| Plant vacuolar membrane H+ -ATPase (V-ATPase) is a multi-subunit protein complex. The most basic functions of V-ATPase were to maintain the pH balance within the cytoplasm (pH value stable state) and to establish the transmembrane electrochemical gradient which provides energy for the related metabolism. Plant V-ATPase shows complexity and diversity under stress conditions. The role of V-ATPase in stress resistance would be achieved by in-depth studying of its important subunits. Some reseachers showed the stress tolerance function of the V-ATPase subunits a, c, G and so on. However, to the best of our knowledge, there are few publish studies on abiotic stress regulation of c" subunit. So, in this study, the gene expression and stress resistance of c" subunit were done. We have obtained the following results:The expression patterns of ThVHAc’’l were analyzed in roots, stems and leaves of T. hispida exposed to 0.4 mol/L NaCl,20% PEQ 0.3 mol/L NaHCO3 or 150 μmol/L CdCl2 treatments. The results showed that ThVHAc’’l can responded to these abiotic stress. And which expression levels were mainly up-regulated in all studied tissues. For example, the expression level of ThVHAc"1 was induced 8.88-fold in leaves after 0.4 mol/L NaCl.In the previous study, ThVHAcl has been vertified to increase the ability of resistent stress in plant. In order to explore whether ThVHAc"1 play role in stress resistance, the ThVHAc"1 gene was transformed into yeast. The survival rate of transgenic yeast and the conrol yeast under various stresses were compared. The results showed that the yeast transformed with ThVHAc"1 gene possessed higher tolerance to salt, peroxide, heavy metals, abnormal temperature and drought stress than the control yeast, indicating that ThVHAc"1 gene was also involved in stress tolerance regulation.For further study on the stress resistance of ThVHAc"l in plant, ThVHAc"l was inserted into a plant expression vector pROKⅡ with control of 35S promoter to form recombinant vector 35S::ThVHAc"l, which was then transformed into Arabidopsis by Agrobacterium-mediated flower dip method. The overexpression of ThVHAc"1 Arabidopsis lines were obtained. The T3 generation of transgenic plants were used to further salt treatment. The germinant rate, growth and related physiological indicators were surveyed and compared between the transgenic lines and control Arabidopsis. The result indicated that Arabidopsis transformed with ThVHAc"1 possessed obviously higher germinate rate and biomass accumulation than the control. The results of histochemical staining (DAB, H2DCF, Evans Blue) assays showed that the reactive oxygen species (ROS) accumulation and cell damage in ThVHAc"l transgenic lines were significantly less and weaker than WT under NaCl treatment. Meanwhile, both transgenic and WT Arabidopsis were not obvious different on protecting enzyms activity, lectrolyte leakages, MDA content under normol condition. However, the activities of SOD, POD, GST of ThVHAc"l transgenic lines were significant higher than WT after NaCl stress. By contraries, the content of H2O2, MDA and lectrolyte leakages were significant less than WT under NaCl stress condition. These results suggested that overexpression of ThVHAc"l significantly enhanced the salt tolerance of transgenic Arabidopsis.In order to test the results of heterologous expression of ThVHAc"l in Arabidopsis, ThVHAc"l was transient overexpressed in T. hispida. The relative index were compared between the transient overexpressed ThVHAc"l T. hispida and the control (transient transfer pROK Ⅱ vector T. hispida). The results showed that the activity of protecting enzyms and level of ROS and cell damage of overexpression lines were obvious better than the control under salt stress, indicating that the expression of ThVHAc"l indeed enhanced the plant tolerance to salt stress.In addition, in order to understand the upstream mechanism of ThVHAc"l on tolerance regulation, the upstream 1033 bp sequence of ThVHAc"l was cloned by using the chromosome walking method. And the promoter contained many important cis-elements, such as G-box, TATA-box, CAAT-box, W-box, etc. The results of yeast one-hybrid assay showed that ThDof8 can specially bind to the DOF element in the ThVHAc"l promoter. And it may be the upstream regulatory gene of ThVHAc"l. Therefore, it was preliminarily confirmed that ThDof8 may be involved in the regulation or co-regulation of plant salt stress tolerance with ThVHAc"l.In a word, this study preliminarily identified that ThVHAc"l contributed to the salt resistance of plant, enhancing our understanding of the subunits genes of V-ATPase and laid a solid foundation for illustrating the participation of V-ATPase into stress resistance. In the future, we will further study on the upstream regulation mechanism of ThVHAc"l and the effect of its expression on V-ATPase. |
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