小麦Na^＋／H^＋反转运蛋白基因的克隆和特性

以水稻 (OryzasativaL .)Na /H 反转运蛋白cDNA片段为探针 ,从小麦盐胁迫cDNA文库中筛选和克隆了 2个小麦Na /H 反转运蛋白基因 ,分别命名为TaNHX1和TaNHX2。序列分析表明TaNHX1为 2 0 2 9bp ,包含一个完整的 16 38bp的ORF ,编码 5 4 6个氨基酸 ,其中含有DIFFIYLLPPI跨膜区。TaNHX2为 16 93bp ,包含部分ORF及 80 8bp的 3′_UTR。这 2个基因与已知的水稻、拟南芥 (Arabidopsisthialiana)和滨藜 (Atriplexgmelini)中的同类基因NHX的相似性约为 70 %。RT_PCR分析表明小麦苗经 4 0 0mmol/LNaCl处理 1h后 ,TaNHX1的转录水平有所提高。     

RLM-RACE法扩增獐茅液泡膜Na+/H+逆向转运蛋白基因5'-cDNA末端序列

根据已获得的盐生植物獐茅(Aeluropus littoralis var.sinensis Debeaux)液泡膜Na+/H+逆向转运蛋白(Na+/H+antiporter)基因部分cDNA片段,设计2条特异引物(GSP1、GSP2),采用RLM-RACE(RNA ligase-mediated rapid amplification of 5'and 3'cDNA ends)法获得了其5'-cDNA末端序列,并找出了转录起始位点.该片段长816 bp,与芦苇液泡膜Na+/H+逆向转运蛋白基因序列同源性最高达91%,与拟南芥、盐角草、水稻、大麦、小麦的同源性分别为81%、82%、86%、87%和81%,为该基因全长的克隆及启动子的研究奠定了基础.与其它测定基因转录起始位点的方法相比,RLM-RACE方法更快捷、简便、准确.     

新疆无苞芥Na~+/H~+逆向转运蛋白基因OpNHX1的克隆、表达分析与功能验证

从耐盐植物无苞芥中克隆获得了1个Na+/H+逆向转运蛋白基因NHX1,命名为OpNHX1(GenBank登录号:KC200248)。OpNHX1基因cDNA全长2 153 bp,包含一个1 605 bp的开放阅读框,编码534个氨基酸。系统进化树分析表明,OpNHX1编码产物与拟南芥、小盐芥亲缘关系较近,属于同一进化分支。实时荧光定量PCR分析表明,该蛋白基因在无苞芥根、茎、叶、花和荚果中均有表达,其中茎中表达量最高。半定量RT-PCR分析表明,该基因受高盐、干旱、低温及ABA的诱导上调表达。进一步将该基因在拟南芥中过量表达,显著提高了转基因植株在盐胁迫下的存活率,说明OpNHX1基因参与了植物的耐盐性。酵母功能互补试验结果显示,该基因转化酵母Δnhx1后可以补充NHX1的缺失,表明OpNHX1参与Na+/H+的转运。     

北美海蓬子SbNHX1基因耐盐性及功能结构域分析

对从北美海蓬子中分离的Na+/H+逆向转运蛋白基因SbNHX1进行了耐盐性及功能结构域分析.利用套叠PCR技术去除SbNHX1基因C末端162个核苷酸,得到SbNHX1-C基因,然后将SbNHX1、SbNHX1-C和拟南芥Na+/H+ 逆向转运蛋白基因AtNHX1分别插入pET22b(+)表达载体,转化大肠杆菌B菌株,进行各种金属盐离子胁迫分析.结果表明,北美海蓬子Na+/H+ 逆向转运蛋白基因SbNHX1只对Na+ 、K+离子有抗性,且耐盐性强于拟南芥Na+/H+ 逆向转运蛋白基因AtNHX1.缺失C末端的SbNHX1-C基因对Na+、K+离子胁迫无抗性,说明北美海蓬子Na+/H+ 逆向转运蛋白基因SbNHX1的耐盐作用与该基因C末端1 353 bp至1 514 bp的序列密切相关.     

