| CMS is a widely observed phenomenon in plant kingdom, which is characterised by the inability to produce functional pollen grains during the reproductive process. But the development of pistil is normal. Based on this characteristic, the CMS line is widely used to produce hybrid seeds, which eliminates the tedious process of manual emasculation. It is generally accepted that CMS is caused by the incompatibility of the nuclear genome and the cytoplasmic genome. Thus CMS is regarded as a good model to study on the nuclear-cytoplasm interactions. In this study, we used the CMS line MeixiangA (MxA) and its maintainer line MeixiangB (MxB) as materials. To study the cellular metabolism and the retrograde signaling during the abortive process, proteomic and miRNA studies are performed on MxA and MxB. The results will give us new insights on the abortive mechanisms as well as the metabolic regulations in this new CMS line. In addition, we have introduced molecular dynamics simulations to study protein-protein interactions in rice.The main results in this research are as follows:1. Cytological studies were performed for MxA and MxB. The pollen of MxA cannot be stained by I2-KI. DAPI staining result implys that MxA is aborted at the early uninucleate stage. A potential sterile gene ZD352was cloned, sequencing result shows that it is highly homologous with WA352with only two nucleotide variance. When it is transformed into the BL21cells, expression of ZD352protein can be induced and not lethal to E. coli. On mRNA level, it is widely expressed in various tissues such as leaf, root, young spikelet. On protein level, it is mainly accumulated in the anthers at the MMC/meiotic stage.2. Based on the results from the cytological study, iTRAQ-labeling quantitative proteomic analysis was performed using the anthers at the uninucleate stage from MxA and MxB to study their protein expression profiles. A total of688proteins were quantitatively identified. Setting the ratio (MxA/MxB)>1.1or<0.9as threshold for a distinct expressed protein,45proteins were found to be significant differentially expressed. In MxA, a number of proteins involved in the ribosome assembly were up-regulated; However, some proteins involved in carbohydrate metabolism, stress response and cellular metabolisms are down-regulated. Among which, two of the calcium-binding proteins were decreased in MxA. Our previous study demonstrated that the concentration of free calcium ions were increased in ZidaoA. These results demonstrated that Ca2+might play regulatory roles during the abortive process in MxA. In addition, the ROS level in MxA anthers is much higher than MxB, which implied that ROS might be also participated in the regulation of nuclear gene expression.3. Reports have shown that miRNAs are involved in the reproductive development in plants. To comprehend the relationship between miRNA and the CMS occurance, the miRNA expression profile between MxA and MxB was examined using high-throughput sequencing approach. Results showed that24miRNAs were differentially expressed, and these miRNAs were involved in various biological metabolic pathways such as reproductive regulation, stress response. We speculate that some of these miRNAs might participate in the regulation of CMS. Besides, miRNA editing events were observed in rice spikelet.4. In most cases, protein complexes are formed to play their various regulatory roles in the cells. In this study, we have tried to use structural biology in combination with molecular dynamics simulations approach to study the binding mechanisms betweeen Hsp90and Sgtl in rice. Molecular dynamics simulations and binding free energy analysis indicate that three robust salt bridges were crucial for maintaining the interactions between Hsp90and Sgtl. In addition, Tyr173of Sgtl played key roles in the interactions, as it was not only participating in the hydrophobic interactions but also involved in a hydrogen bonding with Hsp90. |
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