多浆旱生植物霸王液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达分析
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摘要
盐胁迫是影响作物生长和产量的主要非生物因素之一。土壤中过高的Na~+浓度引起离子毒害、渗透胁迫和K~+/Na~+比率失调使植物新陈代谢紊乱,从而对植物造成伤害。植物抵御盐胁迫的有效策略之一是通过液泡膜Na~+/H~+逆向转运蛋白将细胞质中过多的Na~+区域化在液泡内,以减轻过量Na~+对细胞质的伤害,同时维持细胞的渗透势,从而提高植物的耐盐抗旱性。从极端干旱环境中生存的荒漠植物中克隆和鉴定液泡膜Na~+/H~+逆向转运蛋白基因,对农作物和优良牧草耐盐抗旱性的遗传改良具有重要意义。
本研究以多浆旱生植物霸王(Zygophyllum xanthoxylum)为实验材料,克隆到液泡膜Na~+/H~+逆向转运蛋白基因ZxNHX和Actin片段,并以Actin为内参对ZxNHX在盐处理下的表达进行了研究。主要结果如下:
1.根据已知的液泡膜Na~+/H~+逆向转运蛋白基因的保守区设计简并性引物,采用RT-PCR及RACE方法,从霸王中克隆到液泡膜Na~+/H~+逆向转运蛋白基因的全长cDNA,将其命名为ZxNHX。该cDNA全长2127bp,包括1599bp的开放阅读框(ORF)、213 bp的5'非翻译区(5'UTR)、315 bp的3'非翻译区(3'UTR)和25 bp的poly(A)尾巴。此cDNA编码一个532个氨基酸的多肽,推测分子量为58.8 KDa,等电点为7.23。霸王ZxNHX的氨基酸序列与已报道的胡杨PeNHX2、拟南芥AtNHX1、毛白杨PtNHX1、北滨藜AgNHX1、盐地碱蓬SsNHX1和水稻OsNHX1的同源性较高,分别为81%、80%、79%、78%、74%和73%。ZxNHX蛋白含有12个跨膜区,其中TM3具有高度保守的氨氯吡嗪脒结合位点(~(78)LFFYLLPPI~(87))。此外,ZxNHX含有2个糖基化位点,分别为Asn~(44)、Asn~(287),表明此蛋白是已糖基化的蛋白。ZxNHX已在GenBank注册,登录号为EU103624。
2.根据已知的Actin基因的保守区设计简并性引物,利用RT-PCR方法从霸王中克隆到Actin基因片段,将其命名为ZxACT。该片段大小为598 bp,编码198个氨基酸。序列分析表明,ZxACT与其他植物的Actin基因核苷酸序列的同源性在82%以上;而与氨基酸序列的同源性达91%以上。ZxACT已在GenBank注册,登录号为EU019550。
3.半定量RT-PCR分析结果显示,ZxNHX在叶和根中均表达,但叶中的表达量明显高于根;在高浓度盐处理下(150、200 mmol·L~(-1)NaCl),ZxNHX转录水平明显上调,在处理3、12h后达到最大值。由此表明,ZxNHX表达受盐胁迫的诱导和调节。
Salinity is one of the major abiotic stress factors that adversely effect the crop plant growth and yield.The excessive sodium ions in soil cause ions toxicity,osmotic stresses, and the damage in K~+/Na~+ homeostasis.One of the effective strategies that enable plants to resist salt stress is to compartmentalize Na~+ into the vacuole by the vacuolar Na~+/H~+ antiporter.This process can avert the deleterious effects of Na~+ in the cytosol and maintain osmotic balance by using Na~+ accumulated in the vacuole to drive water into the cells,and improve salt- and drought- tolerance of plants.Therefore,it is important meaning for genetic improvement of salt- and drought- tolerance of the crops and herbages to isolate and characterize the vacuolar Na~+/H~+ antiporter from the desert plants lived in extremely arid environment.
In this study,the vacuolar Na~+/H~+ antiporter gene,ZxNHX,and the fragment of actin gene were cloned from the succulent xerophyte Zygophyllum xanthoxylum;and expression of ZxNHX was analyzed using actin as internal control under salt stress.The main results were as following:
1.Degenerate primers were designed based on the conserved sequences of the vacuolar Na~+/H~+ antiporter genes from other plants.A novel vacuolar Na~+/H~+ antiporter gene was isolated from Z.xanthoxylum using RT-PCR and RACE,and named as ZxNHX.The cDNA is 2127 bp in length including an open reading frame(ORF)of 1558 bp,a 5'untranslated region(5'URT)of 213 bp and a 3'untranslated region(3'URT)of 315 bp with a ploy(A) tail.The ZxNHX cDNA encodes a protein of 532 amino acids with a deduced molecular mass of 58.8 KDa and a pI of 7.23.The ZxNHX protein shares 81%,80%,79%,78%,74% and 73%homology with the six reported other NHXs,namely PeNHX2(DQ414512), AtNHX1(AY685183),PtNHX1(AY660749),AgNHX1(AB038492),SsNHX1(AF370358) and OsNHX1(AY324877),respectively.ZxNHX has twelve putative transmembrane(TM) domains,the TM3 of which contains the conserved amiloride-binding sites (~(78)LFFYLLPPI~(87)).In addition,two potential N-glycosylation sites were found within the ZxNHX protein,at Asn~(44)and Asn~(287),respectively.These results suggested that the ZxNHX protein is glycosylated.The gene has been submitted to GenBank database,the accession number is EU103624.
2.Degenerate primers were designed based on the conserved sequences of the actin genes from other plants.The fragment of actin gene was isolated from Z.xanthoxylum by RT-PCR,and named as ZxACT.The sequencing result revealed that the actin gene fragment from Z.xanthoxylum is 598 bp in length,encoding a protein of 198 amino acids. Homology comparison with other plants actin gene sequences in the GenBank shows that it shares over 82%nucleotide sequence homology and 91%amino acid sequence homology with other plants actins.The gene has been submitted to GenBank database,the accession number is EU019550.
3.Semi-quantitative RT-PCR analysis results showed ZxNHX was expressed strongly in leaves while weakly in roots.The transcript level of ZxNHX in the leaves was up-regulated by 150 or 200 mmol·L~(-1)NaCl,and highest at 3 or 12 h.These results suggested the expression of ZxNHX was induced and regulated by salt stress.
本研究以多浆旱生植物霸王(Zygophyllum xanthoxylum)为实验材料,克隆到液泡膜Na~+/H~+逆向转运蛋白基因ZxNHX和Actin片段,并以Actin为内参对ZxNHX在盐处理下的表达进行了研究。主要结果如下:
1.根据已知的液泡膜Na~+/H~+逆向转运蛋白基因的保守区设计简并性引物,采用RT-PCR及RACE方法,从霸王中克隆到液泡膜Na~+/H~+逆向转运蛋白基因的全长cDNA,将其命名为ZxNHX。该cDNA全长2127bp,包括1599bp的开放阅读框(ORF)、213 bp的5'非翻译区(5'UTR)、315 bp的3'非翻译区(3'UTR)和25 bp的poly(A)尾巴。此cDNA编码一个532个氨基酸的多肽,推测分子量为58.8 KDa,等电点为7.23。霸王ZxNHX的氨基酸序列与已报道的胡杨PeNHX2、拟南芥AtNHX1、毛白杨PtNHX1、北滨藜AgNHX1、盐地碱蓬SsNHX1和水稻OsNHX1的同源性较高,分别为81%、80%、79%、78%、74%和73%。ZxNHX蛋白含有12个跨膜区,其中TM3具有高度保守的氨氯吡嗪脒结合位点(~(78)LFFYLLPPI~(87))。此外,ZxNHX含有2个糖基化位点,分别为Asn~(44)、Asn~(287),表明此蛋白是已糖基化的蛋白。ZxNHX已在GenBank注册,登录号为EU103624。
2.根据已知的Actin基因的保守区设计简并性引物,利用RT-PCR方法从霸王中克隆到Actin基因片段,将其命名为ZxACT。该片段大小为598 bp,编码198个氨基酸。序列分析表明,ZxACT与其他植物的Actin基因核苷酸序列的同源性在82%以上;而与氨基酸序列的同源性达91%以上。ZxACT已在GenBank注册,登录号为EU019550。
3.半定量RT-PCR分析结果显示,ZxNHX在叶和根中均表达,但叶中的表达量明显高于根;在高浓度盐处理下(150、200 mmol·L~(-1)NaCl),ZxNHX转录水平明显上调,在处理3、12h后达到最大值。由此表明,ZxNHX表达受盐胁迫的诱导和调节。
Salinity is one of the major abiotic stress factors that adversely effect the crop plant growth and yield.The excessive sodium ions in soil cause ions toxicity,osmotic stresses, and the damage in K~+/Na~+ homeostasis.One of the effective strategies that enable plants to resist salt stress is to compartmentalize Na~+ into the vacuole by the vacuolar Na~+/H~+ antiporter.This process can avert the deleterious effects of Na~+ in the cytosol and maintain osmotic balance by using Na~+ accumulated in the vacuole to drive water into the cells,and improve salt- and drought- tolerance of plants.Therefore,it is important meaning for genetic improvement of salt- and drought- tolerance of the crops and herbages to isolate and characterize the vacuolar Na~+/H~+ antiporter from the desert plants lived in extremely arid environment.
