【作者】 乔卫华；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 植物学， 2006， 博士

【摘要】 盐胁迫是全世界范围内影响作物产量的最严重的非生物胁迫之一。利用基因工程提高植物的耐盐性,培育出能适合在盐渍化土地上生长的植物品种是有效利用盐渍化土地的主要方法。植物液泡膜Na+/H+反向转运蛋白能利用液泡膜上的H+-ATPase和H+-PPiase所产生的质子动力势将Na+区隔化在液泡内。这不仅能降低细胞质内的Na+浓度,还可以有效利用储存在液泡内的Na+作为渗透剂。已有研究表明过量表达液泡膜Na+/H+反向转运蛋白基因可以提高拟南芥、番茄等植物的耐盐性,说明液泡膜Na+/H+反向转运蛋白基因在植物的耐盐性中起着重要的作用。本研究首次从盐生单子叶植物长穗偃麦草中分离出液泡膜Na+/H+反向转运蛋白基因,并对该基因的表达和功能进行了全面地分析。首次将长穗偃麦草液泡膜Na+/H+反向转运蛋白基因导入禾本科草坪草高羊茅,转基因高羊茅耐盐能力显著提高。主要结果和结论如下:分别从长穗偃麦草的根和叶中克隆出两个Na+/H+反向转运蛋白基因,命名为AeNHX1(Acession Numeber:AF507044)和AeNHX2 (Acession Numeber:AY371178)。氨基酸序列分析和进化树分析表明它们与拟南芥、水稻、小麦等植物的液泡膜Na+/H+反向转运蛋白亲缘关系较近,而与拟南芥质膜Na+/H+反向转运蛋白亲缘关系较远。将AeNHX1-GFP融合蛋白在洋葱表皮细胞内瞬时表达。在洋葱表皮细胞的液泡膜上观察到绿色荧光,表明AeNHX1定位于液泡膜。Southern杂交结果发现AeNHX1在长穗偃麦草基因组中可能存在一个小的基因家族。Northern杂交结果表明AeNHX1基因特异性地在长穗偃麦草根中表达,并且它的表达不受盐胁迫的诱导,是一个组成型表达的基因。将AeNHX1插入酵母表达载体pYES2,转化酵母突变体ATX3,阳性转化菌落比对照菌落耐盐能力提高。AeNHX1还能拮抗Li+与潮霉素对酵母突变体的毒害作用,部分恢复酵母突变体对碱性阳离子敏感缺陷型。表明AeNHX1与酵母液泡膜Na+/H+反向转运蛋白ScNHX1,拟南芥液泡膜Na+/H+反向转运蛋白AtNHX1、AtNHX2具有相似的功更多还原

【Abstract】 Salt tolerance is one of the most important factors that affect productivity of crops in the world. To cope with salt stress, plants have developed a variety of adaptation mechanisms. One of them is the compartmentation of Na+ into the vacuoles by the vacuolar Na+/H+ antiporters. It is reported that overexpressing the vacuolar Na+/H+ antiporter gene can improve the salt tolerance of Arabidopsis and tomato. In this study, we islated a vacuolar Na+/H+ antiporter gene AeNHX1 from Agropyron elongatum, the functional analysis demonstrated that this gene plays an important role in plant salt tolerance. Overexpressing AeNHX1 can remarkably promote the salt tolerance of transgenic Arabidopsis and Festuca arundinacea plants. Our study suggests that AeNHX1 can be used to improve plant salt tolerance by genetic engineering. The main results of this study summarized below: According to the sequences of the vacuolar antiporter proteins in GenBank, we designed degenerate primers to isolate vacuolar antiporter gene. Two genes, which named AeNHX1 (Acession Numeber:AF507044)and AeNHX2 (Acession Numeber:AY371178) were isolated from root and leaf of Agropyron plants. Sequence analysis and phylogenetic tree shows that they belong to a vacuolar antiporter gene family.AeNHX1-GFP fusion protein was constructed and transiently expressed in onion epidermal cells. The green fluorescence was detected on the tonoplast, demonstrating that AeNHX1 subcellular localization on vacuolar membrane. Southern hybridization shows that there are at least seven copies of AeNHX1 in Agropyron genome. Northern hybridization shows that AeNHX1 is constitutively expressed in root tissues, and its expression is not induced by salt stress.Full-lengh cDNA of AeNHX1 was introduced into the yeast Na+/H+ antiporter mutant更多还原