植物中单细胞水平微量生长素的原位实时测定方法研究

项目名称： 植物中单细胞水平微量生长素的原位实时测定方法研究

项目编号： No.91317312

项目类型： 重大研究计划

立项/批准年度： 2014

项目学科： 微生物学、植物学

项目作者： 肖浪涛

作者单位： 湖南农业大学

项目金额： 200万元

中文摘要： 传统IAA测定方法难以满足目前相关研究对生长素测定提出的新要求，建立符合目前植物激素前沿研究需求的单细胞水平微量生长素原位实时测定新方法已成为当务之急。本项目拟在前期研究工作基础上开展以下研究：进一步利用转DR5::GFP的拟南芥IAA低水平合成双突变体yuc2yuc6为研究材料，基于GFP的荧光强度、分子数量和mRNA表达量等多条途径建立IAA浓度与GFP表达量之间的准确数量关系，建立IAA高灵敏原位实时测定方法；在小立碗藓中构建双元（生长素和温度）诱导的可控GFP表达系统，通过与IAA诱导的DII-GFP降解系统进行对比，建立单细胞水平的IAA准确定量方法；基于IAA共受体和二维液相建立微量样品中IAA的高效分离纯化新技术；基于DNA末端保护小分子分析技术建立IAA的高灵敏检测新方法；系统开展所建立新方法的应用技术研究和检测应用示范，以促进我国植物激素检测的技术进步。

中文关键词： IAA；单细胞；原位；测定；方法学

英文摘要： Traditional IAA assays can hardly satisfy the new demands from the frontier in phytohormonal research area for IAA determination. So it is urgent to develop new technology for IAA determination at single cell level. The following approach based on early efforts will be carried out in this project: Arabidopsis auxin biosynthesis defective double mutant yuc2yuc6 will be used as materials after being transformed by DR5::GFP, and highly sensitive in situ real time assay will be established through multiple accurate quantitative relationship of IAA with the fluorescence intensity, GFP molecule number and GFP mRNA expression level based on the expression of reporter gene GFP drived by auxin-induced promoter DR5. Secondly, highly efficient temperature-induced promoter with feasible inducing temperature will be used for the construction of controllable dual-inducing GFP expression system in model plant Physcomitrella patens to easily prepare plenty of homogeneous protoplast, then the accurate assay method of IAA in single plant cell will be achieved through the DR5 driven GFP biosynthesis and IAA induced DII-GFP degradation. Thirdly, based on the DNA terminal protection of exonuclease or nucleic acid polymerase technique, the nucleic acid probe marked by IAA will be protected through competing with IAA from samples for

英文关键词： IAA；Single cell；In-situ；Assay；