SGER: Alkaloid Transport in Nicotiana Tabacum

SGER：烟草中的生物碱转运

• 批准号: 0129943
• 负责人: John Jelesko
• 金额: $ 7万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-11-01 至 2003-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0129943&HistoricalAwards=false
• 关键词: SGER; Alkaloid; Transport; Nicotiana; Tabacum

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.

本项目的目的是表征烟草（Nicotiana tabacum）的膜转运蛋白，该转运蛋白可能负责生物碱从根木质部组织中的生物合成起始位点转运并最终在叶中积累。这将通过使用先前在转录谱实验中分离的基因片段作为杂交探针来分离全长cDNA来实现。有了这个cDNA在手，一个广泛的努力将madeto确定组织和细胞间定位模式的假定生物碱transportprotein，使用RNA酶保护试验和转基因报告基因融合。生物碱转运活性的生物化学确认将通过在非洲爪蟾卵母细胞中异源表达假定的生物碱转运蛋白并直接测量生物碱转运动力学来完成。最后，在体内调节假定的生物碱转运蛋白基因的表达水平，将实现使用过表达和反义转基因毛状根培养，以评估对整体生物碱积累水平的影响。这些实验可能会导致第一次鉴定和分子特征的假定生物碱转运，isresponsible调动生物碱之间的生物合成位点在特定的根细胞类型到他们的网站在叶中的行动。这将是一个重要的贡献，以了解如何以及为什么许多植物次生代谢物的生物合成的网站往往是空间上分开的网站，这些重要的防御化合物的行动。