钙离子是真核细胞中常见的第二信使，在细胞中的胞吐作用、基因转录等过程中起着十分重要的作用。内质网和线粒体是细胞维持是细胞维持正常生理功能和传导钙信号的关键细胞器。荧光钙成像因其可视化，高灵敏性，高时空分辨率等优点成为检测钙信号的有力工具。荧光钙探针发展迅速，尤其是基因编码钙探针（GECI），成为细胞器靶向荧光成像有力工具。荧光钙探针在哺乳动物中的研究最为深入，而在酵母中相对落后。

本研究使用的是基因编码钙探针NEMO系列探针，相比于先应用的GCaMP6系列探针有更快的钙动力学和更大的动态范围。选择定位在内质网上的NEMOer-m和定位在线粒体上的mt-NEMOs两个探针，首先其分别与酵母常用质粒pGBKT7重组，获得重组质粒并转化酵母后，在荧光显微镜下观察基因表达情况。同时，通过营养饥饿培养的实验，评估了两探针的实际应用情况。在荧光显微镜下观察到目的基因成功表达并定位到相应细胞器上；营养饥饿条件下培养的酵母细胞中，基因表达情况与正常培养的酵母细胞中无明显区别。

本研究将新的探针应用在酵母钙信号的研究中，丰富了酵母细胞器靶向探针的类型。尽管针对内质网的探针定位出错，但是线粒体探针表达定位情况良好。酵母钙探针的开发对深入研究钙信号传导过程有重要意义。

Calcium ions are common second messengers in eukaryotic cells, playing a crucial role in processes such as exocytosis and gene transcription. The endoplasmic reticulum and mitochondria are key organelles for maintaining normal physiological functions and conducting calcium signaling in cells. Fluorescent calcium imaging has become a powerful tool for detecting calcium signals due to its advantages such as visualization, high sensitivity, and high spatiotemporal resolution. Fluorescent calcium probes have developed rapidly, especially gene encoded calcium probes (GECI), which have become powerful tools for organelle targeted fluorescence imaging. The research on fluorescent calcium probes is the most in-depth in mammals, but relatively backward in yeast.

This study used the gene encoded calcium probe NEMO series probe, which has faster calcium kinetics and a larger dynamic range compared to the previously applied GCaMP6 series probe. Select two probes, NEMoer-m located on the endoplasmic reticulum and mt-NEMOs located on mitochondria, and first recombine them with the commonly used yeast plasmid pGBKT7. Obtain the recombinant plasmid and transform it into yeast. Observe the gene expression under a fluorescence microscope. Meanwhile, the practical application of the two probes was evaluated through experiments on nutrient starvation cultivation. The target gene was successfully expressed and localized on the corresponding organelles under a fluorescence microscope; There was no significant difference in gene expression between yeast cells cultured under nutritional starvation conditions and those cultured normally.

This study applies new probes to the study of yeast calcium signaling, enriching the types of probes targeting yeast organelles. Although the localization of the probe targeting the endoplasmic reticulum was incorrect, the expression and localization of the mitochondrial probe were good. The development of yeast calcium probes is of great significance for in-depth research on calcium signaling processes.