Isolation And Functional Characterization Of A Cell Cycle Related Gene NtCDC48 From Tobacco

CDC48 is a highly conserved protein in eukaryote which belongs to the AAA (ATPase associated with a variety of cellular activities) superfamily. It can interact with many different co-factors and form some protein complex to play important roles in several cellular processes, such as mitosis regulation, spindle disassembly and membrane fusion.We used the fission yeast, Schizosaccharomyces pombe, in this study to identify a tobacco gene NtCDC48, A CDC48 homologue NtCDC48 which exhibits high identity to Arabidopsis AtCDC48(92%). The full length of NtCDC48 is 2729bp. From its ORF, we predicted that this protein contains 808 amino acids which probably include two typical ATPase modules (one 245-374 and another 518-646).Overexpression of NtCDC48 dramatically increased the mitotic index in BY-2 cells and also changed the pole structure of mitotic spindle patterns which were normally concentrated into two points, but now became extremely loose. The localization experiments show that NtCDC48-GFP fusion proteins exhibit regular changes during the transition of the cell cycle: the NtCDC48-GFP fusion proteins show up in the whole cell at prophase; scatter in cytoplasm far away from cell plate during metaphase; and concentrate on both sides of cell plate where the phragmaplast exists at anaphase. These results indicate that NtCDC48 plays important roles in regulating spindle disassembly.Northern blot show that the NtCDC48 gene constantly expresses during the whole cell cycle in BY-2cell, suggesting that NtCDC48 protein is very important to maintain normal physiology of BY-2 cells.The transgenic tobacco plants of sense and antisense NtCDC48 controlled by of a CaMV 35S promoter show different phenoptypes: Comparing with the antisense NtCDC48 transgenic plants, the sense transgenic plants have fewer flowers, but each silique contains more seeds, also indicating that NtCDC48 protein involves in seed development of tobacco.In summary, we demonstrated that NtCDC48 protein plays very important roles in the cell cycle control and seed development of plant. So, it is necessary to pay more attentions to its role in plant development further.