In this study,the vacuolar Na~+/H~+ antiporter gene,ZxNHX,and the fragment of actin gene were cloned from the succulent xerophyte Zygophyllum xanthoxylum;and expression of ZxNHX was analyzed using actin as internal control under salt stress.The main results were as following:
1.Degenerate primers were designed based on the conserved sequences of the vacuolar Na~+/H~+ antiporter genes from other plants.A novel vacuolar Na~+/H~+ antiporter gene was isolated from Z.xanthoxylum using RT-PCR and RACE,and named as ZxNHX.The cDNA is 2127 bp in length including an open reading frame(ORF)of 1558 bp,a 5'untranslated region(5'URT)of 213 bp and a 3'untranslated region(3'URT)of 315 bp with a ploy(A) tail.The ZxNHX cDNA encodes a protein of 532 amino acids with a deduced molecular mass of 58.8 KDa and a pI of 7.23.The ZxNHX protein shares 81%,80%,79%,78%,74% and 73%homology with the six reported other NHXs,namely PeNHX2(DQ414512), AtNHX1(AY685183),PtNHX1(AY660749),AgNHX1(AB038492),SsNHX1(AF370358) and OsNHX1(AY324877),respectively.ZxNHX has twelve putative transmembrane(TM) domains,the TM3 of which contains the conserved amiloride-binding sites (~(78)LFFYLLPPI~(87)).In addition,two potential N-glycosylation sites were found within the ZxNHX protein,at Asn~(44)and Asn~(287),respectively.These results suggested that the ZxNHX protein is glycosylated.The gene has been submitted to GenBank database,the accession number is EU103624.
2.Degenerate primers were designed based on the conserved sequences of the actin genes from other plants.The fragment of actin gene was isolated from Z.xanthoxylum by RT-PCR,and named as ZxACT.The sequencing result revealed that the actin gene fragment from Z.xanthoxylum is 598 bp in length,encoding a protein of 198 amino acids. Homology comparison with other plants actin gene sequences in the GenBank shows that it shares over 82%nucleotide sequence homology and 91%amino acid sequence homology with other plants actins.The gene has been submitted to GenBank database,the accession number is EU019550.
3.Semi-quantitative RT-PCR analysis results showed ZxNHX was expressed strongly in leaves while weakly in roots.The transcript level of ZxNHX in the leaves was up-regulated by 150 or 200 mmol·L~(-1)NaCl,and highest at 3 or 12 h.These results suggested the expression of ZxNHX was induced and regulated by salt stress.
引文
[1]安静,张荃.拟南芥液泡膜Na~+/H~+逆向转运蛋白的研究进展.生命科学,2006,18(3):273-278.
[2]奥斯伯,布伦特,金斯顿,等.精编分子生物学实验指南(马学军,颜子颖,王海林译).北京:科学出版社,2005,120-126.
[3]包爱科,张金林,郭正刚,等.液泡膜H~+-PPase与植物耐盐性.植物生理学通讯.2006,42(4):777-783.
[4]陈颖,王刚,赵俊霞.高等植物体内的肌动蛋白.生物学报,2003,38(1):13-15.
[5]J 萨姆布鲁克,D W 拉赛尔.分子克隆实验指南(黄培堂译).北京:科学出版社,2002,26-27.
[6]李宏,王新力.植物组织RNA提取的难点及对策.生物技术通报,1999,15(1):36-39.
[7]李三相.Na~+与多浆旱生植物霸王抗旱性研究:[硕士学位论文].甘肃:兰州大学,2006.
[8]刘欣玲.刺槐无性系转化体系的建立及液泡型Na~+/H~+逆向转运蛋白基因的克隆[硕士学位论文].山东:山东农业大学,2006.
[9]李园莉,江元清,赵武玲,等.谷子肌动蛋白基因克隆及序列分析.植物学通报,2002,19(3):310-316.
[10]吕慧颖,李银心,陈华,等.番杏Na~+/H~+逆向转运蛋白基因的克隆及特性分析.高技术通讯,2004,11:26-31.
[11]邱生平,周国安,陆驹,等.一个新的水稻液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达特征.中国水稻科学,2006,20(2):119-124.
[12]沈国新,楼程富,裘晓云,等.桑树中拟南芥同源基因mNHX1的克隆及其功能研究,蚕业科学,2005,31(4):398-403.
[13]孙超,陈维多,林忠平.一种改进的沙蒿总RNA的提取方法.生物技术通报,2004,5:43-44.
[14]田秀红,高建明,肖强,等.水花生Actin基因片段的克隆及序列分析.生物技术,2006,16(3):13-14.
[15]王关林,方宏筠.植物基因工程原理与技术.北京:科学出版社,1998,611-614.
[16]王劲.甘蓝型油菜耐盐基因的克隆及表达研究:[博士学位论文].上海:复旦大学.2004.
[17]伍国强,王强龙,包爱科,等.液泡膜Na~+/H~+逆向转运蛋白与植物耐盐性.中国农业科技导报,2008a,10(2):13-21. 方法.分子植物育种,2008b,6(1):197-200.
[19]伍国强,席杰军,包爱科,等.多浆旱生植物霸王Actin基因片断的克隆及序列分析.生物技术通报,2008c,2:101-105.
[20]肖前谷,叶妙水,钟克亚,等.北美海蓬子Na~+/H~+逆向运输蛋白基因的克隆及拟南芥转化分析.分子植物育种,2007,5(5):619-624.
[21]杨亮,付丽娅,刘仲齐,等.富含多糖番茄果实组织中总RNA的有效提取方法,南开大学学报(自然科学版).2005,38(5):36-39.
[22]杨丽霞,李玲.花生肌动蛋白基因克隆及序列分析.花生学报,2006,35(4):6-9.
[23]严一诺,孙淑斌,徐国华,等.菊芋Na~+/H~+逆向转运蛋白基因的克隆与表达分析.西北植物学报,2007,27(7):1291-1298.
[24]赵一之,朱宗元.亚洲中部荒漠区的植物特有属.云南植物研究.2003,25(3):113-121.
[25]张德强,赵淑堂,卢孟柱,等.杨树Na~+/H~+反向运输蛋白基因(PtNHX1、PtNHX6)的克隆和检测.林业科学,2006,42(11):29-36.
[26]张耿,王赞,关宁,等.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.遗传,2007,29(10):1263-1270.
[27]张今今,王跃进,王西平,等.葡萄总RNA提取方法的研究.果树学报,2003,20(3):178-181.
[28]张俊莲,张金文,陈正华,等.植物Na~+/H~+逆向转运蛋白与植物耐盐性的研究进展.草原与草坪,2005,4:3-8.
[29]张霞,曾幼玲,李金耀,等.胡杨Na~+/H~+反向运输载体(PeNHX2)基因的克隆与序列分析.生物技术,2006,16(3):9-13.
[30]周海飞,赵武玲,阎隆飞.玉兰肌动蛋白基因的克隆及序列分析.农业生物技术学报,2001,9(3):274-278.
[31]庄军平,苏菁,陈维信.一种从香蕉果实提取高质量RNA的方法.分子植物育种,2006,4(1):143-146.
[32]Aharon G S,Apse M P,Duan S,et al..Characterization of a family of vacuolar Na~+/H~+ antiporters in Arabidopsis thaliana.Plant and Soil,2003,00:245-256.
[33]Ainsworth C.Isolation of RNA from floral tissue of Rumex acetosa(Sorrel).Plant Mol Biol Rep,1994,12:198-203.
[34]Apse M P,Aharon G S,Snedden W A,et al..Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiporter in Arabidopsis.Science,1999,285:1256-1258.
