Functional Studies Of The Plasma Membrane Na~+/H~+Antiporter From Bruguiera Gymnorrhiza(L.)LAM

The plasma membrane sodium exchanger SOS1is a critical salt tolerance determinant in plants. Bruguiera gymnorrhiza is one kind of strong halophyte, the seawater salinity of its growth envioronment is up to3.0%～3.2%which is higher than average level. There is no salt gland in Bruguiera gymnorrhiza, but its roots could exclude99%salinity, indicating that the Na+efflux proteins at its plasma membrane may function very well, so Bruguiera gymnorrhiza should be a kind of perfect plant to study the function of SOS1.The main research results were:1.We cloned the BgSOS1gene included3462bp which encodes1153amino acids. Bioinformation analysis showed that the Na+/H+antiporter shared higher identity in amino acids sequences with tomato SOS1(66.61%), rice SOS1(62.18%), wheat SOS1(60.19%), with salt cress SOS1(63.97%) and with poplar SOS1(75.97%). Twelve putative transmembrane domains in N-terminal of the protein were predicated and a long of C-terminal distributes in the cytoplasm. We verified the localization of the BgSOS1by using BgSOS1-GFP fusion protein in yeast cells. The result showed that the location of recombinant yeast was the plasma membrane. Together, these results demonstrated that BgSOS1was maybe the plasma membrane Na+/H+antiporter.2.SOS (Salt Overly Sensitive) signal pathway is the main regulatory mechanism of salt tolerance. There are three main genes:SOS1、SOS2、SOS3. The expression of single BgSOS1gene did not increase the tolerance of yeast strain AXT3K to salt stress, but the salt tolerance of AXT3K co-transformed with BgSOS1, AtSOS2and AtSOS3was significantly higher compared to non-transgenic yeast strain, indicating that Na+/H+antiporter activity of SOS1might be regulated through protein phosphorylation by SOS2/SOS3kinase complex. At the same time in the recombinant yeast SOS2protein kinase could activate the activity of BgSOS1, thus raise the salt tolerance of recombinant yeast.3.To amplify the sequence of Ser1143and Ser1145mutations of BgSOS1(BgSOS1-M) by designing the primers, constituting yeast expression vector was transformed into the salinity sensitive strain AXT3K. The drop test showed that either single AtSOS2or the SOS2-SOS3protein kinase complex both could regulate the activity of SOS1.4.The previous report showed that an auto-inhibitory C-terminal domain existed in SOS1which could keep the transporter in a resting state with basal activity.Because the sequence of BgSOS1are high homology to AtSOS1and the single BgSOS1could not raise the salt tolerance of recombinant yeast, these indicate there is also an auto-inhibitory domain in the BgSOS1C-terminus. To test this idea, by deleting the predictable auto-inhibitory domain, the mutant obviously raised the salt tolerance of recombinant yeast.Under salinity stress, the relative salt tolerance of these transformants are better than the coexpression of SOS1、SOS2and SOS3. This suggests that the incremental salt tolerance level of BgSOS1-3000is maybe correlate with potential economy value.5. Constructing of the plant expression vector pCAMBIA1302-BgSOS1and pCAMBIA1302-BgSOS1-3000, these two plasmids was transferred into Agrobacterium tumefaciens (LBA4404) and getting the transgene strain LBA4404-pCAMBIA1302-BgSOS1and LBA4404-pCAMBIA1302-BgSOS1-3000.This also pave the way for fundamental research of BgSOS1and its hyperactive gene in the area of crop.