GST在蜜蜂中肠细胞感染微孢子虫后的调控功能研究

东方蜜蜂微孢子虫为最常见的成年蜜蜂病原，感染会抑制细胞凋亡，增加谷胱苷肽S-转移酶 (GST) 活性。但蜜蜂GST功能缺乏研究报道。研究已表明，人类癌化细胞的GST活性增加，其中GSTP能直接调控细胞凋亡。昆虫虽无GSTP，但有结构相似的GST，而蜜蜂细胞凋亡与GST的关联尚无研究。因此，微孢子虫病抑制中肠细胞凋亡的现象可作为研究蜜蜂GST功能的模型。本研究将明确微孢子虫影响蜜蜂GST的种类、数量，与翻译后修饰；并确认GST互作蛋白。同时，监控感染中蜜蜂GST基因表达；利用RNAi干扰选定的GST基因；再利用药物干扰GST活性，并以免疫组化验证以上试验结果。通过确认微孢子虫、GST与细胞凋亡间的关系，对未来以GST为抗性或药物靶标靶标，让蜜蜂维持细胞正常凋亡，增加抗病性，控制微孢子虫感染。

Nosema ceranae is the most common pathogen in adult honey bees. N. ceranae needs to largely modulate host cell functions, and the inhibition of apoptosis is the most obvious phenomenon in the infection. N. ceranae alters various host proteins; among them, glutathione-S-transferase (GST; a group of universal genes in eukaryotes) was increased in the quantities and the activities. However, honey bee GSTs have not been well characterized. GSTs are responsible for various cell function modulations in addition to detoxification in human studies. In cancer studies, cancer cells have upregulated GST activities and lowered apoptosis rate. One human GST, GST P, has been demonstrated to directly inhibit apoptosis. Although GST P does not exist in insects and bees, certain insect GST S and GST D showed highly similar structures to that of GST P. This research proposal plans to identify honey bee GST modulation in apoptosis using N. ceranae infection as the research model. First, we will use proteomic methods to identify the specific GST alterations and modifications resulted from N. ceranae infection. The other approach will be carefully identifying each GST gene expression changes during the infection. The identified GSTs will be interfered using RNAi assay. Additional GST inhibition drugs will be included in the functional assays as well. Finally, the changes will be carefully monitored using GST antibodies and cytochemistry with CLARITY method. Bees with N. ceranae resistance showed much higher rate of apoptosis than regular bees. Our results can annotate honey bee GSTs and identify the GST modulating apoptosis. The same GST could be applied as a drug target to treat N. ceranae infection or enhance the resistance to the disease.