The Study On The Function Of Vacuolar H~+-ATPase Subunit B In Arabidopsis Thaliana

Proton pump is an energy system promoting the movement of protons across the membrane, which is widespread in almost all cells. Vacuolar H+-ATPase (V-ATPase or VHA) is one of them. V-ATPase is a membrane-bound multisubunit enzyme complex, and is highly conservative. It is composed of the proton transmembrane transport VO located in the plasma membrane and V1 catalyzed hydrolysis of ATP and located in the cytoplasm. It uses the energy released from ATP hydrolysis to proton pump out extracellular or pump into the intracellular organelles cavity in order to control the pH, which will maintain acidic environment in the extracellular environment or intracellular organelles cavity, so as to maintain relatively stable intracellular pH and to be conducive for the cellular processes.The B subunit is not only the core component of the VO complex, but also the subunit combining microfilaments. It has reported that Arabidopsis V-ATPase have three B subunits (AtVABl, AtVAB2, and AtVAB3), and the N-terminal domain of each subunit contain some motifs which like ABPs profiling. V-ATPase could interact with actin cytoskeleton directly through actin binding sites in the N-terminal domain of B subunit and the two domains of C subunit. Through the vitro biochemical experiments we known that AtVABs combined F-actin, and they may perform some conserved actin-related functions, such as binding, bundling, capping, and stabilizing activities. However, the physiological function of V-ATPase B subunit has not been known. We study on B gene specific expression in organs (GUS staining), at the transcriptional level by RT-PCR, and at the subcellular level. In addition, we get mutants of Arabidopsis B gene and analyze mutants phenotype treated by various abiotic stresses, such as salt, ABA, mannitol, glucose, in order to reveal the tissue localization, subcellular localization and physiological functions of V-ATPase B subunit in Arabidopsis. The main results were summarized as follows:1. Using semi-quantitative PCR analysis and immunohistochemical methods, the analysis of the B subunit expression profiling and results of GUS activity in various tissues of the the transgenic proAtVABs-GUS plants proved that AtVABs genes abundantly express in the cotyledons, roots, flowers, leaves, stems, pollen tubes and other organs.2. Based on the PI staining and plasmolysis experiments, we analysis the subcellular localization of AtVABs genes, and determine that they all located in the plasma membrane, vacuolar membrane and nuclear.3. Studying the expression level of the transgenic plants proAtVABs-GUS treated by abiotic stress (NaCl, mannitol and glucose), the results showed that expression level of AtVABs were up-regulation correspondingly.4. Through the studies of mutant vab3-1 germination and seedling growth treated by abiotic stress (NaCl, ABA, mannitol and glucose), there was no significant difference between mutant vab3-1 and WT.In summary, the expression patterns of AtVABs genes are similar, and AtVABs genes may paly an important role in the regulation of abiotic stress.