The Functional Analysis Of ANK9, A Member Of Putative Ankyrin Protein Family In Arabidopsis Thaliana

ANK protein, a kind of important member of the binding protein family, is characterized by ANK domains that are consisted of ankyrin repeat motifs. The main function of ANK proteins is mediating protein-protein interaction, and participating in the regulation of diverse cellular activities. In Arabidopsis, there are 108 ANK proteins, but the function of these genes are rarely known. Our prevous work has proven that ANK6 played essential role in plant double fertilization. Another ankyrin repeat gene ANK9 showed high homology with ANK6 (76%). The analysis on the function of ANK9 is helpful for elucidating the physiological mechanism of ANK6 in Arabidopsis. The results on ANK9 are shown below.ANK9 has 528 nucleoside acids in its open-reading-frame(ORF). It encodes a putative ankyrin protein contained 175 amino acids, which contains three ANK repeat motifs.ANK9 displays tissue-specific expression in Arabidopsis thaliana. GUS staining showed that ANK9 expressed mainly in the root tip, shoot tip, cotyledons, mature leaves and pollen. ANK9 also expressed in wounds of Arabidopsis.The expression of ANK9-GFP fusion protein was driven by the cauliflower mosaic virus 35S promoter. We determined the subcellular localization of GFP fusion protein by confocal microscopy. We found that ANK9 was expressed in cell with spotted distribution. The number and shape of the fluorescent signals was similar to the nucleus. Furthermore, green GFP fluorescence also existed in the periphery of cells. So we preliminary presume that ANK9 locates in the nucleus and also in the plasma membrane.The T-DNA insertion homozygous mutants of ANK9 gene was screened and analyzed, but without obvious phenotypes. Overexpression plants of ANK9 in Arabidopsis exhibited the bolting time one week earlier than wild type plants. This indicates that ANK9 is related to flowering.Yeast two-hybrid library screen experiment shows that ANK9 interacts with CAT3 which is related to the process of stress and injury, suggesting that ANK9 may play an important role in the stress defense of plants.Detailed studies were conducted on the comparison of ANK9 and ANK6. It found that 75.4% of their gene sequences and 76.1% of their protein sequences are the same. But protein sequence showed that ANK6 had a mitochondrial targeting signal peptides, while ANK9 didn’t. The subcellular localization of GFP showed ANK6 was not only in the nucleus, but also in the mitochondria. Yeast two-hybrid experiment showed that ANK6 also interacted with CAT3. ANK6 plays an essential role in the embryonic development in Arabidopsis. There were many empty seats in the siliques of the ank6/+mutant, but ank9/ank9 mutant didn’t have this phenotype. It demonstrated that the different localizations of two genes may conduce to their different functions. So we speculate that ANK9 and ANK6 are both located in the nucleus and they may be both involved in the regulation of injury stress. Moreover, ANK6 was also located in the mitochondria and it may be involved in embryonic development.In a word, the study on gene ANK9 is important for further studing of the action mechanism of ANK protein-mediated and protein-protein interactions. In addition, the comparative study on the similar gene ANK9 and ANK6 is significant to study the high homology ANK protein. And these studies will also contribute to a new breakthrough of signal transduction in plant.