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

胞间连丝是植物细胞间的细胞质连接，是细胞间通讯、分子和信号交换的重要通道。它们在结构和功能上是高度组织化的，但调节其动态变化和运输活动的分子机制在很大程度上是未知的。我们利用从韧皮部特异性启动子中表达GFP的转基因拟南芥植物建立了T-DNA插入库。在根组织中通过韧皮部后的共塑运输减少GFP运输的筛选中，已经分离出八个突变体。第一个基于pcr的T-DNA插入分析揭示了每个突变的几个候选基因。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。这些基因将通过生成启动子报告细胞系来分析组织特异性表达模式，并通过克隆cdna来生成用于蛋白质定位研究和功能获得分析的结构。将分析功能丧失和功能获得突变体在各种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确定的基因及其预测的结构和功能。

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.