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.     

花烟草NaNHX1基因的克隆及其在非生物胁迫下的表达模式

植物NHX家族基因,在植物的生长发育以及生物与非生物胁迫的应答反应中发挥着十分重要的作用。为了探究花烟草Na+/H+逆向转运蛋白的生理功能,为花烟草耐盐分子机制的研究提供参考。采用同源克隆的方法进行基因克隆,对花烟草进行非生物胁迫,并运用qPCR的方法进行基因表达模式分析。结果表明,从花烟草(Nicotiana alata)中克隆了一个属于Na+/H+逆向转运蛋白家族的基因NaNHX1。该基因的开放阅读框全长为1 599 bp,编码了532个氨基酸残基。生物信息学分析结果表明,该基因编码的蛋白分子量为58.4 kD,等电点为5.66;具有Na+/H+逆向转运蛋白家族典型的保守结构域NhaP2;该蛋白属于疏水性蛋白,包含10个跨膜区。NaNHX1基因主要定位于细胞质膜,并含有多个磷酸化位点。同源性分析的结果显示,NaNHX1基因与美花烟草(Nicotiana sylvestris)、茸毛烟草(Nicotiana tomentosiformis)以及番茄(Solanum lycoperisicum)NHX基因的亲缘关系最近,而与拟南芥的NHX基因同源性最低。NaNHX1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaNHX1的表达呈现3种不同的表达模式。其中,对高盐及低钾胁迫的响应强烈。本研究的结果表明,NaNHX1基因属于Na+/H+逆向转运蛋白家族,可能参与了花烟草高盐和低钾胁迫,以及其它非生物胁迫响应在内的众多生理过程。     

过量表达星星草PtSOS_1提高拟南芥的耐盐性

将星星草中分离的质膜型Na+/H+逆向转运蛋白基因PtSOS1(GenBank登录号EF440291)构建到pGWB2植物表达载体上,转化拟南芥,获得抗卡那霉素的抗性植株.PCR和Northern检测表明,PtSOS1已整合到拟南芥基因组中并过量表达.耐盐性实验表明,PtSOS1过量表达提高了拟南芥植株的耐盐性.盐分测定表明,盐胁迫下PtSOS1转基因植株中Na+积累低于野生型的,K+含量则高于野生型的,转基因植株中K+/Na+比值高于野生型.     

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。     

Na+/H+ 逆向转运蛋白与植物耐盐性关系

Na+/H+ 逆向转运蛋白与植物的耐盐性有密切的关系。在高等植物体内,主要存在两种Na+/H+ 逆向转运蛋白,分别为位于细胞质膜上的逆向转运蛋白SOS1,以及存在于液泡膜上的AtNHX1。质膜Na+/H+ 逆向转运蛋白主要负责Na+ 的外排,液泡膜Na+/H+ 逆向转运蛋白主要负责把Na+ 区隔化入液泡。过量表达质膜Na+/H+ 逆向转运蛋白SOS1或液泡膜Na+/H+ 逆向转运蛋白AtNHX1能够明显提高植物的耐盐性。本文对植物中Na+/H+ 逆向转运蛋白及其与植物耐盐性之间的关系研究最新进展作一概述。     

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.     

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.     

NaCl胁迫对盐芥质膜和液泡膜ATPase活性的影响

以盐生植物盐芥和中生植物拟南芥幼苗为材料,研究了盐胁迫对它们叶片和根质膜、液泡膜H+-ATPase、Ca2+-ATPases和K+-ATPase活性以及H+-ATPase、Na+/H+ 逆向转运蛋白表达的影响.结果显示:在NaCl胁迫下,盐芥叶片和根质膜的H+-ATPase活性分别比对照显著升高41%～212%和35%～53%,液泡膜的H+-ATPase分别显著升高281%～373%和4%～38%,而拟南芥却比相应对照都显著降低;相同盐浓度胁迫下,盐芥叶片的H+-ATPase活性比根部高4～8倍,盐芥根也远高于拟南芥.在NaCl胁迫下,盐芥叶片和根的液泡膜H+-ATPase蛋白质β亚基含量变化与其酶活性变化趋势一致,质膜Na+/H+ 逆向转运蛋白的表达量与Na+含量变化趋势一致.盐胁迫下盐芥根中Ca2+-ATPases和K+-ATPase活性的增加与根中Ca2+和K+含量呈显著正相关.研究发现,在盐胁迫条件下,盐芥能有效增强H+-ATPase蛋白和Na+/H+逆向转运蛋白表达,显著提高其根系与叶片质膜和液泡膜的H+-ATPase、Ca2+-ATPase和K+-ATPase活性,维持细胞质中较高的Ca2+和K+水平,从而缓解盐胁迫的伤害,增强耐盐性.     