[35]Apse M P,Sottosanto J B,Blumwald E.Vacuolar cation/H~+ exchange,ion homeostasis,and leaf development are altered in a T-DNA insertional mutant of AtNHX1,the Arabidopsis vacuolar Na~+/H~+ antiporter.Plant J,2003,36:229-239.
[36]Ballesteros E,Blumwald E,Donaire J P,et al..Na+/H+ antiport activity in tonoplast vesicles isolated from sunflowers roots induced by NaCl stress.Physiologia Plantarum,1997,99:328-334.
[37]Barkla B J,Zingarelli L,Blumwald E,et al..Tonoplast Na~+/H~+ antiport activity and its energization by the vacuolar H+-AYPase in the halophytic plant Mesembryanthemum crystallinum L.Plant Physiol,1995,109:549-556.
[38]Barkla B J,Zingarelli L,Blumwald E,et al..Tonoplast Na+/H+ antiport activity and its energization by the vacuolar H+-ATPase in the halophytic plant Mesembryanthemum crystallinurn.Plant Physiol,1995,109:549-546.
[39]Bartels D,Sunkar R.Drought and salt tolerance in plants.Crit Rev Plant Sci,2005,24:23-58.
[40]Blumwald E,Poole R J.Na~+/H~+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris.Plant Physiol,1985,78:163-167.
[41]Blumwald E.Tonoplast vesicles as a tool in the study of ion transport at the plant vacuole.Physiologia Plantarum,1987,69:731-734.
[42]Blumwald E,Poole R J.Salt Tolerance in suspension cultures of sugar beet:induction of Na~+/H~+ antiport activity at the tonoplast by growth in salt.Plant Physiol,1987,83:884-887.
[43]Boyer J S.Plant productivity and environment.Science,1982,218:443-448.
[44]Bray E A.Plant responses to water deficit.Trends Plant Sci,1997,2:48-54.
[45]Bressan R A,Hasegawa P M,Pardo J M.Plants use calcium to resolve salt stress.Trends Plant Sci,1998,3:411-412.
[46]Brett C L,Donowitz M,Rao R.Evolutionary origins of eukaryotic sodium/proton exchangers.A J P - Cell Physiol,2005,288:C223-C239.
[47]Brini F,Gaxiola R,Berkowitz G,Masmoudi K.Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump.Plant Physiol Biochem,2005,43:347-354.
[48]Brini F,Hanin M,Mezghani I,et al..Overexpression of wheat Na~+/H~+ antiporter TNHX1 and H~+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants.J Exp Bot,2007,58:301-308.
[49]Brownell P F.Sodium as an essential micronutrient element for plants and its possible role in metabolism.Advance in Botanical Research,1979,7:117-224.
[50]Busk P K,Pages M.Regulation of abscisic acid induced transcription.Plant Mol Biol,1998,37:425-435.
[51]Cheng N H,Pittman J K,Zhu J K,et al..The protein kinase SOS2 activates the Arabidopsis H~+/Ca~(2+)antiporter CAX1 to integrate calcium transport and salt tolerance.J Biol Chem,2004,279:2922-2926.
[52]Chen H,An R,Tang J H,et al..Over-expression of a vacuolar Na~+/H~+ antiporter gene improves salt tolerance in an upland rice.Mol Breeding,2007,19:215-225.
[53]Chen L H,Zhang B,Xu Z Q.Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na~+/H~+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum).Transgenic Res,2008,17:121-132.
[54]Chinnusamy V,Jagendorf A,Zhu J K.Understanding and improving salt tolerance in plants.Crop Sci,2005,45:437-448.
[55]Chomczynski P,Sacchi N.Single step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction.Anal Biochem,1987,162:156-159.
[56]Collings DA,Harper JD,Marc J,et al..Life in t he fast lane:Actin based motility of plant peroxisomes.Can J Bot,2002,80:430-441.
[57]Dupont F M.Salt-induced changes in ion transport:Regulation of primary pumps and secondary transporters.In:Cooke D T,Clarkson D T eds.Transport and Receptor Proteins of Plant Membranes.Plenum Press,New York,1992,91-100.
[58]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA,Biotechniques.1992,13:52-57.
[59]Firtel R.Multigene family encoding actin and tubulin.Cell,1981,24:6-7.
[60]Flowers T J,Troke P F,Yeo A R.The mechanisms of salt tolerance in halophytes.Ann Rev Plant Physiol,1977,28:89-121.
[61]Fukuda A,Chiba K,Maeda M,et al..Effect of salt and osmotic stresses on the expression of genes for the vacuolar H~+-pyrophosphatase,H~+-ATPase subunit A,and Na~+/H~+ antiporter from barley.J Exp Bot,2004,55:585-594.
[62]Fukuda A,Nakamura A,Tagiri A,et al..Function,intracellular localization and the importance in salt tolerance of a vacuolar Na~+/H~+ antiporter from rice.Plant Cell Physiol,2004,45:146-159.
[63]Fukuda A,Nakamura A,Tanaka Y.Molecular cloning and expression of the Na~+/H~+exchanger gene in Oryza sativa.Biochimica et Biophysica Acta,1999,1446:149-155.
[64]Fukuda A,Tanaka Y.Effects of ABA,auxin,and gibberellin on the expression of genes for vacuolar H~+-inorganic pyrophosphatase,H~+-ATPase subunit A,and Na~+/H~+antiporter in barley.Plant Physiol Biochem,2006,44:351-358.
[65]Fukuda A,Yazaki Y,Ishikawa T,et al..Na~+/H~+ antiporter in tonoplast vesicles from rice roots.Plant Cell Physiol.1998,39:196-201.
[66]Garbarino J,Dupont F M.NaCl induced a Na~+/H~+ antiport in tonoplast vesicles from barley roots.Plant Physiol,1988,86:231-236.
[67]Garbarino J,Dupont F M.Rapid induction of Na~+/H~+ exchange activity in barley root tonoplast.Plant Physiol.1989,89:1-4.
[68]Gaxiola R A,Li J S,Undurraga S,et al..Drought- and salt-tolerant plants result from overexpression of the AVP1 H~+-pump.Proc Natl Acad Sci USA,2001,98:1444-11449.
[69]Gaxiola R A,Rao R,Sherman A,et al..The Arabidopsis thaliana proton transporters:AtNhx1 and Avpl,can function in cation detoxification in yeast.Proc Natl Acad Sci USA,1999,96:1480-1485.
[70]Geourjon C,Deleage G.SOPMA:Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments.Cabios.1995,11:681-684.
[71]Glenn E,Brown J J,Blumwald E.Salt-tolerant mechanisms and crop potential of halophytes.Crit Rev Plant Sci,1999,8:227-255.
[72]Guo Y,Halfter U,Ishitani M,Zhu J K.Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance.Plant Cell,2001,13:1383-1399.
[73]Guren J,Mathieu Y,Kurkdjian A.Regulation of vacuolar pH in plant cells.Plant Physiol,1989,89:27-36.
[74]Hamada A,Shono M,Xia T,et al..Isolation and characterization of a Na~+/H~+antiporter gene from the halophyte Atriplex gmelini.Plant Mol Biol,2001,46:35-42.
[75]Hanana M,Cagnac O,Yamaguchi T,et al..A grape berry(Vitis vinifera L.)cation/proton antiporter is associated with berry ripening.Plant Cell Physiol,2007,48(6):804-811.
[76]Hasegawa P M,Bressan R A,Zhu J K,et al..Plant cellular molecular responses to high salinity.Annu Rev Plant Physiol Plant Mol Biol.2000,51:463-499.
[77]Hassidim M,Braun Y,Lemer H R,et al..Na~+/H~+ and K~+/H~+ antiport in root membrane vesicles isolated from the halophyte Atriplex and the glycophyte cotton.Plant Physiol,1990,94:1795-1801.
[78]He C X,Yan J Q,Shen G X,et al..Expression of an Arabidopsis vacuolar sodium/proton antiporter gene in cotton improves photosynthetic performance under salt conditions and increases fiber yield in the field.Plant Cell Physiol,2005,46: 1848-1854.
[79]Hernandez J A,Ferrer M A,Jimenez A,et al..Antioxidant systems and O2~+/H_2O_2production in the apoplast of pea leaves.Its relation with salt-induced necrotic lesions in minor veins.Plant Physiol.2001.127:817-831.
