Molecular Genetic Analysis of the Arabidopsis AB12 Locus

该项目的长期目标是识别与脱落酸(ABA)作用相关的蛋白质，并通过生理、生化和分子遗传学分析来研究它们的功能。在拟南芥中，通过分离和鉴定对ABA敏感性改变的突变体，已经鉴定了ABA作用所需的基因。芬克尔斯坦博士之前的研究表明，三个ABI基因座(abi1、abi2和abi3)在重叠的ABA反应途径中发挥作用，这两个途径的功能丧失在花粉中是致命的。她将通过克隆相关的基因座来扩展这些研究，要么通过染色体行走，要么通过基因组减去，重点放在ABI2上。具体目标是：1)研究abi2abi3花粉活力丧失的时间和原因，2)分离新的辐射诱变ABI突变体，3)分离重叠的酵母人工染色体，跨越ABI1基因座两侧最紧密连锁的限制性片段长度多态标记之间的区域。可选地，如果任何辐射诱导的突变是abi2的等位基因，她将使用基因组减法来克隆在突变体中缺失的区域，4)通过用一组重组家族对这些克隆进行RFLP精细定位来识别与abi2最紧密连锁的基因组克隆的子集，5)通过突变表型的互补来识别这个子集内的abi2基因，6)分离并鉴定与ABI2基因相对应的cDNA克隆。

The long term objective of this project is to identify proteins involved in abscisic acid (ABA) action and study their function by physiological, biochemical and molecular genetic analysis. Genes required for ABA action have been identified by isolating and characterizing mutants with altered sensitivity to ABA in Arabidopsis. Dr. Finkelstein's previous studies indicated that three ABI loci (abi1,abi2 and abi3) function in overlapping pathways of ABA response and the loss of function in both pathways was lethal in pollen. She will extend these studies by cloning the relevant loci, either by chromosome walking or genomic subtraction, focussing on ABI2. The specific objectives are: 1) Investigate timing and basis of loss of viability in abi2abi3 pollen, 2)Isolate new radiation induced abi mutants, 3) Isolate overlapping yeast artificial chromosomes spanning the region between the most tightly linked restriction fragment length polymorphism markers flanking the ABI1 locus. Alternatively, if any of the radiation induced mutations are allelic to abi2, she will use genomic subtraction to clone the region deleted in the mutant, 4) Identify the subset of genomic clones showing tightest linkage to ABI2 by RFLP fine mapping these clones with a panel of recombinant families, 5) Identify the ABI2 gene within this subset by complementation of the mutant phenotype, and 6) Isolate and characterize cDNA clones corresponding to the ABI2 gene.