胡杨Na+/H+反向运输载体(PeNHX2)基因的克隆与序列分析

植物液泡膜上Na /H 反向运输载体(Na /H antiporterNHX)已被证明在盐胁迫中发挥关键作用,该研究旨在从胡杨中克隆获得耐盐相关的的完整cDNA序列,为进一步比较胡杨的耐盐基因的差异,探讨胡杨的耐盐分子机理奠定了基础,并为通过转基因手段提高木本植物的耐盐能力提供侯选基因。根据已发表NHX的同源基因序列,采用特异性引物扩增核心片段,并结合5’与3’RACE技术,成功地从胡杨(populus euphratica)克隆获得PeNHX2,其cDNA全长1638bp。测序和序列分析结果表明该基因与已发表毛白杨(Populus tomentosa)PtNHX基因在核酸水平及蛋白质水平上同源性均达到最高,依次为90.6%和88.25%。利用DNAMAN软件进一步分析得知,该基因与新疆盐生植物中所克隆获得的的犁苞滨藜(Atriplex dimorphostegia)AdNHX1、灰绿藜(Chenopodium glaucum)CgNHX1、盐爪爪(Kalidium foliatum)KfNHX同源性也较高,核酸水平上依次为75.1%、74.8%、74.3%,蛋白质水平依次为78.3%、79.0%、78.3%。PeNHX2与NCBI已注册的部分PeNHX1序列,在核酸或蛋白质水平同源性(72.34%和73.72%)均较低,分析它们可能同属于胡杨NHX基因家族,相关生物学功能有待于进一步研究。     

抗盐胡杨Na+/H+逆向转运蛋白基因PeNhaD1的功能

将胡杨Na /H 逆向转运蛋白基因PeNhaD1,分别转入对盐敏感的缺失质膜和缺失液泡膜Na /H 逆向转运蛋白基因的酵母突变菌株ANT3和GX1中。结果表明,在pH6.0、Na 浓度为80mmol/L(固体培养基)或400mmol/L(液体培养基)的条件下,转化具有目的基因的酵母ANT3具有更高的耐盐性,而将目的基因转化到突变株GX1时,却不能提高其耐盐性。实验结果说明PeNhaD1可能是通过编码质膜Na /H 逆向转运蛋白而提高酵母的耐盐性的,推测其在胡杨耐盐机制中的作用可能是提高拒盐性。     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

植物Na+/H+反向转运蛋白的研究进展

盐胁迫是限制植物生长发育的主要因素之一,植物Na+/H+反向转运蛋白可通过将Na+逆向转运出细胞外或将Na+区隔化于液泡中来抵制环境中过高的Na+浓度.植物中Na+/H+反向转运蛋白存在于细胞质膜和液泡膜上,现在已得到多种编码这些Na+/H+反向转运蛋白的基因,对其结构功能特性进行了大量研究,并发现将这些基因转入非抗盐植物中过量表达可提高转基因植物的抗盐性.概述了Na+/H+反向转运蛋白及其编码基因的最新研究进展.     

Na+/H+ Antiporter in Tonoplast Vesicles from Rice Roots

The Na⁺/H ⁺ antiporter in vacuolar membranes transports Na⁺from the cytoplasm to vacuoles using a pH gradient generatedby proton pumps; it is considered to be related to salinitytolerance. Rice (Oryza sativa L.) is a salt-sensitive crop whosevacuolar antiporter is unknown. The vacuolar pH of rice roots,determined by ³¹P-nuclear magnetic resonance (NMR), increasedfrom 5.34 to 5.58 in response to 0.1 M NaCl treatment. Transportof protons into the tonoplast vesicles from rice roots was fluorometricallymeasured. Efflux of protons was accelerated by the additionof Na⁺. Furthermore, the influx of ²²Na⁺ into the tonoplastvesicles was accelerated by a pH gradient generated by proton-translocatingadenosine 5'-triphosphatase (H⁺-ATPase) and proton-translocatinginorganic pyro-phosphatase (H⁺-PPase). We concluded that thisNa⁺/H⁺antiporter functioned as a Na⁺ transporter in the vacuolarmembranes. The antiporter had a K_m of 10 mM for Na⁺ and wascompetitively inhibited by amiloride and its analogues. TheKⁱ values for 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyI)-amiloride(EIPA), and 5-(N, N-hexamethylene)-amiloride (HMA) were 2.2,5.9, and 2.9 µ M, respectively. Unlike barley, a salt-tolerantcrop, NaCl treatment did not activate the antiporter in riceroots. The amount of antiporter in the vacuolar membranes maybe one of the most important factors determining salt tolerance. ¹This work was supported by a grant from Bio-Media Project ofthe Japanese Ministry of Agriculture, Forestry and Fisheries(BMP96-III-1).     

Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter

Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na⁺/H⁺ antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial F_oF₁-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na⁺.     

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.     

Distinct patterns of natural selection in Na+/H+ antiporter genes in Populus euphratica and Populus pruinosa

Salt tolerance genes constitute an important class of loci in plant genomes. Little is known about the extent to which natural selection in saline environments has acted upon these loci, and what types of nucleotide diversity such selection has given rise to. Here, we surveyed genetic diversity in three types of Na⁺/H⁺ antiporter gene (SOS, NhaD, and NHX, belonging to the cation/proton antiporter 1 family), which have well ‐ characterized essential roles in plant salt tolerance. Ten Na⁺/H⁺ antiporter genes and 16 neutral loci randomly selected as controls were sequenced from 17 accessions of two closely related members of the genus Populus, Populus euphratica and Populus pruinosa, section Turanga, which are native to northwest China. The results show that salt tolerance genes are common targets of natural selection in P. euphratica and P. pruinosa. Moreover, the patterns of nucleotide variation across the three types of Na⁺/H⁺ antiporter gene are distinctly different in these two closely related Populus species, and gene flow from P. pruinosa to P. euphratica is highly restricted. Our results suggest that natural selection played an important role in shaping the current distinct patterns of Na⁺/H⁺ antiporter genes, resulting in adaptive evolution in P. euphratica and P. pruinosa.     

水稻 Ca2+/H+ 反向转运体 OsCAX3 的功能分析和亚细胞定位研究

Ca2+/H+ 反向转运体作为一类 Ca2+外向转运器,在植物的营养和信号转导中起着非常重要的作用 . 克隆了水稻 Ca2+/H+ 反向转运体基因 OsCAX3 ,序列分析表明 OsCAX3 具有 11 个跨膜区,其中在第 6 和第 7 个跨膜区之间有一个 17 个氨基酸组成的酸性基序 (acid motif) ,功能互补实验证明 OsCAX3 具有转运 Ca2+ 的功能,并且其 N 端 26 个氨基酸序列对转运 Ca2+ 具有一定的抑制作用 . RT-PCR 分析表明 OsCAX3 的表达受到外源 Ca2+ 的诱导 . 利用 PSORT prediction 进行亚细胞定位分析,和利用 OsCAX3-GFP 融合蛋白瞬时表达分析证明, OsCAX3 定位于细胞质膜 . 以上结果表明, OsCAX3 是一种定位于细胞质膜上的 Ca2+/H+ 反向转运体 .     

Molecular characterization of PeSOS1: the putative Na+/H+ antiporter of Populus euphratica

Populus euphratica is a salt-tolerant tree species growing in semi-arid saline areas. A Na⁺/H⁺ antiporter gene was successfully isolated from this species through RACE cloning, and named PeSOS1. The isolated cDNA was 3665 bp long and contained a 3438 bp open reading frame that was predicted to encode a 127-kDa protein with 12 hypothetical transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The amino acid sequence of this PeSOS1 gene showed 64% identity with the previously isolated SOS1 gene from the glycophyte Arabidopsis thaliana. The level of protein expressed by PeSOS1 in the leaves of P. euphratica was significantly up-regulated in the presence of high (200 mM) concentrations of NaCl, while the mRNA level in the leaves remained relatively constant. Immunoanalysis suggested that the protein encoded by PeSOS1 is localized in the plasma membrane. Expression of PeSOS1 partially suppressed the salt sensitive phenotypes of the EP432 bacterial strain, which lacks the activity of the two Na⁺/H⁺ antiporters EcNhaA and EcNhaB. These results suggest that PeSOS1 may play an essential role in the salt tolerance of P. euphratica and may be useful for improving salt tolerance in other tree species. Yuxia Wu and Nan Ding contributed equally to this work.     

拟南芥内膜Na+,K+/H+反向转运体研究进展

拟南芥内膜Na,K~+/H~+反向转运体(Endosomal NHX)的亚细胞定位、离子转运特性及生物学功能阐释取得了重要进展。拟南芥内膜Na~+,K~+/H~+反向转运体包含AtNHX5和AtNHX6两个成员,它们的氨基酸序列相似性为78.7%。研究表明,AtNHX5和AtNHX6具有功能冗余,它们都定位在高尔基体(Golgi)、反面高尔基体管网状结构(TGN)、内质网(ER)和液胞前体(PVC),参与调控耐盐胁迫、pH平衡和K~+平衡等。有报道显示内膜NHXs跨膜结构域存在能够调控自身离子活性的酸性保守氨基酸残基,对其自身功能具有决定性作用。最新研究结果表明,拟南芥内膜NHXs影响囊泡运输和蛋白存贮,并参与生长素介导的植物生长和发育。文中主要对拟南芥内膜NHXs的亚细胞定位、离子转运、功能及应用进展进行了概述。     

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.     

