Functional Characterization Of Tonoplast Na～+/H～+ Antiporter (NHX1) From Chenopodium Glaucum And Comparative Study Of The Putative Regulation Region Of K～+ Or Na～+ Transportation In C-terminus Between CgNHX1 And OsNHX1 (Oryza Sativa)

With the successful accomplishment of the human genome project and plant genome project such as Arabidopsis thaliana and rice, as well as profound recognization on genes, the study of plant genomics has been transferred from the structural genomics which focuses on determining the complete sequences of the genome to the functional genomics which elucidates the biological function of genes.So various techniques of reverse genetics being used for research on gene function will be the key point of life science research accompany with the arrival of time of post genome.High salinity is one of primary factors limits the development and growth of plant. Overly high soil salinity can cause salina and limit the utility of soil. Up to date, 20% of the world's cultivated land and nearly half of all irrigated land are affected by salinity. High concentration of sodium ions caused ion toxicity, osmotic stress, and K~+/Na~+ ratio imbalance, which resulting in deleterious effect on critical biochemical process of plant.That is why plant either halophyte or glycophyte has to reduce cellular Na~+ and keep K~+/Na~+ balance when exposed to salt stress. Advances in molecular genetics and plant transformation have made it feasible to assess biotechnological strategies in improving the salt tolerance of crops, keeping steady-state growth and increasing the output in the saline environment. NHX1 , a tonoplast Na~+/H~+ antiporter energized by the ?pH across the tonoplast, facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance, an active antiporter would function to sequester Na~+ into the vacuole, which results in avoidance of cytoplasmic Na~+ toxicity and maintenance of a high cytoplasmic K~+/Na~+ ratio. In parallel, vacuolar Na~+ would serve as an osmoticum necessary for cellular H2O homeostasis. Overexpressing AtNHX1 can accumulate the large numbers of antiporter in the transgenic Arabadopsis, oilseed rape, tomato and cotton with increased salt tolerance. Based on the cloned cDNA sequence of tonoplast Na~+/H~+ antiporter gene CgNHX1(AY371319) from Chenopodium glaucum in Xinjiang, Our study showed that overexpression CgNHX1 could enhance the capacity of salt-tolerance in transgenic Arabidopsis thaliana. Meanwhile, the C terminus of NHX1 from both Chenopodium glaucum and Oryza sativa (AB 021878) were compared and the recombinant plant expression-vector containing full length NHX1 gene or NHX1 gene deleted the C terminal region were constructed . After transformation into Arabidopsis thaliana, the T1 transgenic plants overexpressing CgNHX1-histag, OsNHX1-histag, dcCgNHX1-histag and dcOsNHX1-histag were obtained respectively. Furthermore, fusion expression vector pCAMBIA1301-1-CgNHX1-GFP and pCAMBIA1301-1-CgVP1-GFP were successfully constructed for subcellular localization of protein and The detection results of instantaneous expression showed that CgNHX1 may express in membrane system.At first, a pair of primers were designed according to the sequence of CgNHX1 and were used to amplify the coding frame of CgNHX1 gene from Chenopodium glaucum total RNA by RT-PCR. A binary expression vector pCAMBIA1301-1-CgNHX1 harboring with CgNHX1 was constructed and introduced into Arabidopsis thaliana bv Agrobacterium-mediated method.T2 homogenous plantlets were obtained from hygromycin-resistant seedlings. PCR and RT-PCR analysis indicated that CgNHX1 was integrated into the genome of Arabidopsis thaliana and expressed at transcriptional level. The Na~+ content of both wt and transgenic plants increased when exposed to 50 and 100mM NaCl. Although not significant, the Na~+ concentration in transgenic plants was higher than that of wt . Although the relative growth rate of transgenic plants and wild-type (wt ) both decreased, the root length of transgenic plants was significantly longer than that of wt under 150mM NaCl treatment. However, the Na~+ content of transgenic plants was lower than that of wt when exposed to 150mM NaCl. Our data showed that overexpression of CgNHX1 could enhance the capacity of salt-tolerance in transgenic Arabidopsis thaliana.The C terminus of AtNHX1 was reported to play an important role in the regulation of cation transportation of Na~+/H~+ antiporter. In order to compare the different function of C terminus of NHX1 between halophyte and glycophyte, specific primers were designed to amplify the coding frame of NHX1 gene which were deleted about 300 base pairs corresponding to about 100 amnio acid of hydrophilic C terminus of NHX1 protein from Chenopodium glaucum and Oryza sativa by RT-PCR. Then the binary expression vector harboring with full length NHX1gene or NHX1 gene deleted the C terminus with six histidine tags were constructed and introduced into Agrobacterium tumefacience. After transformation by floral dip method, the transgenic plants overexpressing CgNHX1-histag, OsNHX1-histag, dcCgNHX1-histag and dcOsNHX1-histag were obtained respectively and the CgNHX1-histag and OsNHX1-histag transgenic plants were identified by PCR using specific primers. Meanwhile, the primary conditions of growth and transformation for Arabidopsis thaliana were examined.The subcellular localization of protein acts an important part in its function and critical biochemical process. A 756bp GFP fragment was amplified from pCAMBIA1302 vector with two restriction site Salâ… a nd Pstâ… and cloned into pCAMBIA1301-1 vector named as pCAMBIA1301-1-GFP. According to the sequence of CgNHX1 and CgVP1 (DQ 443731) cDNA sequence cloned by our laboratory , specific primers were designed to amplify the coding frame of CgNHX1 and CgVP1 genes by RT-PCR, in which the terminator sequence was deleted. Recovered RT-PCR products were digested with BamHâ… a nd Salâ… or Kpnâ… and Salâ… a nd ligated with pCAMBIA1301-1-GFP. The results of digestions and PCR analysis showed that we had got the fusion expression vector pCAMBIA1301-1-CgNHX1-GFP and pCAMBIA1301-1-CgVP1-GFP and the sequencing results indicated that the two fused genes were in the same reading frame. The detection results of instantaneous expression showed that CgNHX1 may express in membrane system.