[80]Hofmann K,Stoffel W.Tmbase-A database of membrane spanning proteins segments.Biol Chem Hoppe-Seyler,1993,374:166.
[81]Jia W,Wang Y,Zhang S,Zhang J.Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots.J Exp Bot,2002,53:2201-2206.
[82]Kagami T,Suzuki M..Molecular and functional analysis of a vacuolar Na~+/H~+antiporter gene of Rosa hybrida.Genes Genet Syst,2005,80:121-128.
[83]Knight H.Calcium signalling during abiotic stress in plants.International Review of Cytology,2000,195:269-325.
[84]Knight H,Trewavas A J,Knight M R.Calcium signaling in Arabidopsis thaliana responding to drought and salinity.Plant J,1997,12:1067-1078.
[85]Li B,Wang B,Tang K,et al..A simple and convenient approach for isolating RNA from highly viscous plant tissue rich in polysaccharides.Colloids Surf B:Biointerfaces,2006,49(2):101-105.
[86]Li J,Jiang G,Huang P,et al..Overexpression of the Na~+/H~+ antiporter gene from Suaeda salsa confers cold and salt tolerance to transgenic Arabidopsis thaliana.Plant Cell Tiss Organ Cult,2007,90:41-48.
[87]Li J,Yang H,Peer W A,et al..Arabidopsis H+-PPase A VP1 regulates auxin-mediated organ development.Science,2005,310:121-125.
[88]Liu J,Zhu J K.A calcium sensor homolog required for plant salt tolerance.Science,1998,280:1943-1945.
[89]Li Y W,Wong F L,Tsai S N,et al..Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow(BY)-2 cells.Plant Cell Environ,2006,9(29):1122-1137.
[90]Lu S Y,Jing Y X,Shen S H,et al..Antiporter gene from Hordum brevisubulatum (Trin.)link and its overexpression in transgenic tobaccos.Journal of Integrative Plant Biology,2005,47:343-349.
[91]Mange A,Meagher R B.The molecular evolution of actin.Gentics,1986,114:315-332.
[92]Martinoia E,Maeshima M,Neuhaus H E.Vacuolar transporters and their essential role in plant metabolism.J Exp Bot,2007,58(1):83-102.
[93]Matoh T,Watanabe J,Takahashi E.Sodium,potassium,chloride,and betaine concentrations in isolated vacuoles from salt-grown Atriplex gmelini leaves.Plant Physiol,1987,84:173-177.
[94]Ma X L,Zhang Q,Shi H Z,et al..Molecular cloning and different expression of a vacuolar Na~+/H~+ antiporter gene in Suaeda salsa under salt stress.Biologia Plantarum,2004,48:219-225.
[95]Mennen H,Jacoby B,Marschner H.Is sodium proton antiport ubiquitous in plant cells ? J Plant Physiol,1990,137:180-183.
[96]Moffat A S.Plant genetics:Finding new ways to protect drought-stricken plants.Science,2002,296:1226-1229.
[97]Nass R,Cunningham KW,Rao R.Intracellular sequestration of sodium by a novel Na~+/H~+ exchanger in yeast is enhanced by mutations in the plasma membrane H~+-ATPase:insights into mechanisms of sodium tolerance.J Biol Chem,1997,272:26145-26152.
[98]Niu X,Bressan R A,Hasegawa P M,et al..Ion homeostasis in NaCI stress environment.Plant Physiol,1995,109:735-742.
[99]Ohnishi M,Fukada-Tanaka S,Hoshino A,et al..Characterization of a novel Na~+/H~+antiporter gene InNHX2 and comparison of InNHX2 with InNHX1,which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory.Plant Cell Physiol,2005,46:259-267.
[100]Orlowski J,Grinstein S.Na~+/H~+ exchanger of mammalian cells.J Biol Chem,1997,272:22373-22376.
[101]Orlowski J.Heterologous expressiln and functional properties of amiloride high affinity(NHE-1)and low affinity(NHE-3)isoforms of the rat Na~+/H~+ exchanger.J Biol Chem,1993,268:16369-16377.
[102]Page R D.TREEVIEW:An application to display phylogenetic trees on personal computers.Comput Appl Bio,1996,12:357-358.
[103]Pardo J M,Cubero B,Leidi E O,et al..Alkali cation exchangers:roles in cellular homostasis and stress tolerance.J Exp Bot,2006,57:1181-1199.
[104]Popova O V,Golldack D.In the halotolerant Lobularia maritime(Brassicaceae)salt adaptation correlates with activation of the vacuolar H~+-ATPase and the vacuolar Na~+/H~+ antiporter.J Plant Physiol,2007,164:1278-1288.
[105]Porat R,Pavoncello D,Ben-Hayyim G,et al..A heat treatment induced the expression of a Na~+/H~+ antiport gene(cNHX1)in citrus fruit.Plant Sci,2002,162:957-963.
[106]Putney L K and Barber D L.Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition.J Biol Chem,2003,278:44645-44649.
[107]Qiao W H,Zhao X Y,Li W,et al..Overexpression of AeNHX1,a root-specific vacuolar Na~+/H~+ antiporter from Agropyron elongatum,confers salt tolerance to Arabidopsis and Festuca plants.Plant Cell Rep,2007,26(9):1663-1672.
[108]Qiu N,Chen M,Guo J,et al..Coordinate up-regulation of V-H~+-ATPase and vacuolar Na~+/H~+ antiporter as a response to NaCl treatment in a C3 halophyte Suaeda salsa.Plant Sci,2007,173(5):487-494.
[109]Qiu Q S,Guo Y,Quintero F J,et al..Regulation of vacuolar Na~+/H~+ exchange in Arabidopsis thaliana by the salt-overly-sensitive(SOS)pathway.J Biol Chem,2004,279:207-215.
[110]Quesada V,Ponce M R,Micol J L.Genetic analysis of salt tolerant mutants in Arabidopsis thaliana.Genetics.2000,154:421-436.
[111]Rajagopal D,Agarwal P,Tyagi W,et al..Pennisetum glaucum Na~+/H~+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea.Mol Breeding,2007,19:137-151.
[112]Sahi C,Singh A,Kumar K,et al..Salt stress response in rice:genetics,molecular biology,and comparative genomics.Funct Integr Genomics,2006,6:263-284.
[113]Sanders D,Brownlee C,Harper J F.Communicating with calcium.Plant Cell,1999,11:691-706.
[114]Sardet C,Franchi A,Pouyssegur J.Molecular cloning,primary structure and expression of the human growth factor-actiatable Na+/H+ antiporter.Cell,1989,56:271-280.
[115]Sato Y,Sakaguchi M.Topogenic properties of transmembrane segments of Arabidopsis thaliana NHX1 reveal a common topology model of the Na~+/H~+exchanger family.J Biochem,2005,138:425-431.
[116]Sauter A,Dietz K J,Hartung W.A possible stress physiological role of abscisic acid conjugates in root-to-shoot signalling.Plant Cell Environ,2002,25:223-228.
[117]Schroeder J I,Kwak J M,Allen G J.Guard cell abscisic acid signalling and engineering drought hardiness in plants.Nature,2001,410:327-330.
[118]Sheterline P,Sharrow JC.Protein profile:Actin,New York:Academic Press,1994.
[119]Shi H,Lee B H,Wu S J,et al..Overexpression of a plasma membrane Na~+/H~+antiporter gene improves salt tolerance in Arabidopsis thaliana.Nat Biotechnol,2003,21:81-85.
[120]Shi H,Zhu J K.Regulation of expression of the vacuolar Na~+/H~+ antiporter gene AtNHX1 by salt stress and abscisic acid.Plant Mol Biol,2002,50:543-550.
[121]Shinozaki K,Yamaguchi-Shinozaki K.Gene expression and signal transduction in water-stress response.Plant Physiol,1997,115:327-334.
[122]Smirnoff N.The role of active oxygen in the response of plants to water deficit and desiccation.New Phytol,1993,125:27-58.
[123]Staal M,Maathuis F J,Elzenga J T,et al..Na~+/H~+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the saltsensitive Plantago media.Physiologia Plantarum 1991,82:179-184.
[124]Sun Y X,Wang D,Bai Y L,et al..Studies on the overexpression of the soybean GmNHX1 in Lotus corniculatus:The reduced Na~+ level is the basis of the increased salt tolerance.Chinese Science Bulletin,2006,51:1306-1315.
[125]Yesniere C,Vayda M E.Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates,Plant Mol Biol Rep,1991,9(3):242-251.
