【作者】 伍国强；

【导师】 王锁民；

【作者基本信息】 兰州大学 ， 草业科学， 2008， 硕士

【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(⁷⁸LFFYLLPPI⁸⁷)。此外,ZxNHX含有2个糖基化位点,分别为Asn⁴⁴、Asn²⁸⁷,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^-1NaCl),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。更多还原

【Abstract】 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 (⁷⁸LFFYLLPPI⁸⁷).In addition,two potential N-glycosylation sites were found within the ZxNHX protein,at Asn⁴⁴and Asn²⁸⁷,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^-1NaCl,and highest at 3 or 12 h.These results suggested the expression of ZxNHX was induced and regulated by salt stress.更多还原