Development of a new analytical tool for elucidating nitrate signaling networks

该项目旨在开发一种新的荧光指示剂，用于指示活植物根系中硝酸盐浓度的变化。该指示剂依赖于两种荧光蛋白之间的福斯特共振能量转移（FRET），因此硝酸盐水平的变化通过两种蛋白的发射的比率变化来显示。聚球藻PCC 7924蓝藻含有高亲和力的硝酸盐结合蛋白NrtA，编码该蛋白的基因被用于新的指示剂。将NrtA克隆到合适的载体中后，将该基因融合在编码CFP和YFP的基因之间。新的融合蛋白成功地表现出FRET活性，当仅CFP蛋白被激发时，来自CFP和YFP的双发射。当蛋白质暴露于较高的硝酸盐浓度时，也观察到FRET比率略有增加。然而，即使在低硝酸盐水平下也存在高FRET比率，并且比率增加不足以用于植物。使用定点突变鉴定并修饰NrtA中的保守残基。这成功地降低了高的初始FRET比率，但没有导致更大的硝酸盐响应。进一步优化有望为植物研究提供新的实用指标。

This project aimed to develop a new fluorescent indicator of changes in nitrate concentration within living plant roots. This indicator relies on Forster Resonance Energy Transfer (FRET) between two fluorescent proteins, such that a change in nitrate level is shown by a ratio change in emission from the two proteins.Synechococcus PCC 7924 cyanobacterium contains a high-affinity nitrate binding protein, NrtA, and the gene encoding this protein was used for the new indicator. After the NrtA was cloned into the appropriate vectors, the gene was fused between genes encoding CFP and YFP. The new fusion protein successfully showed FRET activity, and dual emission from both CFP and YFP when only the CFP protein was excited. A slight increase in the FRET ratio was also observed when the protein was exposed to higher nitrate concentrations. However, a high FRET ratio was present even at low nitrate levels, and the ratio increase was not enough for use in plants. Conserved residues in NrtA were identified and modified using site-directed mutagenesis. This successfully reduced the high initial FRET ratio but did not result in a greater nitrate response. Further optimization is expected to provide the new practical indicator for plant research.