[126]Thompson J D,Gibson T J,Plewniak F,et al..The Clustal X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucl.Acids Res.1997,25:4876-4882.
[127]Vasekina A V,Yershov P V,Reshetova O S,et al..Vacuolar Na~+/H~+ antiporter from Barley:identification and response to salt stress.Biochemistry(Moscow),2005,70,(1):100-107.
[128]Venema V,Belver A,Marin-Manzano M C,et al..A novel intracellular K~+/H~+antiporter related to Na~+/H~+ antiporters is important for K~+ ion homeostasis in plants.J Biol Chem,2003,278:22453-22459.
[129]Verma D,Singla-Pareek S L,Rajagopal D,et al..Functional validation of a novel isoform of Na~+/H~+ antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice.JBiosci,2007,32(3):1-7.
[130]Verslues P E,Zhu J K.Before and beyond ABA:upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress.Biochem Soc Trans,2005,33:375-379.
[131]Vidali,L,Helper,P K.Actin and pollen tube growth.Protoplasma,2001,215:64-76.
[132]Wang J,Zuo K,Wu W,et al..Expression of a novel antiporter gene from Brassica napus resulted in enhanced salt tolerance in transgenic tobacco plants.Biologia Plantarum,2004,48:509-515.
[133]Wang J,Zuo K,Wu W,et al..Molecular cloning and characterization of a new Na~+/H~+ antiporter gene from Brassica napus.DNA Seq,2003,14:351-358.
[134]Wang S M,Wan C G,Wang Y R,et al.The characteristics of Na~+,K~+ and free proline distribution in several drought-resistant plants of the Alxa Desert China.J Arid Environ,2004,56(3):525-539.
[135]Woodhead M,Taylor M A,Davies H V,et al..Isolation of RNA from blackcurrant (Ribes nigrum L.)fruit.Mol Biotechnol,1997,7(1):1-4.
[136]Wu C A,Yang G D,Meng Q W,et al..The cotton GhNHX1 gene encoding a novel putative tonoplast Na~+/H~+ antiporter plays an important role in salt stress.Plant Cell Physiol,2004,45:600-607.
[137]Wu S J,Ding L,Zhu J K.Sosl,a genetic locus essential for salt tolerance and potassium acquisition.Plant Cell,1996,8:617-627.
[138]Xia T,Apse M P,Aharon G S,et al..Identification and characterization of a NaCl-inducible vacuolar Na~+/H~+ antiporter in Beta vulgaris.Physiol Plant,2002,116:206-212.
[139]Xiong L,Zhu J K.Regulation of abscisic acid biosynthesis.Plant Physiol,2003,133:29-36.
[140]Xue Z Y,Zhi D Y,Xue G P,et al..Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.)expressing a vacuolar Na~+/H~+ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na~+.Plant Sci,2004,167(4):849-859.
[141]Yamaguchi T,Aharon G S,Sottosanto J B,et al..Vacuolar Na~+/H~+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca~(2+)-and pH-dependent manner.Proc Natl Acad Sci USA,2005,102:16107-16112.
[142]Yamaguchi T,Apse M P,Shi H,et al..Topological analysis of a plant vacuolar Na~+/H~+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity.Proc Natl Acad Sci USA,2003,100:12510-12515.
[143]Yamaguchi T,Blumwald E.Developing salt-tolerant crop plants:challenges and opportunities.Trends Plant Sci,2005,10:615-620.
[144]Yamaguchi T,Fukada-Tanaka S,Inagaki Y,et al..Genes encoding the vacuolar Na~+/H~+ exchanger and flower coloration.Plant Cell Physiol,2001,42:451-61.
[145]Yin XY,Yang A F,Zhang KW,et al..Production and analysis of transgenic maize with improved salt tolerance by the introduction of AtNHX1 gene.Acta Botanica Sinica,2004,46:854-861.
[146]Yokoi S,Quintero F J,Cubero B,et al..Differential expression and function of Arabidopsis thaliana NHX Na~+/H~+ antiporters in the salt stress response.Plant J,2002,30:529-539.
[147]Yoshida K,Kawachi M,Mori M,et al..The involvement of tonoplast proton pumps and Na~+(K~+)/H~+ exchangers in the change of petal color during flower opening of morning glory,Ipomoea tricolor cv.heavenly blue.Plant Cell Physiol,2005,46:407-415.
[148]Yu J N,Huang J,Wang Z N,et al..A vacuolar Na~+/H~+ antiporter from wheat plays an important role in stress tolerance.J Biosci,2007,32:1153-1161.
[149]Zhang G H,Su Q,An L J,et al..Characterization and expression of a vacuolar Na~+/H~+ antiporter from the monocot halophyte Aeluropus littoralis.Plant Physiol Biochem,2008,46:117-126.
[150]Zhang H X,Blumwald E.Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit.Nature Biotechnology,2001,19:765-768.
[151]Zhang H X,Joanna N H,John P M,et al..Engineering salt tolerant Brassica plant:characterization of yield and seed oil quality in transgenic plants with increase vacuolar sodium accumulation.Proc Natl Acad Sci USA,2001,98:12832-12836.
[152]Zhao F Y,Zhang X J,Li P H,et al..Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1.Mol Breeding,2006,17(4):341-353.
[153]Zhen R G,Kim E J,Rea P A.Acidic residues necessary for pyrophosphate energized pumping and inhibition of the vacuolar H~+-pyrophyosphatase by N,N-dicyclohex -ylcarbodiimide.J Biol Chem,1997,29:22340-22348.
[154]Zhou S,Chen X,Zhang X,et al..Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene BADH and a vacuolar Na~+/H~+antiporter gene SeNHX1.Biotechnol Lett,2008,30:369-376.
[155]Zhu J K.Plant salt tolerance.Trends Plant Sci,2001,6:66-71.
[156]Zorb C,Noll A,Karl S,et al..Molecular characterization of Na~+/H~+ antiporters (ZmNHX)of maize(Zea mays L.)and their expression under salt stress.J Plant Physiol,2005,162:55-66.
[2]奥斯伯,布伦特,金斯顿,等.精编分子生物学实验指南(马学军,颜子颖,王海林译).北京:科学出版社,2005,120-126.
[3]包爱科,张金林,郭正刚,等.液泡膜H~+-PPase与植物耐盐性.植物生理学通讯.2006,42(4):777-783.
[4]陈颖,王刚,赵俊霞.高等植物体内的肌动蛋白.生物学报,2003,38(1):13-15.
[5]J 萨姆布鲁克,D W 拉赛尔.分子克隆实验指南(黄培堂译).北京:科学出版社,2002,26-27.
[6]李宏,王新力.植物组织RNA提取的难点及对策.生物技术通报,1999,15(1):36-39.
[7]李三相.Na~+与多浆旱生植物霸王抗旱性研究:[硕士学位论文].甘肃:兰州大学,2006.
[8]刘欣玲.刺槐无性系转化体系的建立及液泡型Na~+/H~+逆向转运蛋白基因的克隆[硕士学位论文].山东:山东农业大学,2006.
[9]李园莉,江元清,赵武玲,等.谷子肌动蛋白基因克隆及序列分析.植物学通报,2002,19(3):310-316.
[10]吕慧颖,李银心,陈华,等.番杏Na~+/H~+逆向转运蛋白基因的克隆及特性分析.高技术通讯,2004,11:26-31.
[11]邱生平,周国安,陆驹,等.一个新的水稻液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达特征.中国水稻科学,2006,20(2):119-124.
[12]沈国新,楼程富,裘晓云,等.桑树中拟南芥同源基因mNHX1的克隆及其功能研究,蚕业科学,2005,31(4):398-403.
[13]孙超,陈维多,林忠平.一种改进的沙蒿总RNA的提取方法.生物技术通报,2004,5:43-44.
[14]田秀红,高建明,肖强,等.水花生Actin基因片段的克隆及序列分析.生物技术,2006,16(3):13-14.
[15]王关林,方宏筠.植物基因工程原理与技术.北京:科学出版社,1998,611-614.
[16]王劲.甘蓝型油菜耐盐基因的克隆及表达研究:[博士学位论文].上海:复旦大学.2004.
[17]伍国强,王强龙,包爱科,等.液泡膜Na~+/H~+逆向转运蛋白与植物耐盐性.中国农业科技导报,2008a,10(2):13-21. 方法.分子植物育种,2008b,6(1):197-200.
[19]伍国强,席杰军,包爱科,等.多浆旱生植物霸王Actin基因片断的克隆及序列分析.生物技术通报,2008c,2:101-105.
