【作者】 王蕾；

【导师】 邱全胜；

【作者基本信息】 兰州大学 ， 植物学， 2012， 硕士

【摘要】 钾离子是植物生长发育必需的营养元素。钾离子吸收及其在植物组织器官中的分配以及钾离子在植物细胞内各个细胞器之间的运输都是由钾离子转运蛋白完成的。因此,研究钾离子转运蛋白对于阐明植物钾营养代谢机理,揭示植物生长发育规律以及提高植物钾离子利用效率等都有着极其重要的理论和实践意义。拟南芥AtKEA基因序列与细菌K+/H+反向转运体的同源性很高,因此,拟南芥AtKEA可能也编码K+/H+反向转运体。然而,目前没有任何有关AtKEA生物功能的实验报道。本文研究了AtKEAl和AtKEA2基因在K+转运,盐胁迫和渗透胁迫中的生物功能。研究结果总结如下：1.鉴定T-DNA插入突变体并进行生长表型分析。结果表明,Atkeal、Atkea2单突变体与野生型在NaCl胁迫,KC1亏缺,甘露醇处理下无明显表型差异,有可能其它基因互补了该基因的作用。2.制备AtkealAtkea2双突变体。AtkealAtkea2双突变体植株相比野生型和单突变体植株明显矮小,并且叶片发黄,生长发育缓慢。3.在0-10mM不同浓度的KC1处理下,AtkealAtkea2双突变体与单突变体和野生型植株无明显表型差异；当KCl浓度增加到20mM时,双突变体的根长明显短于单突变体和野生型植株,表明AtKEAl和AtKEA2参与细胞钾离子转运,AtKEA1和AtKEA2编码钾离子转运蛋白(可能属于低亲和钾离子转运蛋白)。4. Atkeal Atkea2双突变体生长受介质pH的影响,在pH4.5时双突变体的根长明显短于单突变体和野生型植株,而在pH8.4时则无明显差异。然而,当进行低钾(1mM KCl), pH8.4处理时,双突变体的根长短于单突变体和野生型植株；进行高钾(50-100mM KC1), pH4.5处理时,双突变体与单突变体和野生型植株则无明显差异,表明AtKEA1和AtKEA2可能编码K+/H+反向转运蛋白。5.用不同浓度NaCl处理时,随着NaCl浓度增加,双突变体与单突变体和野生型植株之间的生长差异逐渐缩小；当NaCl浓度为200mM以上时,双突变体与单突变体和野生型植株则无明显差异,表明双突变体对高浓度的Na+不敏感,暗示AtKEA1和AtKEA2可能不参与植物耐盐胁迫作用。同样,双突变体对高浓度甘露醇处理不敏感,表明AtKEAl和AtKEA2可能不参与植物渗透胁迫作用。6.为了分析AtKEA1和AtKEA2在液胞融合中的作用,采用酵母双杂交方法检测了AtKEA1和AtKEA2与液胞融合有关的SNAREs组分之间的相互作用。结果显示,AtKEA1和AtKEA2与Vamp727、Syp2、Vps45都有相互作用。由于Vamp727、Syp2、Vps45都是液胞前体(PVC)上调节液胞融合的SNAREs蛋白,推测AtKEA1和AtKEA2可能参与液胞融合的调节过程。更多还原

【Abstract】 K+is an essential nutrient for plant growth. K+absorption and distribution in plants and its transport between organelles within cells are mediated by K+transport proteins. Therefore, investigating the functions and regulatory mechanisms of the potassium transporters is critical for the understanding of the molecular mechanisms controlling cellular K+homeostasis, plant growth and development, and improving K+utilization efficiency in plants.Based on sequence similarity, the Arabidopsis AtKEA gene family is predicted to encode K+/H+antiporters. However, there has no experimental report about their biological functions in plants. We studied the functions of AtKEA1and AtKEA2in K+transport, and salt and osmotic stress. The results are described below.1. The T-DNA mutants were screened and the growth phenotype was analyzed. The results showed that the single mutants Atkeal and Atkea2displayed no obvious phenotype compared with the wild type plants under treatments of NaCl stress, low KC1and mannitol stress. This might due to the complement of their functions by other genes.2. The double mutant AtkealAtkea2was generated. The AtkealAtkea2double mutant was significant smaller than the wild type plants and single mutants. The double mutants had yellow leaves and grew slower relative to the wild type plants.3. When growing on media containing KC1ranging from0-10mM, The AtkealAtkea2double mutants had no obvious phenotype compared with the single mutants and wild type plants. However, when KC1was increased to20mM, the roots of the double mutants are shorter than the single mutants and wild type plants, showing that AtKEA1and AtKEA2mediate cellular K+transport and might encode K+transport proteins (might be a low affinity K+transporter).4. The growth of Atkeal Atkea2double mutants was affected by the pH of media. When pH was4.5, the roots of the double mutants were significantly shorter than the single mutants and wild type plants; but no difference was observed when pH was8.4. However, when growing on media with pH8.4and low K+(1mM KC1), the roots of the double mutants were shorter than the single mutants and wild type plants; on the other hand, when growing on media with pH4.5and high K+(50-100mM KC1), no obvious difference was observed, indicating that AtKEAl and AtKEA2might encode K+/H+antiporters.5. Increasing NaCl concentration gradually reduced the differences between the double mutants and the single mutants and wild type plants, no difference was observed when NaCl was above200mM, indicating that the double mutants are not sensitive to high salt stress. This result suggests that AtKEAl and AtKEA2might not function in salt tolerance in plants. Similarly, the double mutants were not sensitive to high mannitol treatment, indicating that AtKEAl and AtKEA2might not function in osmotic stress as well.6. The role of AtKEAl and AtKEA2in vacuole fusion was analyzed. The interactions between AtKEAl/AtKEA2and SNAREs involved in vacuole fusion were determined by a yeast two-hybrid analysis. The result showed that AtKEAl/AtKEA2interacted with Vamp727, Syp21and Vps45. Vamp727, Syp21and Vps45are SNARE proteins mediating vacuole fusion, therefore, it is reasonable to believe that AtKEAl and AtKEA2might involve in vacuolar fusion as well.更多还原