【作者】 全先庆；

【导师】 李法曾； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2003， 硕士

【摘要】 土壤盐渍化是影响农业生产和生态环境的一个非常重要的非生物胁迫因素，是现代生物科学迫切需要解决的问题。研究真盐生植物的耐盐机理、利用遗传王程育种提高植物的耐盐性已成为当前的研究热点之一。 盐芥（Thellungiella salsuginea）是一种真盐生植物，在进化中形成了特殊的耐盐机制，能在含100mMNaCl的土壤中正常完成生活史。许多研究表明，植物的耐盐性常与其有效的抗氧化系统呈正相关，植物的抗氧化系统可分为酶促反应系统和非酶促反应系统。盐生植物和甜土植物在活性氧的解毒机制上可能存在显著的差异，因此从盐芥中克隆活性氧清除蛋白的基因并分析其功能，有助于理解植物中盐胁迫的耐受机制。金属硫蛋白是一种盐诱导表达蛋白，是很好的OH^-自由基清除蛋白，其清除能力主要来源于蛋白中处于还原态的巯基。本实验第一部分从用200mmo]．L^-1NaCl处理的盐芥地上部分构建的λZap-cDNA文库中克隆了编码金属硫蛋白的基因TsMT（GeneBank号为AF499726.1），并分别对其序列特征、基因组结构和在盐胁迫下的表达特性进行了分析，结果表明TsMT编码盐芥金属硫蛋白，Southern结果显示TsMT在盐芥基因组中只有一个拷贝。用200mmol．L^-1NaCl处理盐芥，随时间延长，TsMT基因在盐芥中的表达量增加，在处理48h时表达量最大。由实验结果推测，TsMT基因可能在保护盐芥免受氧化损伤过程中起到一定作用。 盐胁迫条件下，植物细胞渗透均衡和离子正常均衡状态被破坏，特别是胞质中过多的Na⁺渗透调节对植物细胞的新陈代谢产生毒害，影响到植物细胞的调节机制。植物细胞适应胁迫环境形成了一系列适应机制，其中许多盐生植物可通过Na⁺外排、Na⁺区隔化或调整K⁺／Na⁺比率保持细胞内较低的Na⁺浓度，消除Na⁺的毒害，其中Na⁺／H⁺反向转运蛋白起着重要作用。大豆是公认的难转化植物，利用大豆子叶节再生系统及农杆菌介导进行转化是目前大豆转基因的首选方法。本实验第二部分通过农杆菌介导法将含NPT-Ⅱ和盐地碱蓬Na⁺／H⁺反向转运蛋白基因（SsNHX1）的表达载体pROK2导入大豆子叶节中，经过含Km的筛选培养基连续筛选，获得了SsNHX1转基因植株，筛选剂卡那霉素的适宜浓度是50mg.L^-1。农杆菌侵染时间和共培养时间对大豆的转化有明显的影响，侵染时间以15min为宜，共培养时间以3d为宜。在农杆菌侵染和共培养阶段的乙酰丁香酮（acetosyringane，AS）浓度和pH值对大豆转化频率有明显的影响，AS的浓度为150μmol.L^-1左右、pH值以5.2为宜。更多还原

【Abstract】 The soil salination is a significant abiotic stress for agricultural production and ecological environment and the research on salt tolerance represents an important part for basic plant biology. Studying the salt-tolerance mechanism of euhalophytes and improving plant ability of salt tolerance by genetic engineering breeding has become one of the central issues.Thellungiella salsuginea is an euhalophyte that may have evolved the unique salt-tolerance mechanism and can complete its life history normally in soil containing 100mmol . L-1.NaCl.The results of many studies suggest that plant salt tolerance is usually correlated with a more efficient antioxidant system. Plant has evolved protection mechanism which contains enzymatic reaction system and no-enzymatic reaction system. There may have distinct difference on the scavenging of reactive oxygen species (ROS) between euhalophyte and glycophyte. So cloning those genes that encode proteins engaged in scavenging ROS from Thellungiella salsuginea may attribute to get insight into the mechanism of plant salt tolerance. Metallothionein(MT) is a salt-induced protein and a good scavenger of hydroxyl radical. Its ability of scavenging hydroxyl radical derives from reduced - SH group in MT. In the first part of this experiment, we isolated the TsMT gene ( GeneBank accession number :AF499726. 1 )from a A Zap-cDNA library constructed from a 200mmolL-1 NaCl-treated library of Thellungiella salsuginea and analyzed its sequence characterization, genomic organization and the differential expression in response to salt stress. TsMT encodes metallothionein and there is only one copy in Thellungiella salsuginea genome according to its Southern Blotting. The expression of TsMT was slightly induced by saltstress and the activity of MT increased gradually in response to salt stress .The result indicates that TsMT may play an important role in protecting Thellungiella salsuginea from the oxidation damage.The maladies caused by salt stress arise from the disruption of cellular aqueous and ionic equilibrium. Na+ accumulates excessively in the cytoplasm can be toxic to the metabolism and affects the regulation mechanism of the plant cell. To cope with salt stress, plants have developed a variety of adaptation mechanisms. The mechanisms of ion homeostasis of some halophytes include extrusing Na+, sequestering Na+ into the vacuolar and regulating the ratio of KV Na+, and Na+/H+antiporter plays an important role in these processes. Soybean is a generally acknowledged recalcitrant crop for transformation. The regeneration system of soybean cytoledon node and Agrobacteriunr mediated transformation method is the first selection at present. In the second part of this experiment, the expression vector pROK2 containing NPT II and SsNHX1 (Na+/H+ antiporter) gene from suaeda salsa was introduced into soybean cytoledon nodes by gene transformation mediated by Agrobacterium tumefaciens , and kanamycin resistant transgenic plants were obtained by screening in selective condition. The suitable concentration of the selective agent kanamycin is 50mg. L-1. The duration of infection and co-culture with Agrobacterium influences soybean transformation evidently. The suitable infection duration is about 15 minutes, and the appropriate co-culture duration is 3 days. Both the concentration of acetosyringane and pH value at stages of infection and co-culture influence the transformation frequency of soybean. The suitable pH value is 5. 2 and the optimum concentration of acetosyringane is 150 mol.L-1.更多还原