SGER: Alkaloid Transport in Nicotiana Tabacum

本项目的目的是表征一种烟草膜转运蛋白，这种转运蛋白可能负责将生物碱从根的最初生物合成位点运输到叶质部组织，并最终在叶片中积累。这将通过使用先前在转录分析实验中分离的基因片段作为杂交探针来分离全长cDNA来完成。有了这一cDNA，我们将利用RNase保护实验和转基因报告基因融合来确定假定的生物碱转运蛋白的组织和细胞间定位模式。生物碱转运活性的生化证实将通过推测的生物碱转运蛋白在爪蟾卵母细胞中的异源表达和直接测量生物碱转运动力学来完成。最后，将利用过表达和反义转基因毛状根培养来实现对生物碱转运基因表达水平的体内调节，以评估对总体生物碱积累水平的影响。这些实验可能导致首次鉴定和分子表征假设的生物碱转运体，该转运体负责将生物碱从特定根细胞类型的生物合成位点转移到它们在叶片中的作用位点。这将对理解许多植物次生代谢物的生物合成位点如何以及为什么经常在空间上与这些重要防御化合物的作用位点分离做出重大贡献。

The objective of this project is to characterize a Nicotiana tabacum membrane transporter thatmay be responsible for the transport of alkaloids from their initial site of biosynthesis in roots toxylem tissue and for their final accumulation in leaves. This will be accomplished by using a genefragment previously isolated during transcriptional profiling experiments as a hybridizationprobe to isolate the full-length cDNA. With this cDNA in hand, an extensive effort will be madeto determine the tissue and intercellular localization patterns of the putative alkaloid transportprotein, using both RNase protection assays and transgenic reporter gene fusions. Biochemicalconfirmation of alkaloid transport activity will be accomplished by heterologous expression ofthe putative alkaloid transporter in Xenopus oocytes and directly measuring alkaloid transportkinetics. Finally, in vivo modulation of the putative alkaloid transporter gene expression levelswill be achieved using overexpression and antisense transgenic hairy root cultures to assess theeffect on overall alkaloid accumulation levels. These experiments may lead to the firstidentification and molecular characterization of a postulated alkaloid transporter that isresponsible for mobilizing alkaloids between their site of biosynthesis in specific root cell typestowards their site of action in the leaves. This would be a significant contribution tounderstanding both how and why the site of biosynthesis of many plant secondary metabolites isoften spatially separated from the site of action of these important defense compounds.