中间偃麦草Na+/H+逆向转运蛋白的分子克隆及生物信息学分析

根据小麦液泡膜Na /H 逆转运蛋白基因TaNHX1的全长序列设计引物,通过RT-PCR直接扩增的方法从中间偃麦草(Elytrigia intermedia)中克隆到了TaNHX1的同源基因,命名为TiNHX1(Acession Numeber:EF409418).TiNHX1最大开放阅读框为1 641 bp,编码含有546个氨基酸残基、分子量为59.8 kDa的蛋白,预测等电点8.0.TiNHX1含有38个碱性氨基酸,36个酸性氨基酸,256个疏水氨基酸及129个极性氨基酸.二级结构预测表明该蛋白含约44%的a-螺旋、21%的p-折叠、4%的p-转角和29%的不规则卷曲.亲疏水性分析显示,TiNHX1含有12个连续的疏水片断,其中10个可能构成穿膜螺旋.序列分析显示,TiNHX1与小麦(Triticum aestivum)、长穗偃麦草(Elytrigia elongate)、水稻(Oryza sativa)、小盐芥(Thellungiella halophila)、拟南芥(Arabidopsis thaliana)等植物的液泡膜Na /H 逆向转运蛋白高度同源,序列相似性分别为97%、96%、85%、68%、67%.序列比对结果以及进化树分析均表明TiNHX1应为定位于中间偃麦草液胞膜上的Na /H 逆向转运蛋白.     

黄花草木樨MoSOS1基因克隆及表达分析

植物质膜Na+/H+逆向转运蛋白基因SOS1是植物耐盐性必需的基因之一,在抵御盐胁迫过程中发挥十分重要的作用。以黄花草木樨叶片总RNA为模板,通过RT-PCR结合RACE方法克隆得到黄花草木樨MoSOS1基因全长序列,命名为MoSOS1。序列分析表明该基因全长为3 931 bp,开放阅读框(ORF)为2 874 bp,编码957个氨基酸,分子量为112.8 k D,等电点为5.31。TMHAM软件跨膜区的预测分析表明,黄花草木樨MoSOS1蛋白具有8个跨膜结构区域,N端和C端都位于细胞外。氨基酸序列分析表明,MoSOS1蛋白含有1个Na+/H+Exchanger superfamily和一个c NMP(Cyclic nucleotide-monophosphate)结合位点以及1个CAP_ED(Catabolite gene activator protein-effector domain)superfamily结构域。生物信息预测显示,MoSOS1的编码蛋白为不稳定酸性蛋白,不存在信号肽,二级结构多为α-螺旋和无规则卷曲。荧光实时定量RT-PCR分析表明:随着Na Cl浓度的增加,黄花草木樨地上部和根中MoSOS1基因表达水平呈增加趋势,根中表达量大于地上部,表明MoSOS1基因的表达受盐胁迫诱导和调节。     

长叶红砂液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达特性

为研究长叶红砂(Reaumuria trigyna)离子转运分子机制,利用RT-PCR和RACE技术,克隆到其液泡膜Na+/H+逆向转运蛋白基因(NHX1)的全长cDNA片段,命名为RtNHX1(NCBI序列号为KR919802)。结果表明:RtNHX1的cDNA片段全长2 622bp,开放阅读框1 662bp,5′非编码区509bp,3′非编码区451bp,编码553个氨基酸,推测分子量为60.91kD。该蛋白含有12个跨膜结构域,为疏水蛋白,与其他植物液泡膜Na+/H+逆向转运蛋白NHX1的亲缘关系较近。实时荧光定量PCR对其在NaCl胁迫下的表达检测显示,不同时间和不同浓度NaCl胁迫下,RtNHX1表达量变化均呈先升高后降低趋势,在100mmol/L NaCl胁迫6h和200mmol/L NaCl胁迫后达到最高,表达量分别超过或约是对照的3倍,一定程度反应出RtNHX1参与长叶红砂的盐胁迫应答,是该植物离子转运体系的重要元件。     

三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析

为研究Na+/H+-exchanger基因在三疣梭子蟹（Portunus trituberculatus）盐度胁迫过程中的功能作用,克隆了三疣梭子蟹Na+/H+-exchanger基因并进行表达分析。结果显示,Na+/H+-exchanger基因（GenBank:KU519329）全长4233 bp,5和3非编码区（UTR）长分别为519和753 bp,开放阅读框（ORF）长2961 bp。编码986个氨基酸,预测蛋白质分子量和等电点分别为110.8 kD和7.42,具有信号肽和典型的Na+/H+-exchanger蛋白结构域,含12个跨膜螺旋;三疣梭子蟹Na+/H+-exchanger基因与普通滨蟹（Carcinus maenas）同源性最高,达到87.2%,系统进化分析也显示该序列与普通滨蟹聚为一支;表达分析显示,三疣梭子蟹Na+/H+-exchanger基因在鳃中表达量最高;在低盐（盐度5、10和20）胁迫过程中,Na+/H+-exchanger基因在0-12h上调表达明显,在24-168h间表达量呈下降趋势;在高盐（盐度50）胁迫初期（0-12h）,该基因表达量相对稳定,之后（24-168h）显著下调表达。研究表明低盐显著诱导Na+/H+-exchanger基因的高表达,推测三疣梭子蟹Na+/H+-exchanger基因在低盐环境下发挥重要的渗透调节功能。     

