Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

Importance of the seryl and threonyl residues of the fifth transmembrane domain to the substrate specificity of yeast plasma membrane Na+/H+ antiporters

The Zygosaccharomyces rouxii Na⁺/H⁺ antiporter Sod2-22p is a member of the subfamily of yeast plasma membrane Nha/ Sod antiporters that do not recognize potassium as their substrate. A functional study of two ZrSod2-22p mutated versions that improved the tolerance of a S. cerevisiae alkali-metal-cation sensitive strain to high extracellular concentration of KCl identified two polar non-charged amino-acid residues in the fifth transmembrane domain, Thr141 and Ser150, as being involved in substrate recognition and transport in yeast Nha/Sod antiporters. A reciprocal substitution of amino-acid residues with a hydroxyl group at these positions, T141S or S150T, produced a broadened cation selectivity of the antiporter for K⁺, in addition to Na⁺ and Li⁺. Site-directed mutagenesis of Ser150 showed that while the replacement of Ser150 with a small hydrophobic (valine) or negatively charged (aspartate) amino acid did not produce a significant change in ZrSod2-22p substrate specificity, the introduction of a positive charge at this position stopped the activity of the antiporter. This data demonstrates that the amino-acid composition of the fifth transmembrane domain, mainly the presence of amino acids containing hydroxyl groups in this part of the protein, is critical for the recognition and transport of substrates and could participate in conformational movements during the binding and/or cation transport cycle in yeast plasma membrane Na⁺/H⁺ antiporters.     

The Mrp system: a giant among monovalent cation/proton antiporters?

Mrp systems are a novel and broadly distributed type of monovalent cation/proton antiporter of bacteria and archaea. Monovalent cation/proton antiporters are membrane transport proteins that catalyze efflux of cytoplasmic sodium, potassium or lithium ions in exchange for external hydrogen ions (protons). Other known monovalent cation antiporters are single gene products, whereas Mrp systems have been proposed to function as hetero-oligomers. A mrp operon typically has six or seven genes encoding hydrophobic proteins all of which are required for optimal Mrp-dependent sodium-resistance. There is little sequence similarity of Mrp proteins to other antiporters but three of these proteins have significant sequence similarity to membrane embedded subunits of ion-translocating electron transport complexes. Mrp antiporters have essential roles in the physiology of alkaliphilic and neutralophilic Bacillus species, nitrogen-fixing Sinorhizobium meliloti and in the pathogen Staphylococcus aureus, although these bacteria contain multiple monovalent cation/proton antiporters. The wide distribution of Mrp systems leads to the anticipation of important roles in an even wider variety of pathogens, extremophiles and environmentally important organisms. Here, the distribution, established physiological roles and catalytic activities of Mrp systems are reviewed, hypotheses regarding their complexity are discussed and major open questions about their function are highlighted.     

A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

Functional Analysis of the Na+/H+ Antiporter Encoding Genes of the Cyanobacterium Synechocystis PCC 6803

The role of putative Na⁺/H⁺ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na⁺/H⁺ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na⁺/H⁺ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na⁺/H⁺ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na⁺/H⁺ antiporters was studied. No induction of any Na⁺/H⁺ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na⁺/H⁺ antiporter encoding genes showed induction. These results might indicate that some of Na⁺/H⁺ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.     

Gene structure of three kinds of vacuolar-type Na+/H+ antiporters including TaNHX2 transcribed in bread wheat

The vacuolar-type sodium/proton antiporter is considered to play an important role in withstanding salt stress by transporting sodium ions into vacuoles. In this study, the gene structures of three kinds of vacuolar-type antiporters transcribed in bread wheat under salt stress were analyzed. After spraying 0.5 M NaCl to seedlings of wheat cultivar Chinese Spring, 1,392~1,400 bp cDNA fragments were isolated by RT-PCR using primers designed from common regions in rice OsNHX1 and Atriplex subcordata AgNHX1. Next, the entire structure of the genomic DNA and cDNA were determined via CapFishing-5’ Rapid Amplification of cDNA Ends (RACE), 3’RACE, and genomic PCR cloning. As a result, 3 kinds of vacuolar-type Na⁺/H⁺ antiporter genes, TaNHXa (genome DNA 4,255 bp, cDNA 2,414 bp, 539 a.a.), TaNHXb (gDNA 4,167 bp, cDNA 1,898 bp, 539 a.a.) and TaNHXc (gDNA 4,966 bp, cDNA 1,928 bp, 547 a.a.), were identified. They encode 12 transmembrane domains containing third domain’s amyloid binding sites (FFIYLLPP), characteristic of the vacuolar-type Na⁺/H⁺ antiporter, binding to the cell vacuolar membrane. TaNHXa, b and c consisting of 14 exons and 13 introns were 22~55 % longer than A. thaliana AtNHX1 in total length. TaNHXa (TaNHX2) showed homogeneity with OsNHX1, while TaNHXb and c were phylogenetically independent.     

Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli

Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal N a⁺/H⁺ antiporters, the result was a Li⁺ sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na⁺/H⁺ antiporters. These cells became more resistant to external LiCl, but only in Na⁺ containing media. In the absence of external Na⁺, the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na⁺/H⁺ antiporter in conjunction with different housekeeping systems of E. coli host cells.     