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。     

拟南芥液泡膜Na+/H+逆向转运蛋白的研究进展

拟南芥液泡膜Na /H 逆向转运蛋白是由AtNHX1基因编码的一个在盐胁迫中起重要作用的蛋白。本文综述了AtNHX1的基本结构、功能及作用机制,展望其作为有效植物耐盐基因的前景,并对拟南芥液泡膜Na /H 逆向转运蛋白基因家族其他成员的研究,也做了相应的概括。     

Ectopic Expression of a Bacterium NhaD-type Na~+/H~+ Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

AaNhaD,a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica,encodes a Na+/H+ antiporter crucial for the bacterium’s resistance to salt/alkali stresses.However,it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses.To investigate the use of extremophile genetic resources in higher plants,transgenic tobacco BY-2 cells and plants harboring AaNhaD were generated and their stress tolerance was evaluated.Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner.Compared to wild-type controls,the transgenic cells exhibited increased Na+concentrations and pH levels in the vacuoles.Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts.Similar to the transgenic BY-2 cells,AaNhaD-overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil.These results indicate that AaNhaD functions as a pH-dependent tonoplast Na+/H+antiporter in plant cells,thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.     

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants. Salt tolerance assays followed by non-stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild-type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.     

水稻胁迫相关基因OsPM1启动子的克隆与分析

依据NCBI数据库OsPM1的序列信息,采用PCR技术扩增获取OsPM1的2 100bp的启动子序列。利用PLACE预测启动子的顺式作用元件分析表明,启动子内含有大量与胁迫相关的顺式作用元件,主要有ABA响应相关元件、脱水响应元件、低温响应元件、热激响应元件和转录因子结合元件。构建OsPM1的启动子和GUS基因融合表达载体,转入拟南芥。组织化学染色分析结果显示,非生物胁迫处理前,幼苗中GUS基因表达水平很低;干旱、低温、高盐等胁迫处理后,GUS基因表达量显著升高。研究表明,OsPM1的启动子能够显著提高在干旱、高盐和低温处理后下游基因的表达水平。     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

拟南芥AtNHX2启动子的克隆及表达模式分析

AtNHX2基因是拟南芥NHX基因家族的一员,编码了一种液泡膜中的Na+/H+反向运输体并对拟南芥的耐盐能力起着重要的作用.采用PCR扩增的方法克隆了拟南芥AtNHX2基因启始密码子上游约2.8kb的DNA片段,并将其克隆到植物表达载体pCAMBIA1301-1中,通过基因枪轰击洋葱表皮瞬时表达的方法,初步检测启动子的活性.将重组质粒pCAMBIA1301-1/AtNHX2 promoter转化拟南芥并筛选纯合子.AtNHX2 promoter-GUS分析显示AtNHX2在所有的组织中均有表达,包括根尖.在保卫细胞中检测到了强烈的GUS表达,这一结果表明,AtNHX2对特殊细胞的pH调控和K+自身稳定方面起着重要的作用.AtNHX2启动子的活性可被NaCl抑制,并且抑制的强度和NaCl的浓度成正相关.300 mmol/L KCl处理可增强启动子的活性,说明NaCl和KCl是在转录水平上调控AtNHX2的表达.在老叶中GUS活性比在新叶中GUS活性强,这说明了AtNHX2优先将有毒的离子积累在老叶中,从而有利于植物的正常发育.在根毛细胞中也观测到了强烈的GUS活性,这就暗示了AtNHX2在扩大的液泡中储存Na+.     

Overexpression of <Emphasis Type="Italic">OsVP1</Emphasis> and <Emphasis Type="Italic">OsNHX1</Emphasis> Increases Tolerance to Drought and Salinity in Rice

Drought and salinity are major abiotic stresses affecting rice production. To improve plant tolerance to salinity and drought, we overexpressed rice Na⁺/H⁺ exchangers (OsNHX1) and H⁺-pyrophosphatase in tonoplasts (OsVP1) in a japonica elite rice cultivar, Zhonghua 11. Compared with our wild-type control, transgenic plants overexpressing both genes incurred less damage when exposed to long-term treatment with 100 mM NaCl or water deprivation. Under high-saline conditions, the transformants accumulated less Na⁺ and malondialdehyde in the leaves, thereby allowing the plants to maintain a low level of leaf water potential and reduce stress-induced damage. Those transgenics also had higher photosynthetic activity during the stress period. Under those conditions, they also showed an increase in root biomass, which enabled more water uptake. These results suggest that OsVP1 and OsNHX1 improve the tolerance of rice crops against drought and salt by employing multiple strategies in addition to osmotic regulation.