Functional analysis of genes controlling protein trafficking through plasmodesmata using Arabidopsis thaliana T-DNA insertion mutants

使用拟南芥 T-DNA 插入突变体对通过胞间连丝控制蛋白质运输的基因进行功能分析

• 批准号: 80422275
• 负责人: Dr. Magdalena Weingartner
• 金额: --
• 依托单位: Department Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/80422275?language=en
• 关键词: Functional; analysis; genes; controlling; protein

Plasmodesmata are cytoplasmic connections between plants cells which are essential for intercellular communication and exchange of molecules and signals. They are highly organised in their structure and function but molecular mechanisms regulating their dynamic changes and transport activities are largely unknown. We have generated a T-DNA insertion bank using transgenic Arabidopsis thaliana plants expressing GFP from a phloem specific promoter. In a screen for reduced trafficking of GFP by symplastic post-phloem transport in the root tissue eight mutants have been isolated. A first PCR-based analysis of the T-DNA insertions has revealed several candidate genes for each mutant. We will identify the genes responsible for the phenotypes and confirm these results by a second mutant allele for each gene and complementation tests. The genes will be characterised by generating promoterreporter lines to analyse the tissue specific expression patterns and by cloning the cDNAs to generate constructs for protein localisation studies and gain-of-function analyses. The loss-of-function and gain-of-function mutants will be analysed for anatomical phenotypes and for GFP movement phenotypes in various tissues and at all developmental stages. Further functional tests will depend on the identified genes and their predicted structure and function.

胞间连丝是植物细胞间的胞质连接，对细胞间的通讯和分子与信号的交换至关重要。它们在结构和功能上高度组织化，但调节它们动态变化和运输活动的分子机制在很大程度上是未知的。我们已经用表达韧皮部特定启动子的绿色荧光蛋白的转基因拟南芥植株建立了T-DNA插入库。在根组织中通过共生体后韧皮部运输减少GFP运输的筛查中，已经分离出8个突变体。对T-DNA插入片段的首次基于聚合酶链式反应的分析揭示了每个突变体的几个候选基因。我们将确定负责表型的基因，并通过每个基因的第二个突变等位基因和互补测试来确认这些结果。这些基因的特征将是通过产生启动子报告器线来分析组织特定的表达模式，并通过克隆cDNA来产生用于蛋白质定位研究和功能增益分析的结构。将分析功能丧失和功能获得突变体在不同组织和所有发育阶段的解剖表型和GFP运动表型。进一步的功能测试将取决于已识别的基因及其预测的结构和功能。