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation

The Na⁺/H⁺ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na⁺/H⁺ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na⁺/H⁺ exchanger were examined for their ability to translocate protons as assessed by a cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na⁺/H⁺ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na⁺/H⁺ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na⁺/H⁺ exchangers.     

Respiration-dependent contraction of swollen heart mitochondria: Participation of the K+/H+ antiporter

Respiration-dependent contraction of heart mitochondria swollen passively in K⁺ nitrate is activated by the ionophore A23187 and inhibited by Mg²⁺. Ion extrusion and osmotic contraction under these conditions are strongly inhibited by quinine, a known inhibitor of the mitochondrial K⁺/H⁺ antiporter, as measured in other systems. The inhibition by quinine is relieved by the exogenous antiporter nigericin. Respiration-dependent contraction is also inhibited by dicyclohexylcarbodiimide (DCCD) when reacted under conditions known to inhibit K⁺/H⁺ antiport (Martinet al., J. Biol. Chem. 259, 2062–2065, 1984). These studies strongly support the concept that K⁺ is extruded from the matrix by the endogenous K⁺/H⁺ antiporter and that inhibition of this component by quinine or DCCD inhibits respiration-dependent contraction. The extrusion of K⁺ nitrate is accompanied by a respiration-dependent efflux of a considerable portion of the endogenous Mg²⁺. This Mg²⁺ efflux does not occur in the presence of nigericin or when the mitochondrial Na⁺/H⁺ antiporter is active. Mg²⁺ efflux may take place on the K⁺/H⁺ antiporter. DCCD, reacted under conditions that do not result in inhibition of the K⁺/H⁺ antiporter, blocks a monovalent cation uniport pathway. This uniport contributes to futile cation cycling at elevated pH, and its inhibition by DCCD stimulates respiration-dependent contraction.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

Conformational changes in NhaA Na+/H+ antiporter

Abstract

Na⁺/H⁺ antiporters play a primary role in Na⁺/H⁺ homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a ‘pH sensor' – a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 Å removed from the pH sensor. Alternative accessibility of the binding site to either side of the membrane, i.e., functional-dynamics, is the essence of secondary transport mechanism. Because NhaA is tightly pH regulated, structures of the pH-activated and ligand-activated NhaA conformations are needed to identify its functional-dynamics. However, as these are static snapshots of a dynamic protein, the dynamics of the protein both in vitro and in situ in the membrane are also required as reviewed here in detail. The results reveal two different conformational changes characterizing NhaA: One is pH-induced for NhaA activation; the other is ligand-induced for antiport activity.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini

With a homologous gene region we successfully isolated a Na⁺/H⁺ antiporter gene from a halophytic plant, Atriplex gmelini, and named it AgNHX1. The isolated cDNA is 2607 bp in length and contains one open reading frame, which comprises 555 amino acid residues with a predicted molecular mass of 61.9 kDa. The amino acid sequence of the AgNHX1 gene showed more than 75% identity with those of the previously isolated NHX1 genes from glycophytes, Arabidopsis thaliana and Oryza sativa. The migration pattern of AgNHX1 was shown to correlate with H⁺-pyrophosphatase and not with P-type H⁺-ATPase, suggesting the localization of AgNHX1 in a vacuolar membrane. Induction of the AgNHX1 gene was observed by salt stress at both mRNA and protein levels. The expression of the AgNHX1 gene in the yeast mutant, which lacks the vacuolar-type Na⁺/H⁺ antiporter gene (NHX1) and has poor viability under the high-salt conditions, showed partial complementation of the NHX1 functions. These results suggest the important role of the AgNHX1 products for salt tolerance.     

Functional and regulatory analysis of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> CAX2 cation transporter

The vacuolar sequestration of metals is an important metal tolerance mechanism in plants. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified in a Saccharomyces cerevisiae suppression screen as Ca²⁺/H⁺ antiporters. CAX2 has a low affinity for Ca²⁺ but can transport other metals including Mn²⁺ and Cd²⁺. Here we demonstrate that unlike cax1 mutants, CAX2 insertional mutants caused no discernable morphological phenotypes or alterations in Ca²⁺/H⁺ antiport activity. However, cax2 lines exhibited a reduction in vacuolar Mn²⁺/H⁺ antiport and, like cax1 mutants, reduced V-type H⁺-ATPase (V-ATPase) activity. Analysis of a CAX2 promoter -glucoronidase (GUS) reporter gene fusion confirmed that CAX2 was expressed throughout the plant and strongly expressed in flower tissue, vascular tissue and in the apical meristem of young plants. Heterologous expression in yeast identified an N-terminal regulatory region in CAX2, suggesting that Arabidopsis contains multiple cation/H⁺ antiporters with shared regulatory features. Furthermore, despite significant variations in morphological and biochemical phenotypes, cax1 and cax2 lines both significantly alter V-ATPase activity, hinting at coordinate regulation among transporters driven by H⁺ gradients and the V-ATPase.     

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。