钙离子是细胞内一种重要的第二信使，它调节许多生物学功能，例如基因转录、细胞增殖与凋亡、免疫反应等。在质膜及细胞器膜上的许多钙泵、钙通道及钙交换器，还有钙结合蛋白，通过对Ca2+的摄取、释放和结合，产生不同的钙信号，维持细胞钙稳态。若钙稳态失调，会破坏细胞正常功能，损坏机体生长发育，进而导致相关疾病的发生。内质网在蛋白质和脂质的合成中起重要作用，也是细胞内最大的钙库。因此，探究内质网钙稳态维持的分子机制至关重要。在实验室前期工作中，通过全基因组水平的基因敲除，获得了影响内质网静息钙水平的候选基因。本研究选取45个敲除后内质网静息钙水平上升的候选基因，构建过表达质粒进行功能验证，得到5个显著降低内质网静息钙水平的基因。通过文献调研，选取关键基因TSSK6，设计实验，猜测降低内质网静息钙水平的机制。本研究主要使用了实验室前期开发的可见光激发的比值型钙指示剂TuNer，以及活细胞钙成像技术。

Calcium ion is an important second messenger in cells, which regulates many biological functions, such as gene transcription, cell proliferation and apoptosis, and immune response. There are many calcium pumps, calcium channels, calcium exchangers in the plasma membrane and organelle membranes, and calcium-binding proteins, which generate different calcium signals through the uptake, release, and binding of Ca2+ to maintain cellular calcium homeostasis. If calcium homeostasis is dysregulated, it can disrupt normal cellular function and damage the growth and development of the organism, which can lead to the development of related diseases. The endoplasmic reticulum plays an important role in protein and lipid synthesis and is also the largest intracellular calcium reservoir. Therefore, it is crucial to investigate the molecular mechanisms underlying the maintenance of ER calcium homeostasis. In the laboratory’s previous work, candidate genes that affect the resting calcium level of the ER were obtained through genome-wide gene knockout. In this study, 45 candidate genes whose ER resting calcium level increased after knockout were selected, overexpression plasmids were constructed for functional verification, and 5 genes that significantly decreased ER resting calcium level were obtained. The key gene TSSK6 was selected through literature research, and experiments were designed to guess the mechanism of reducing the resting calcium level of the ER. The main techniques of this study are TuNer, a visible light-excited ratio-type calcium indicator developed in the laboratory’s previous work, and live cell calcium imaging.