Subcellular Localization Analysis of Rice Protein Kinase Gene OsCIPK5 and Generation of Its RNAi Transgenic Plants

Plant CBL-interacting protein kinases (CIPKs) play an important role in stress signaling transduction and enhancing plant stress tolerance. However, the functions of OsCIPK5 in the rice (Oryza sativa) have not been studied. In order to study the functions of OsCIPK5, RT-PCR method was used to clone OsCIPK5 gene from rice leaf (Oryza sativa Japonica Group cultivar Nipponbare).The bioinformatics analysis showed that OsCIPK5 contained two functional domains, N-terminal kinase activation loop domain and C-terminal NAF domain, which were the same as other CIPK that had been reported.OsCIPK5 was highly homologous in amino acid with other five plant species, especially with Oryza brachyantha, which was about 94%. By Real-time PCR approach the expression pattern of OsCIPK5 was detected in the rice seedlings responses to potassium. The result showed that the expression of OsCIPK5 in root was up-regulated under low potassium treatment, but not in leaf. The recombinant expression plasmid pEarleyGate101-OsCIPK5 contained yellow fluorescent protein (YFP) was transformed into GV3101 and then the positive clones were infiltrated into the epidermal leaves of Nicotiana benthamiana. The results revealed that the OsCIPK5 protein localized on the nucleus, cytolemma and cytoplasm. The RNAi vector containing OsCIPK5 specific gene was constructed and transferred into EHA105 (Agrobacterium tumefaciens), then rice transformation was conducted through agrobacterium-mediated system. T₀ RNAi transgenic seedlings of OsCIPK5 were obtained and 26 plants were identified to be positive.The expression of OsCIPK5 in transgenic plants was obviously reduced compared with that in wild type by Real-time PCR analysis.These results including bioinformatics analysis, expression pattern, subcellular localization analysis and transgenic rice may be useful in study on the important role of OsCIPK5 in the process of plant growth.