulating plant flowering time in photoperiod pathway.