新疆盐生植物车前PmNHX1基因的克隆及生物信息学分析

盐分对植物的伤害主要是Na+引起的,而Na+/H+逆向运输蛋白催化Na+/H+逆向跨膜运输,从而使质膜上Na+运出细胞和液泡膜中的Na+区隔化。这是植物尤其是盐生植物抵御盐胁迫的主要方式之一。根据不同植物编码液泡膜逆向运输蛋白基因的保守序列,设计简并引物,采用RT-PCR和RACE技术,首次从新疆盐生植物车前（Plantago maritima）中克隆到Na+/H+逆向运输蛋白基因的cDNA全长2464 bp,命名为PmNHX1(GenBank登录号：EU233808),该基因编码区长为1 662bp,编码553个氨基酸,理论分子量为61.16kDa,等电点为7.22。数据分析结果显示,该蛋白质主要定位于液泡膜上,由12个序列保守的跨膜结构域组成,其中TM3跨膜结构域上存在“LFFIYLLPPI”-氨氯吡嗪咪结合域,并且该位点与Na+有竞争作用。PmNHX1逆向运输蛋白与其他植物逆向运输蛋白的氨基酸同源性为64％~80％。通过生物信息学方法对其理化性质和功能分析进行预测,这为进一步研究转耐盐基因PmNHX1及其功能鉴定奠定了基础。     

梭梭Na+/H+逆向转运蛋白基因克隆及分析

采用RT-PCR、RACE方法从超旱生、耐盐植物梭梭中扩增出Na+/H+逆向转运蛋白基因的开放阅读框架,其核苷酸序列长1 683bp,推测的氨基酸序列全长为560个氨基酸残基。含有多个物种Na+/H+逆向转运蛋白基因的高度保守序列氨氯砒嗪脒的结合位点(LFFIYLIPPI)。序列一致性分析结果显示,该cDNA片段与同科植物NHX基因的一致性为70%～80%,但与不同科植物的一致性较低,仅为60%,表明该基因在进化上存在多样性,但它们都具有氨氯砒嗪脒结合位点,对Na+具有高度专一性,对植物的耐盐性起着重要作用。     

Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa.

Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes. We isolated a rice cDNA clone the deduced amino acid sequence of which had homology with a putative Na+/H+ exchanger in Saccharomyces cerevisiae, NHX1. The sequence contains 2330 bp with an open reading frame of 1608 bp. The deduced amino acid sequence is similar to that of NHX1 and NHE isoforms in mammals, and shares high similarity with the sequences within predicted transmembrane segments and an amiloride-binding domain. The expression of the gene was increased by salt stress. These results suggest that the product of the novel gene, OsNHX1, functions as a Na+/H+ exchanger, and plays important roles in salt tolerance of rice.     

An Na+/H+ antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na+/H+ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5'-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (deltanhx1), TaNHX2 suppressed the salt sensitivity of the mutant,which was deficient in vacuolar Na+/H+ antiporter, and caused partial recovery of growth of delta nhx1 in NaCl and LiCl media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.     

Isolation and characterization of a new Na+/H+ antiporter gene OsNHA1 from rice (Oryza sativa L.).

The full-length cDNA (3612 bp) of OsNHA1 was cloned by RT-PCR approach from rice (Oryza sativa L.), which encodes a putative plasma membrane Na+/H+ antiporter. Its deduced protein, OsNHA1, has 11 transmembrane domains and a significant similarity to a plasma membrane Na+/H+ antiporter AtNHA1 from Arabidopsis thaliana. Phylogenetic analysis showed that the OsNHA1 clusters with the plasma membrane Na+/H+ antiporters from various organisms. The semi-quantitative RT-PCR assay revealed that the expression of OsNHA1 was up-regulated in both shoots and roots of rice seedlings under salt stress, whereas it was not induced in the rice seedlings treated by drought stress.     

Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes

Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of alpha-aminoisobutyric acid (AIB), methyl-AIB, proline, and leucine in rat thymocytes. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl-AIB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+ -dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: 1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5-(N,N-disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and 2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related.     

Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter

We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.