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。细胞质中钠的积累导致许多酶系统的抑制，是盐分造成的损害的主要原因，无论是自然的还是由于农田灌溉造成的。因此，对Na+转运机制的了解不仅对全面了解植物的功能很重要，而且还可以提高我们培育更耐盐碱的作物的能力。该项目将使用两种轮藻，它们对盐碱条件的耐受能力不同。Na+通过质膜输出从细胞质中重新转运是实现耐盐性的重要途径，有证据表明这种转运可能与ATPase有关，包括生物物理证据表明Na+/H+逆向转运不足以维持所测量的Na+梯度，免疫学证据表明盐生藻类中存在异常的P型ATPase，以及耐盐藻类中异常P型ATPase的分子证据。这项研究的目的是从耐盐物种长叶轮藻中分离并测序一个不寻常的ATPase的全长克隆。利用Northern印迹分析确定该基因在适应盐碱和淡水介质以及适应盐胁迫的长叶沙枣中的表达。以确定盐敏感物种珊瑚藻是否有类似的基因，如果有，它在什么条件下表达。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，或者通过向节间微量注射mRNA在轮状体内过表达来确定ATPase基因的运输功能。最终，基于这项研究的信息可以用来通过基因操作改变轮藻和其他植物的耐盐性，以增强细胞质中的钠输出。这项研究将在POWRE赠款计划下进行，因为它将帮助作者在植物盐度研究领域保持竞争力。过去，生物物理技术已经被成功地用于研究这一领域的重要问题，但为了保持竞争力，必须开发该项目的分子方面。

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.