Function Analysis Of OsMKK4in Rice

The mitogen-activated protein kinase (MAPK) cascade is composed of mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase kinase kinase (MAPKKKs), which widely exist in plants, mammals and yeasts. The intracellular signals were relayed and amplified through sequential phosphorylations to the target proteins. Studies indicate that:MAPK cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses.OsMKK4was isolated previously in our laboratory, belonging to MAPKKs family. In present study, the protein structure, the protein subcellular localization and biological functions were studied.Bioinformatics analysis indicated that the full-length cDNA sequence was1879bp and encodes a369amino acid labile protein with a predicted molecular mass of39.9434kD and a PI of9.45. Homologous sequence alignment revealed that OsMKK4has the ALKV conserved domain and the S/TxxxxxS/T motif as the phosphorylation site like other plant MAPKKs. In addition, a putative MAPK-docking domain RRRPDLTLPL and a NES sequence exist in the N-terminal extension of OsMKK4sequence. It also indicates that OsMKK4belongs to group C and share very high similarity with BdMKK4, ZmMKK4, Sb04g035370, AtMKK4and AtMKK5.Upon semi-quantitative RT-PCR and real time PCR, we analyzed the expression of OsMKK4in different organs (root, stem, leaf, sheath, spikelet). The result suggests that OsMKK4may play an important role in all parts of rice growth and development because its expression pattern is not tissue-specific, only with higher expression in leaf and sheath.An interference plant expression vector towards OsMKK4was constructed, and then introduced into9522(a japonica variety) by Agrobacterium-mediated transformation. The results show that the transgenic plants have lower expression of OsMKK4, and shorter grain length, shoter plant height as expected like the mutants, suggesting that OsMKK4functions in the rice development, including plant height and grain traits.TargetP1.1, WOLF PSORT, Subloc v1.0and BaCelLo are selected to predict the subcellular localization of OsMKK4, and the results indicate the protein location is uncertain. The fusion protein of OsMKK4-eGFP is transformed into tobacco by the Agrobacterium-mediated method. LSCM (Laser Scanning Confocal Microscope) clearly indicate that OsMKK4expresses in tobacco mesophyll cells both in cytoplasm and nucleus. The elucidation of localization of OsMKK4will be helpful to understand their in vivo functions.