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

对拟南芥完整序列的初步注释表明，在发现的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，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。 本项目的重点是研究拟南芥质膜H ~+-ATP酶基因家族（Arabidopsis plasma membrane H ~+-ATPase，AHA）中的单个基因家族成员在植物体内的功能，AHA基因编码的蛋白质将ATP水解与质子跨质膜转运偶联。由ATP酶产生的质子动力然后驱动溶质通过载体和通道的运输。在拟南芥中，质子泵包括具有至少12种基因同种型的基因家族。这些泵在不同的组织中表达，为植物的各种基本过程提供潜在的能量，如细胞内稳态、矿物质和代谢物运输、细胞生长和植物形态建成，以及对环境的各种响应，包括气孔控制、向性响应和防御相关机制。作为确定AHA基因功能的第一步，我们筛选了超过80，000个拟南芥T-DNA插入系，并鉴定了29个突变体。 这个突变体的集合包括12个AHA基因家族成员中的10个，具有许多同种型的多个等位基因。对现有的80，000个品系进行彻底的筛选和对最近可用的品系进行筛选，应该可以达到为每个基因家族成员找到多个等位基因的目标。为了理解这些基因单独和在基因家族中的作用，我们将采取以下方法：-我们将分离，异交和制备aha突变株系用于分析和分配到拟南芥生物资源中心，哥伦布，OH 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）以揭示基因家族中的遗传冗余。 - 我们将测试条件表型的突变体AHA线比较突变体野生型植物的条件下，旨在揭示不同的发展，对环境刺激的反应，营养吸收等-我们将建立一个在线数据库的实验条件，设计了广泛的表型分析。初步方案将在第一年第一季度提供。 详细的方案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可用性为AHA基因和基因家族功能的实质性进展提供了原材料。该资源在主要本科院校的可用性提供了将基因组规模研究与本科生培训相结合的手段。AHA基因可以通过登录号在http：//www.ncbi.nlm.nih.gov：80/Database/index.html上引用：At2g18960、AT4g30190、MJB 24.16、T17F15.180、At2g24520、At2g07560、F27H5.120、T12K4.90、F23A5.1、F20D23.4、MRG 21.9、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.