[20]肖前谷,叶妙水,钟克亚,等.北美海蓬子Na~+/H~+逆向运输蛋白基因的克隆及拟南芥转化分析.分子植物育种,2007,5(5):619-624.
[21]杨亮,付丽娅,刘仲齐,等.富含多糖番茄果实组织中总RNA的有效提取方法,南开大学学报(自然科学版).2005,38(5):36-39.
[22]杨丽霞,李玲.花生肌动蛋白基因克隆及序列分析.花生学报,2006,35(4):6-9.
[23]严一诺,孙淑斌,徐国华,等.菊芋Na~+/H~+逆向转运蛋白基因的克隆与表达分析.西北植物学报,2007,27(7):1291-1298.
[24]赵一之,朱宗元.亚洲中部荒漠区的植物特有属.云南植物研究.2003,25(3):113-121.
[25]张德强,赵淑堂,卢孟柱,等.杨树Na~+/H~+反向运输蛋白基因(PtNHX1、PtNHX6)的克隆和检测.林业科学,2006,42(11):29-36.
[26]张耿,王赞,关宁,等.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.遗传,2007,29(10):1263-1270.
[27]张今今,王跃进,王西平,等.葡萄总RNA提取方法的研究.果树学报,2003,20(3):178-181.
[28]张俊莲,张金文,陈正华,等.植物Na~+/H~+逆向转运蛋白与植物耐盐性的研究进展.草原与草坪,2005,4:3-8.
[29]张霞,曾幼玲,李金耀,等.胡杨Na~+/H~+反向运输载体(PeNHX2)基因的克隆与序列分析.生物技术,2006,16(3):9-13.
[30]周海飞,赵武玲,阎隆飞.玉兰肌动蛋白基因的克隆及序列分析.农业生物技术学报,2001,9(3):274-278.
[31]庄军平,苏菁,陈维信.一种从香蕉果实提取高质量RNA的方法.分子植物育种,2006,4(1):143-146.
[32]Aharon G S,Apse M P,Duan S,et al..Characterization of a family of vacuolar Na~+/H~+ antiporters in Arabidopsis thaliana.Plant and Soil,2003,00:245-256.
[33]Ainsworth C.Isolation of RNA from floral tissue of Rumex acetosa(Sorrel).Plant Mol Biol Rep,1994,12:198-203.
[34]Apse M P,Aharon G S,Snedden W A,et al..Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiporter in Arabidopsis.Science,1999,285:1256-1258.
[35]Apse M P,Sottosanto J B,Blumwald E.Vacuolar cation/H~+ exchange,ion homeostasis,and leaf development are altered in a T-DNA insertional mutant of AtNHX1,the Arabidopsis vacuolar Na~+/H~+ antiporter.Plant J,2003,36:229-239.
[36]Ballesteros E,Blumwald E,Donaire J P,et al..Na+/H+ antiport activity in tonoplast vesicles isolated from sunflowers roots induced by NaCl stress.Physiologia Plantarum,1997,99:328-334.
[37]Barkla B J,Zingarelli L,Blumwald E,et al..Tonoplast Na~+/H~+ antiport activity and its energization by the vacuolar H+-AYPase in the halophytic plant Mesembryanthemum crystallinum L.Plant Physiol,1995,109:549-556.
[38]Barkla B J,Zingarelli L,Blumwald E,et al..Tonoplast Na+/H+ antiport activity and its energization by the vacuolar H+-ATPase in the halophytic plant Mesembryanthemum crystallinurn.Plant Physiol,1995,109:549-546.
[39]Bartels D,Sunkar R.Drought and salt tolerance in plants.Crit Rev Plant Sci,2005,24:23-58.
[40]Blumwald E,Poole R J.Na~+/H~+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris.Plant Physiol,1985,78:163-167.
[41]Blumwald E.Tonoplast vesicles as a tool in the study of ion transport at the plant vacuole.Physiologia Plantarum,1987,69:731-734.
[42]Blumwald E,Poole R J.Salt Tolerance in suspension cultures of sugar beet:induction of Na~+/H~+ antiport activity at the tonoplast by growth in salt.Plant Physiol,1987,83:884-887.
[43]Boyer J S.Plant productivity and environment.Science,1982,218:443-448.
[44]Bray E A.Plant responses to water deficit.Trends Plant Sci,1997,2:48-54.
[45]Bressan R A,Hasegawa P M,Pardo J M.Plants use calcium to resolve salt stress.Trends Plant Sci,1998,3:411-412.
[46]Brett C L,Donowitz M,Rao R.Evolutionary origins of eukaryotic sodium/proton exchangers.A J P - Cell Physiol,2005,288:C223-C239.
[47]Brini F,Gaxiola R,Berkowitz G,Masmoudi K.Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump.Plant Physiol Biochem,2005,43:347-354.
[48]Brini F,Hanin M,Mezghani I,et al..Overexpression of wheat Na~+/H~+ antiporter TNHX1 and H~+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants.J Exp Bot,2007,58:301-308.
[49]Brownell P F.Sodium as an essential micronutrient element for plants and its possible role in metabolism.Advance in Botanical Research,1979,7:117-224.
[50]Busk P K,Pages M.Regulation of abscisic acid induced transcription.Plant Mol Biol,1998,37:425-435.
[51]Cheng N H,Pittman J K,Zhu J K,et al..The protein kinase SOS2 activates the Arabidopsis H~+/Ca~(2+)antiporter CAX1 to integrate calcium transport and salt tolerance.J Biol Chem,2004,279:2922-2926.
[52]Chen H,An R,Tang J H,et al..Over-expression of a vacuolar Na~+/H~+ antiporter gene improves salt tolerance in an upland rice.Mol Breeding,2007,19:215-225.
[53]Chen L H,Zhang B,Xu Z Q.Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na~+/H~+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum).Transgenic Res,2008,17:121-132.
[54]Chinnusamy V,Jagendorf A,Zhu J K.Understanding and improving salt tolerance in plants.Crop Sci,2005,45:437-448.
[55]Chomczynski P,Sacchi N.Single step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction.Anal Biochem,1987,162:156-159.
[56]Collings DA,Harper JD,Marc J,et al..Life in t he fast lane:Actin based motility of plant peroxisomes.Can J Bot,2002,80:430-441.
[57]Dupont F M.Salt-induced changes in ion transport:Regulation of primary pumps and secondary transporters.In:Cooke D T,Clarkson D T eds.Transport and Receptor Proteins of Plant Membranes.Plenum Press,New York,1992,91-100.
[58]Fang G,Hammar S,Grumet R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA,Biotechniques.1992,13:52-57.
[59]Firtel R.Multigene family encoding actin and tubulin.Cell,1981,24:6-7.
[60]Flowers T J,Troke P F,Yeo A R.The mechanisms of salt tolerance in halophytes.Ann Rev Plant Physiol,1977,28:89-121.
[61]Fukuda A,Chiba K,Maeda M,et al..Effect of salt and osmotic stresses on the expression of genes for the vacuolar H~+-pyrophosphatase,H~+-ATPase subunit A,and Na~+/H~+ antiporter from barley.J Exp Bot,2004,55:585-594.
[62]Fukuda A,Nakamura A,Tagiri A,et al..Function,intracellular localization and the importance in salt tolerance of a vacuolar Na~+/H~+ antiporter from rice.Plant Cell Physiol,2004,45:146-159.
[63]Fukuda A,Nakamura A,Tanaka Y.Molecular cloning and expression of the Na~+/H~+exchanger gene in Oryza sativa.Biochimica et Biophysica Acta,1999,1446:149-155.
[64]Fukuda A,Tanaka Y.Effects of ABA,auxin,and gibberellin on the expression of genes for vacuolar H~+-inorganic pyrophosphatase,H~+-ATPase subunit A,and Na~+/H~+antiporter in barley.Plant Physiol Biochem,2006,44:351-358.
[65]Fukuda A,Yazaki Y,Ishikawa T,et al..Na~+/H~+ antiporter in tonoplast vesicles from rice roots.Plant Cell Physiol.1998,39:196-201.
[66]Garbarino J,Dupont F M.NaCl induced a Na~+/H~+ antiport in tonoplast vesicles from barley roots.Plant Physiol,1988,86:231-236.
[67]Garbarino J,Dupont F M.Rapid induction of Na~+/H~+ exchange activity in barley root tonoplast.Plant Physiol.1989,89:1-4.
