Arabidopsis 2010: RUI: Genetic and Physiological Characterization of Arabidopsis Plasma Membrane H+-ATPase Mutants

对拟南芥全序列的初步注释表明，在已发现的11,601种不同的基因类型中，只有35%在基因组中由一个基因代表，而37%由具有五个或更多不同成员的基因家族代表。对于大多数较大的基因家族来说，编码蛋白质的分子功能是已知的。然而，单个基因家族成员在特定的生理或发育途径中的作用往往是不确定的。本项目的重点是建立描述AHA(拟南芥质膜H+-ATPase)基因家族中单个基因家族成员的植物内功能的方案。AHA基因编码将ATP水解酶与跨植物细胞质膜的质子运输偶联的蛋白质。然后，ATPase产生的质子动力通过载体和通道驱动溶质的运输。在拟南芥中，质子泵包括一个至少有12个基因异构体的基因家族。泵在不同的组织中表达，为各种基本的植物过程提供能量，如细胞动态平衡、矿物质和代谢物运输、细胞生长和植物形态建成，以及各种对环境的反应，包括气孔控制、热带反应和防御相关机制。作为确定AHA基因功能的第一步，我们已经筛选了8万多个拟南芥T-DNA插入系，并鉴定了29个突变体。该突变体包括12个AHA基因家族成员中的10个，具有多个亚型的多个等位基因。对现有的80,000个品系进行详尽的筛选，以及对最近可用的品系进行筛选，应该能够达到为每个基因家族成员找到多个等位基因的目标。为了了解这些基因各自和在基因家族中的作用，我们将采取以下方法：-我们将分离、异交和准备aha突变系，用于分析和分配给俄亥俄州哥伦布市的拟南芥生物资源中心，邮编：43210。-我们将进行遗传杂交并创建多个插入系(即双重、三重突变体等)。以发现基因家族中的遗传冗余。-我们将通过将突变株与野生型植物在旨在揭示发育差异、对环境刺激的反应、养分吸收等条件下的比较来测试突变AHA系的条件表型。-我们将建立一个在线实验条件数据库，旨在进行广泛的表型分析。初步方案将在第一年第一季度提供。详细的协议和相关数据将在生成时添加。29个AHA基因家族突变等位基因的出现，为进一步研究AHA基因及其家族的功能提供了原料。AHA基因在http://www.ncbi.nlm.nih.gov:80/Database/index.html，上的登录号为：AT2G18960、AT4G30190、MJB24.16、T17F15.180、AT2G24520、AT2G07560、F27H5.120、T12K4.90、F23A5.1、F20D23.4、MRG21.9、AT4G11730。

The initial annotation of the complete sequence of Arabidopsis thaliana indicates that of the 11,601 distinct gene types found, only 35% are represented in the genome by one gene, while 37% are represented by gene families with five or more different members. For a majority of the larger gene families, the molecular function of the encoded proteins is known. However, the role of individual gene family members in specific physiological or development pathways is often uncertain. The focus of this project is to develop protocols to assist in describing the in planta function of individual gene family members in the AHA, Arabidopsis plasma membrane H+-ATPase, gene family.The AHA genes code for proteins that couple ATP hydrolysis to proton transport across the plasma membrane of plant cells. The proton-motive force produced by the ATPases then drives the transport of solutes via carriers and channels. In Arabidopsis, the proton pumps comprise a gene family with at least twelve gene isoforms. Expressed in different tissues, the pumps provide the underlying energy for a variety of fundamental plant processes such as cellular homeostasis, mineral and metabolite transport, cell growth and plant morphogenesis, and a variety of responses to the environment including stomatal control, tropic responses, and defense related mechanisms. As a first step towards determining the function of the AHA genes, we have screened over 80,000 Arabidopsis T-DNA insertion lines and have identified twenty-nine mutants. This collection of mutants includes ten of twelve AHA gene family members, with multiple alleles for many isoforms. Exhaustive screening of the current 80,000 lines and screening of recently available lines should make it possible to reach the goal of finding multiple alleles for each gene family member. To understand the role these genes individually and in the gene family, we will take the following approaches: - We will isolate, outcross and prepare aha mutant lines for analysis and distribution to the Arabidopsis Biological Resource Center, Columbus, OH 43210. - We will perform genetic crosses and create multiple insert lines (i.e., double, triple mutants, etc.) in order to uncover genetic redundancy in the gene family. - We will test for conditional phenotypes in the mutant AHA lines by comparing mutants to wild-type plants under conditions designed to reveal differences in development, responses to environmental stimuli, nutrient uptake, etc. - We will establish an on-line database of experimental conditions designed for a broad range of phenotypic analysis. Preliminary protocols will be available Year 1, first quarter. Detailed protocols and associated data will be added as it is generated. The availability of twenty-nine AHA gene-family mutant alleles provides the raw materials for substantial progress into the function of the AHA genes and gene family. The availability of this resource at a predominantly undergraduate institution provides a means of integrating genomic scale research with undergraduate training.The AHA genes can be referenced at http://www.ncbi.nlm.nih.gov:80/Database/index.html, via the accession numbers: At2g18960, AT4g30190, MJB24.16, T17F15.180, At2g24520, At2g07560, F27H5.120, T12K4.90, F23A5.1, F20D23.4, MRG21.9, AT4g11730.