POWRE: Characterization of a Novel ATPase

所有植物细胞都必须主动地从细胞质中输出钠。钠在细胞质中的积累导致了许多酶系统的抑制，这是造成盐分损害的主要原因，无论是自然的还是农田灌溉造成的。因此，了解Na+转运的机制不仅对了解植物的一般功能很重要，而且有助于我们开发出更耐盐碱化的作物。该项目将使用两种绿藻，它们对盐水环境的耐受能力不同。通过跨质膜出口从细胞质中去除Na+对于实现耐盐性很重要，有证据表明atp酶可能参与了这种运输，包括生物物理证据表明Na+/H+反端口不足以维持测量的Na+梯度，免疫证据表明盐培养藻类中存在不寻常的p型atp酶，分子证据表明耐盐藻类中存在不寻常的p型atp酶。本研究的目的是从耐盐植物Chara longifolia中分离出一种不寻常的atp酶并对其进行全长克隆测序。利用northern blot分析测定该基因在适应盐水和淡水介质以及适应盐胁迫的长叶参中的表达。为了确定对盐敏感的珊瑚藻是否有类似的基因，如果有，在什么条件下表达。长期目标包括通过在缺乏Na+或H+输出能力的酵母突变体中表达ATPase基因，或通过在Chara中微量注射mRNA到节间来过表达ATPase基因，以确定其转运功能。最终，基于本研究的信息可用于通过基因操作改变Chara和其他植物的耐盐性，以增加细胞质中的钠输出。这项研究将在POWRE资助计划下进行，因为它将帮助作者在植物盐度研究领域保持竞争力。过去，生物物理技术已经成功地应用于研究这一领域的重要问题，但为了保持竞争力，必须发展该项目的分子方面。

All plant cells must actively export sodium from the cytoplasm. Accumulation of sodium in the cytoplasm results in inhibition of a number of enzyme systems, and is a major cause of damage due to salinity, whether natural or due to irrigation of agricultural fields. A knowledge of the mechanisms of Na+ transport is therefore not only important in general understanding of the function of plants, but also enhances our ability to develop crop plants which are more resistant to salinization. This project will use two species of Charophyte algae that differ in their ability to tolerate saline conditions. Re-moval of Na+ from the cytoplasm by export across the plasma mem-brane is important in achieving salt tolerance, and there is evidence that an ATPase may be involved in this transport, including biophysical evidence that a Na+/H+ antiport is not sufficient to maintain the Na+ gradients measured, immunological evidence of an unusual P-type ATPase in salt-cultured algae, and molecular evidence for an unusual P-type ATPase in the salt-tolerant algae. The objectives of the proposed research are To isolate and sequence a full-length clone for an unusual ATPase from the salt-tolerant species, Chara longifolia. To use northern blot analysis to determine expression of this gene in C. longifolia adapted to saline and freshwater media, and undergoing adaptation to salt stress. To determine whether the salt-sensitive species, C. corallina, has a similar gene and, if so, under what conditions it is expressed. Long-term goals include determining the transport function of the ATPase gene either by expres-sion in yeast mutants lacking Na+ or H+ export capability or by overexpression in Chara by micro-injection of mRNA into the internode. Ultimately, information based on this research could be used to alter the salt tolerance of Chara and other plants by genetic manipulation to enhance sodium export from the cytoplasm. This research will be performed under the POWRE grant program because it will aid the author in remaining competitive in the field of salinity studies in plants. Bio-physical techniques have been successfully employed in the past to investigate important ques-tions in this field, but molecular aspects of the project must be developed in order to remain competitive.