[68]Gaxiola R A,Li J S,Undurraga S,et al..Drought- and salt-tolerant plants result from overexpression of the AVP1 H~+-pump.Proc Natl Acad Sci USA,2001,98:1444-11449.
[69]Gaxiola R A,Rao R,Sherman A,et al..The Arabidopsis thaliana proton transporters:AtNhx1 and Avpl,can function in cation detoxification in yeast.Proc Natl Acad Sci USA,1999,96:1480-1485.
[70]Geourjon C,Deleage G.SOPMA:Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments.Cabios.1995,11:681-684.
[71]Glenn E,Brown J J,Blumwald E.Salt-tolerant mechanisms and crop potential of halophytes.Crit Rev Plant Sci,1999,8:227-255.
[72]Guo Y,Halfter U,Ishitani M,Zhu J K.Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance.Plant Cell,2001,13:1383-1399.
[73]Guren J,Mathieu Y,Kurkdjian A.Regulation of vacuolar pH in plant cells.Plant Physiol,1989,89:27-36.
[74]Hamada A,Shono M,Xia T,et al..Isolation and characterization of a Na~+/H~+antiporter gene from the halophyte Atriplex gmelini.Plant Mol Biol,2001,46:35-42.
[75]Hanana M,Cagnac O,Yamaguchi T,et al..A grape berry(Vitis vinifera L.)cation/proton antiporter is associated with berry ripening.Plant Cell Physiol,2007,48(6):804-811.
[76]Hasegawa P M,Bressan R A,Zhu J K,et al..Plant cellular molecular responses to high salinity.Annu Rev Plant Physiol Plant Mol Biol.2000,51:463-499.
[77]Hassidim M,Braun Y,Lemer H R,et al..Na~+/H~+ and K~+/H~+ antiport in root membrane vesicles isolated from the halophyte Atriplex and the glycophyte cotton.Plant Physiol,1990,94:1795-1801.
[78]He C X,Yan J Q,Shen G X,et al..Expression of an Arabidopsis vacuolar sodium/proton antiporter gene in cotton improves photosynthetic performance under salt conditions and increases fiber yield in the field.Plant Cell Physiol,2005,46: 1848-1854.
[79]Hernandez J A,Ferrer M A,Jimenez A,et al..Antioxidant systems and O2~+/H_2O_2production in the apoplast of pea leaves.Its relation with salt-induced necrotic lesions in minor veins.Plant Physiol.2001.127:817-831.
[80]Hofmann K,Stoffel W.Tmbase-A database of membrane spanning proteins segments.Biol Chem Hoppe-Seyler,1993,374:166.
[81]Jia W,Wang Y,Zhang S,Zhang J.Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots.J Exp Bot,2002,53:2201-2206.
[82]Kagami T,Suzuki M..Molecular and functional analysis of a vacuolar Na~+/H~+antiporter gene of Rosa hybrida.Genes Genet Syst,2005,80:121-128.
[83]Knight H.Calcium signalling during abiotic stress in plants.International Review of Cytology,2000,195:269-325.
[84]Knight H,Trewavas A J,Knight M R.Calcium signaling in Arabidopsis thaliana responding to drought and salinity.Plant J,1997,12:1067-1078.
[85]Li B,Wang B,Tang K,et al..A simple and convenient approach for isolating RNA from highly viscous plant tissue rich in polysaccharides.Colloids Surf B:Biointerfaces,2006,49(2):101-105.
[86]Li J,Jiang G,Huang P,et al..Overexpression of the Na~+/H~+ antiporter gene from Suaeda salsa confers cold and salt tolerance to transgenic Arabidopsis thaliana.Plant Cell Tiss Organ Cult,2007,90:41-48.
[87]Li J,Yang H,Peer W A,et al..Arabidopsis H+-PPase A VP1 regulates auxin-mediated organ development.Science,2005,310:121-125.
[88]Liu J,Zhu J K.A calcium sensor homolog required for plant salt tolerance.Science,1998,280:1943-1945.
[89]Li Y W,Wong F L,Tsai S N,et al..Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow(BY)-2 cells.Plant Cell Environ,2006,9(29):1122-1137.
[90]Lu S Y,Jing Y X,Shen S H,et al..Antiporter gene from Hordum brevisubulatum (Trin.)link and its overexpression in transgenic tobaccos.Journal of Integrative Plant Biology,2005,47:343-349.
[91]Mange A,Meagher R B.The molecular evolution of actin.Gentics,1986,114:315-332.
[92]Martinoia E,Maeshima M,Neuhaus H E.Vacuolar transporters and their essential role in plant metabolism.J Exp Bot,2007,58(1):83-102.
[93]Matoh T,Watanabe J,Takahashi E.Sodium,potassium,chloride,and betaine concentrations in isolated vacuoles from salt-grown Atriplex gmelini leaves.Plant Physiol,1987,84:173-177.
[94]Ma X L,Zhang Q,Shi H Z,et al..Molecular cloning and different expression of a vacuolar Na~+/H~+ antiporter gene in Suaeda salsa under salt stress.Biologia Plantarum,2004,48:219-225.
[95]Mennen H,Jacoby B,Marschner H.Is sodium proton antiport ubiquitous in plant cells ? J Plant Physiol,1990,137:180-183.
[96]Moffat A S.Plant genetics:Finding new ways to protect drought-stricken plants.Science,2002,296:1226-1229.
[97]Nass R,Cunningham KW,Rao R.Intracellular sequestration of sodium by a novel Na~+/H~+ exchanger in yeast is enhanced by mutations in the plasma membrane H~+-ATPase:insights into mechanisms of sodium tolerance.J Biol Chem,1997,272:26145-26152.
[98]Niu X,Bressan R A,Hasegawa P M,et al..Ion homeostasis in NaCI stress environment.Plant Physiol,1995,109:735-742.
[99]Ohnishi M,Fukada-Tanaka S,Hoshino A,et al..Characterization of a novel Na~+/H~+antiporter gene InNHX2 and comparison of InNHX2 with InNHX1,which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory.Plant Cell Physiol,2005,46:259-267.
[100]Orlowski J,Grinstein S.Na~+/H~+ exchanger of mammalian cells.J Biol Chem,1997,272:22373-22376.
[101]Orlowski J.Heterologous expressiln and functional properties of amiloride high affinity(NHE-1)and low affinity(NHE-3)isoforms of the rat Na~+/H~+ exchanger.J Biol Chem,1993,268:16369-16377.
[102]Page R D.TREEVIEW:An application to display phylogenetic trees on personal computers.Comput Appl Bio,1996,12:357-358.
[103]Pardo J M,Cubero B,Leidi E O,et al..Alkali cation exchangers:roles in cellular homostasis and stress tolerance.J Exp Bot,2006,57:1181-1199.
[104]Popova O V,Golldack D.In the halotolerant Lobularia maritime(Brassicaceae)salt adaptation correlates with activation of the vacuolar H~+-ATPase and the vacuolar Na~+/H~+ antiporter.J Plant Physiol,2007,164:1278-1288.
[105]Porat R,Pavoncello D,Ben-Hayyim G,et al..A heat treatment induced the expression of a Na~+/H~+ antiport gene(cNHX1)in citrus fruit.Plant Sci,2002,162:957-963.
[106]Putney L K and Barber D L.Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition.J Biol Chem,2003,278:44645-44649.
[107]Qiao W H,Zhao X Y,Li W,et al..Overexpression of AeNHX1,a root-specific vacuolar Na~+/H~+ antiporter from Agropyron elongatum,confers salt tolerance to Arabidopsis and Festuca plants.Plant Cell Rep,2007,26(9):1663-1672.
[108]Qiu N,Chen M,Guo J,et al..Coordinate up-regulation of V-H~+-ATPase and vacuolar Na~+/H~+ antiporter as a response to NaCl treatment in a C3 halophyte Suaeda salsa.Plant Sci,2007,173(5):487-494.
[109]Qiu Q S,Guo Y,Quintero F J,et al..Regulation of vacuolar Na~+/H~+ exchange in Arabidopsis thaliana by the salt-overly-sensitive(SOS)pathway.J Biol Chem,2004,279:207-215.
[110]Quesada V,Ponce M R,Micol J L.Genetic analysis of salt tolerant mutants in Arabidopsis thaliana.Genetics.2000,154:421-436.
[111]Rajagopal D,Agarwal P,Tyagi W,et al..Pennisetum glaucum Na~+/H~+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea.Mol Breeding,2007,19:137-151.
