Isolation of Arabidopsis Genes by Phenotype Rescue of Seedling Transformants.

通过幼苗转化体的表型拯救来分离拟南芥基因。

• 批准号: 9103733
• 负责人: Robert Ludwig
• 金额: $ 10万
• 依托单位: University of California-Santa Cruz
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-15 至 1993-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9103733&HistoricalAwards=false
• 关键词: Isolation; Arabidopsis; Genes; Phenotype; Rescue

We have constructed a transformation-competent, Arabidopsis ecotype Columbia strain GH50 nuclear genomic library in Agrobacterium host strain AGL1 and cosmid vector pLZO3, both specially engineered for these purposes. This genomic library comprises some 22,000 random clones gridded onto 225 96-well microtiter dishes and, if random, carries some five Arabidopsis genomic equivalents. Arabidopsis strain GH50 carries a dominant acetolactate synthase (als) allele conferring chlorsulfuron resistance. We shall develop high efficiency Arabidopsis whole-plant DNA transformation of newly germinated seedlings as a tool for gene rescue. By a "successive approximation" method, we shall conduct seedling transformation so as to be able to infer individual clones in the genomic library giving rise to transformants. We shall use the transformation- competent Arabidopsis GH50 genomic library constructed in Agrobacterium to rescue the ahs1-1 allele conferring resistance to the sulfonylurea herbicide chlorsulfuron. If successfully developed, this technology will allow the expeditions isolation of genes central to plant growth and development. This gene rescue technology will allow us to efficiency integrate Arabidopsis physical genomic and genetic maps.***//

我们在农杆菌宿主菌株AGL1和cosmid载体pLZO3中构建了具有转化能力的拟南芥生态型哥伦比亚菌株GH50核基因组文库。这个基因组文库包括22000个随机克隆，网格分布在225个96孔微滴皿中，如果随机的话，携带大约5个拟南芥基因组等价物。拟南芥菌株GH50携带一个优势的乙酰乳酸合成酶（als）等位基因，赋予氯磺隆抗性。我们将开发高效的拟南芥新发芽幼苗全株DNA转化技术，作为基因拯救的工具。通过“逐次逼近”的方法，我们将进行幼苗转化，从而能够推断出基因组文库中产生转化子的单个克隆。我们将利用在农杆菌中构建的具有转化能力的拟南芥GH50基因组文库，挽救对磺酰脲类除草剂氯磺隆具有抗性的ahs1-1等位基因。如果开发成功，这项技术将使探险队能够分离出对植物生长和发育至关重要的基因。这项基因拯救技术将使我们能够高效地整合拟南芥物理基因组图谱和遗传图谱