植物Na+/H+逆向转运蛋白研究进展

盐胁迫主要由Na+引起,过高的Na+浓度引起的离子毒害,渗透胁迫和K+/Na+比率的不平衡使植物新陈代谢异常,这是对大多数器官造成伤害的原因。植物抵御盐胁迫的主要方式是将细胞内过多的Na+从质膜向细胞外排放和将Na+在液泡中区隔化,这一过程是由Na+/H+ 逆向转运蛋白完成的。本文概述了植物中Na+/H+ 逆向转运蛋白的发现、特征、分子生物学方面的研究,以及Na+/H+ 逆向转运蛋白在植物耐盐性中的重要作用。     

植物Na+/H+逆向转运蛋白研究进展

盐胁迫主要由Na 引起,过高的Na 浓度引起的离子毒害,渗透胁迫和K ／Na 比率的不平衡使植物新陈代谢异常,这是对大多数器官造成伤害的原因。植物抵御盐胁迫的主要方式是将细胞内过多的Na 从质膜向细胞外排放和将Na 在液泡中区隔化,这一过程是由Na ／H 逆向转运蛋白完成的。本文概述了植物中Na ／H 逆向转运蛋白的发现、特征、分子生物学方面的研究,以及Na ／H 逆向转运蛋白在植物耐盐性中的重要作用。     

盐爪爪Na^＋/H^＋逆向转运蛋白和焦磷酸酶的亚细胞定位

将盐爪爪Na+/H+逆向转运蛋白基因(KfNHX1)和焦磷酸酶基因(KfVP1)分别构建至植物表达载体,利用基因枪介导的方法转化洋葱表皮细胞,通过荧光显微镜观察研究其亚细胞定位.结果表明,转化了KfNHX1(或KfVP1)-GFP融合蛋白的洋葱表皮细胞仅膜系统散发荧光,而对照组即未转入KfNHX1(或KfVP1)基因的细胞则整体均匀发出荧光.说明KfNHX1和KfVP1可能定位于细胞的膜系统,作为跨膜转运蛋白在离子的调控运输中发挥重要作用.     

拟南芥液泡膜Na+/H+逆向转运蛋白研究进展

盐分是植物生长发育的主要限制因素之一,而离子在胞内区室之间的选择性运动对提高植物耐盐性是至关重要的。来自于拟南芥(Arabidopsis thaliana)的AtNHX1基因可编码Na /H 逆向转运蛋白,而Na /H 逆向转运蛋白AtNHX1可将细胞质中多余的Na 排进液泡来消除Na 的毒害,维持细胞的渗透平衡,提高植物的耐盐性。简要综述了AtNHX1基因及Na /H 逆向转运蛋白AtNHX1的特征,AtNHX1的耐盐机制以及植物耐盐基因工程改良等方面的研究进展。     

质膜Na^＋／H^＋逆向转运蛋白与植物耐盐性

土壤盐碱化是造成农作物减产的主要原因之一。质膜Na^＋／H^＋逆向转运蛋白能够介导植物根部Na^＋的外排和体内Na^＋的长距离运输, 并能够调控细胞K＋的稳态平衡及细胞内pH值和Ca^2＋的转运, 因此其在植物耐盐性方面具有重要作用。该文概述了植物质膜Na^＋／H^＋逆向转运蛋白的分子结构、功能、表达调控及其与植物耐盐性关系等方面的研究进展, 并对今后有关该蛋白的主要研究方向作了分析和展望。     

菊芋Na+/H+逆向转运蛋白基因的克隆与表达分析

根据同源序列设计简并引物,通过RT-PCR及RACE的方法从菊芋中克隆了Na /H 逆向转运蛋白基因。序列分析表明,该基因全长2148 bp,开放读码框为1647 bp,可编码长549个氨基酸的多肽,与其它植物已克隆的Na /H 逆向转运蛋白具有很高的同源性。系统发育分析表明该蛋白(HtNHX1)与液泡型Na /H 逆向转运蛋白的亲缘关系较近,与质膜型Na /H 逆向转运蛋白亲缘关系较远。NaCl胁迫条件下RT-PCR检测结果表明,HtNHX1随NaCl浓度增加和处理时间延长表达持续增强,但到了第3天表达量开始下降。HtNHX1逆向转运蛋白基因的转录调控可能是决定菊芋耐盐能力的一个重要因素。     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.