[112]Sahi C,Singh A,Kumar K,et al..Salt stress response in rice:genetics,molecular biology,and comparative genomics.Funct Integr Genomics,2006,6:263-284.
[113]Sanders D,Brownlee C,Harper J F.Communicating with calcium.Plant Cell,1999,11:691-706.
[114]Sardet C,Franchi A,Pouyssegur J.Molecular cloning,primary structure and expression of the human growth factor-actiatable Na+/H+ antiporter.Cell,1989,56:271-280.
[115]Sato Y,Sakaguchi M.Topogenic properties of transmembrane segments of Arabidopsis thaliana NHX1 reveal a common topology model of the Na~+/H~+exchanger family.J Biochem,2005,138:425-431.
[116]Sauter A,Dietz K J,Hartung W.A possible stress physiological role of abscisic acid conjugates in root-to-shoot signalling.Plant Cell Environ,2002,25:223-228.
[117]Schroeder J I,Kwak J M,Allen G J.Guard cell abscisic acid signalling and engineering drought hardiness in plants.Nature,2001,410:327-330.
[118]Sheterline P,Sharrow JC.Protein profile:Actin,New York:Academic Press,1994.
[119]Shi H,Lee B H,Wu S J,et al..Overexpression of a plasma membrane Na~+/H~+antiporter gene improves salt tolerance in Arabidopsis thaliana.Nat Biotechnol,2003,21:81-85.
[120]Shi H,Zhu J K.Regulation of expression of the vacuolar Na~+/H~+ antiporter gene AtNHX1 by salt stress and abscisic acid.Plant Mol Biol,2002,50:543-550.
[121]Shinozaki K,Yamaguchi-Shinozaki K.Gene expression and signal transduction in water-stress response.Plant Physiol,1997,115:327-334.
[122]Smirnoff N.The role of active oxygen in the response of plants to water deficit and desiccation.New Phytol,1993,125:27-58.
[123]Staal M,Maathuis F J,Elzenga J T,et al..Na~+/H~+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the saltsensitive Plantago media.Physiologia Plantarum 1991,82:179-184.
[124]Sun Y X,Wang D,Bai Y L,et al..Studies on the overexpression of the soybean GmNHX1 in Lotus corniculatus:The reduced Na~+ level is the basis of the increased salt tolerance.Chinese Science Bulletin,2006,51:1306-1315.
[125]Yesniere C,Vayda M E.Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates,Plant Mol Biol Rep,1991,9(3):242-251.
[126]Thompson J D,Gibson T J,Plewniak F,et al..The Clustal X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucl.Acids Res.1997,25:4876-4882.
[127]Vasekina A V,Yershov P V,Reshetova O S,et al..Vacuolar Na~+/H~+ antiporter from Barley:identification and response to salt stress.Biochemistry(Moscow),2005,70,(1):100-107.
[128]Venema V,Belver A,Marin-Manzano M C,et al..A novel intracellular K~+/H~+antiporter related to Na~+/H~+ antiporters is important for K~+ ion homeostasis in plants.J Biol Chem,2003,278:22453-22459.
[129]Verma D,Singla-Pareek S L,Rajagopal D,et al..Functional validation of a novel isoform of Na~+/H~+ antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice.JBiosci,2007,32(3):1-7.
[130]Verslues P E,Zhu J K.Before and beyond ABA:upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress.Biochem Soc Trans,2005,33:375-379.
[131]Vidali,L,Helper,P K.Actin and pollen tube growth.Protoplasma,2001,215:64-76.
[132]Wang J,Zuo K,Wu W,et al..Expression of a novel antiporter gene from Brassica napus resulted in enhanced salt tolerance in transgenic tobacco plants.Biologia Plantarum,2004,48:509-515.
[133]Wang J,Zuo K,Wu W,et al..Molecular cloning and characterization of a new Na~+/H~+ antiporter gene from Brassica napus.DNA Seq,2003,14:351-358.
[134]Wang S M,Wan C G,Wang Y R,et al.The characteristics of Na~+,K~+ and free proline distribution in several drought-resistant plants of the Alxa Desert China.J Arid Environ,2004,56(3):525-539.
[135]Woodhead M,Taylor M A,Davies H V,et al..Isolation of RNA from blackcurrant (Ribes nigrum L.)fruit.Mol Biotechnol,1997,7(1):1-4.
[136]Wu C A,Yang G D,Meng Q W,et al..The cotton GhNHX1 gene encoding a novel putative tonoplast Na~+/H~+ antiporter plays an important role in salt stress.Plant Cell Physiol,2004,45:600-607.
[137]Wu S J,Ding L,Zhu J K.Sosl,a genetic locus essential for salt tolerance and potassium acquisition.Plant Cell,1996,8:617-627.
[138]Xia T,Apse M P,Aharon G S,et al..Identification and characterization of a NaCl-inducible vacuolar Na~+/H~+ antiporter in Beta vulgaris.Physiol Plant,2002,116:206-212.
[139]Xiong L,Zhu J K.Regulation of abscisic acid biosynthesis.Plant Physiol,2003,133:29-36.
[140]Xue Z Y,Zhi D Y,Xue G P,et al..Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.)expressing a vacuolar Na~+/H~+ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na~+.Plant Sci,2004,167(4):849-859.
[141]Yamaguchi T,Aharon G S,Sottosanto J B,et al..Vacuolar Na~+/H~+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca~(2+)-and pH-dependent manner.Proc Natl Acad Sci USA,2005,102:16107-16112.
[142]Yamaguchi T,Apse M P,Shi H,et al..Topological analysis of a plant vacuolar Na~+/H~+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity.Proc Natl Acad Sci USA,2003,100:12510-12515.
[143]Yamaguchi T,Blumwald E.Developing salt-tolerant crop plants:challenges and opportunities.Trends Plant Sci,2005,10:615-620.
[144]Yamaguchi T,Fukada-Tanaka S,Inagaki Y,et al..Genes encoding the vacuolar Na~+/H~+ exchanger and flower coloration.Plant Cell Physiol,2001,42:451-61.
[145]Yin XY,Yang A F,Zhang KW,et al..Production and analysis of transgenic maize with improved salt tolerance by the introduction of AtNHX1 gene.Acta Botanica Sinica,2004,46:854-861.
[146]Yokoi S,Quintero F J,Cubero B,et al..Differential expression and function of Arabidopsis thaliana NHX Na~+/H~+ antiporters in the salt stress response.Plant J,2002,30:529-539.
[147]Yoshida K,Kawachi M,Mori M,et al..The involvement of tonoplast proton pumps and Na~+(K~+)/H~+ exchangers in the change of petal color during flower opening of morning glory,Ipomoea tricolor cv.heavenly blue.Plant Cell Physiol,2005,46:407-415.
[148]Yu J N,Huang J,Wang Z N,et al..A vacuolar Na~+/H~+ antiporter from wheat plays an important role in stress tolerance.J Biosci,2007,32:1153-1161.
[149]Zhang G H,Su Q,An L J,et al..Characterization and expression of a vacuolar Na~+/H~+ antiporter from the monocot halophyte Aeluropus littoralis.Plant Physiol Biochem,2008,46:117-126.
[150]Zhang H X,Blumwald E.Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit.Nature Biotechnology,2001,19:765-768.
[151]Zhang H X,Joanna N H,John P M,et al..Engineering salt tolerant Brassica plant:characterization of yield and seed oil quality in transgenic plants with increase vacuolar sodium accumulation.Proc Natl Acad Sci USA,2001,98:12832-12836.
[152]Zhao F Y,Zhang X J,Li P H,et al..Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1.Mol Breeding,2006,17(4):341-353.
[153]Zhen R G,Kim E J,Rea P A.Acidic residues necessary for pyrophosphate energized pumping and inhibition of the vacuolar H~+-pyrophyosphatase by N,N-dicyclohex -ylcarbodiimide.J Biol Chem,1997,29:22340-22348.
[154]Zhou S,Chen X,Zhang X,et al..Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene BADH and a vacuolar Na~+/H~+antiporter gene SeNHX1.Biotechnol Lett,2008,30:369-376.
[155]Zhu J K.Plant salt tolerance.Trends Plant Sci,2001,6:66-71.
[156]Zorb C,Noll A,Karl S,et al..Molecular characterization of Na~+/H~+ antiporters (ZmNHX)of maize(Zea mays L.)and their expression under salt stress.J Plant Physiol,2005,162:55-66.