Clone, Expression And Characterization Of Arabidopsis AtCCX1

Salt stress is one of the major abiotic stresses that depress plant growth and drastically limit crop production. Finding and improving salt tolerance genes are of great signifiance to increase crop production. The plant vacuole plays an important role in the sequestration of various compounds. Vacuole transporters play an important role in sequestration mechanism of various ions. CAXs transporters are regarded as vacuole H+/Ca²⁺ exchangers. Recently, phylogenetic analyses suggested that CAX7 to CAX11 should be reclassified as a new family CCXs, because they show more homologs with mammalian NCKX6. Howecer, experimental evidences still are needed. In this research, the gene AtCCX1 was cloned and expressed into yeast; the function of AtCCX1 was studied, localization information of AtCCX1 in tissues and yeast as well as substrate transported by AtCCX1 was indentified. These results support that CCXs is a new family. The main results were showed below:Firstly, the gene AtCCX1 was cloned from Arabidopsis. Full length of the gene is 1713bp, encoding 570 amino acid residues, and the product of the gene was predicted 61.8KD. AtCCX1 has similar primary structure with AtCCX3、AtCCX4, and contains almost absolutely same twoα-repeat domains. The predicted model for protein secondary structure showed that AtCCX1 had 10 transmembranes and a short N-terminal. The structure does not support AtCCX1 has N-terminal autoinhibitory domain.Secondly, expression of AtCCX1 was found in root, leaf, stem and flower. After treatment on Arabidopsis by 100mmol·L^-1 KCl、100mmol·L^-1 NaCl、20%PEG6000, the expression of AtCCX1 were enhanced.Thirdly, after expression of AtCCX1, tolerance to sodium salt of yeast was enhanced significantly; yeast grew better in acidic environments, and the best growth pH became 6 from 8; in sodium and potassium salt medium, the yeast expressing AtCCX1 grew better than normal type.Fourthly, various cation contents in yeast were tested by ICP-MS. Na⁺ in variant was high above 121.79% than wild type yeast; K⁺ and Cu²⁺ in variant were low about 33.4% and 13.82%. Fifthly,EGFP sequence was linked to the end of C-terminal of AtCCX1, then was expressed in yeast. Fluorescent pictures were obtained by Olimpus fluorescence microscope. AtCCX1 was located in tonoplast.Sixthly, culturing yeast cell in K⁺ gradient medium, content change of K⁺ and Na⁺ was tested with ICP-MS. In variants expressing AtCCX1, companying increasing K⁺ concentration in medium, K⁺ increased quickly; but in wildtype yeast cells, companying increasing K⁺ concentration in medium, K⁺ kept in a steady level. In yeast cells no matter whether AtCCX1 was expressed or not, ompanying increasing K⁺ concentration in medium, Na⁺ has the same change curve, but each level of K⁺ gradient medium, Na⁺ in variant expressing AtCCX1 was higher than wildtype.Seventhly, culturing yeast cell in pH gradient medium, K⁺、Na⁺、Ca²⁺、Mn²⁺、Fe³⁺、Cu²⁺、Zn²⁺ change in yeast was test by ICP-MS. Na⁺ has significantly positive change and K⁺, Cu²⁺ have significantly negative change. The change was reached to max on pH 6. The results shows that Na⁺、K⁺、Cu²⁺ are main substrates, the transport process is related to pH. Eighthly, SBFⅠand PBFⅠ,which were fluorescent indicators for sodium and potassium respectively, were used in this experiment. Without Na⁺ in the solution, a steady-state PBFⅠfluorescence ratio level was observed. While 20mmol·L^-1 Na⁺ was added into the solution, PBFⅠfluorescence ratio began to reduce sharply. However, if vanadate,a H+-ATPase inhibitor,was contained in the solutions, PBFⅠfluorescence ratio kept in a steady level all the time, no matter whether Na⁺ was added. Without K⁺ in the solution, a steady-state SBFⅠfluorescence ratio level was observed. While 20mmol·L^-1 K⁺ was added into the solution, SBFⅠfluorescence ratio rised up dramatically. However, if vanadate was contained in the solution, PBFⅠfluorescence ratio kept in a steady level all the time, no matter whether K⁺ was added.As a conclusion, Na⁺ and K⁺ are main substates of AtCCX1 transpoter. AtCCX1 gene is expressed throughout the plant, located in vacuole, increase Na⁺ accumulation and decrease K⁺, Cu²⁺ accumulation in yeast. Dynamic transport assays suggested that AtCCX1 facilitate H+ dependent Na⁺ influx and K⁺ efflux. The results provide the evidence for AtCCX1’s positive effect on maintaing cytosolic high K⁺/Na⁺.