大豆球蛋白调控鱼肠ROS产生的NOX信号通路作用机制研究

大豆蛋白是鱼类优质的植物蛋白源，但易引起鱼肠结构破坏、功能异常，这与其含有的大豆球蛋白（11S）有关；11S能引起ROS产生破坏鱼肠结构，但作用机制不清楚。NOX在调控人ROS产生中起关键作用，但调控机制在不同物种间有较大差异，而鱼上缺乏研究。因此，本项目拟在鉴定鱼肠NOX亚型及相关信号分子的基础上，利用基因沉默技术率先探明NOX亚型在调控鱼肠ROS产生中的作用；利用荧光标记技术探索11S是否通过促进p22phox结合NOX亚型形成复合物调控ROS产生；利用基因重组等技术进一步研究11S是否通过PKCs亚型调控p47phox（NOXO1）磷酸化后与p67phox（NOXA1）结合进行细胞膜转位后激活p22phox/NOX亚型复合物调控ROS产生；通过系列研究揭示11S调控鱼肠ROS产生的p47phox（NOXO1）/p67phox（NOXA1）/p22phox/NOX亚型信号通路作用机制。

Soybean protein is an excellent plant protein source for fish feed. However, high levels of soybean protein could cause damage to the structural integrity of the intestine, resulting in its dysfunction. These negative influences are partially related to the anti-nutritional factor glycinin present in soybean protein. Our previous study firstly showed that soybean glycinin could induce reactive oxygen species (ROS) production to damage the structural integrity of the intestine, but the mechanisms are still unknown. Previous researches in humans revealed that NADPH oxidase (NOX) isoform signaling plays an important role in ROS production, but this signaling pathway is large difference in various species. Unfortunately, there lacks of information on this signaling pathways in fish. Therefore, this project will be the first time to investigate the NOX isoform signaling pathways-regulated the ROS production in fish. Firstly, we will identify the NOX isoforms and the related signaling factors in the intestine of fish. Base on this, we will be the first time to study the roles of NOX isoforms to regulation of intestinal ROS production by using gene silence technology. Then, we will further explore whether soybean glycinin-regulated ROS production is related to the promotion of p22phox binding NOX to formation of a complex located in the cell membrane by using a fluorescent labeling technology. After that, we will use recombinant gene, fluorescence labeling, gene mutation / deletion technologies to further study whether glycinin influences PKCs isoforms to regulate the phosphorylation of p47phox (NOXO1), promoting p47phox (NOXO1) combined with and p67phox (NOXA1) and then translocation to the cell membrane, further binding and activating the cell membrane p22phox/NOX isoform complexes to promote ROS production. Through a series of studies, we will reveal the possible mechanism of glycinin regulation of intestinal ROS production through regulation of p47phox (NOXO1) /p67phox (NOXA1, DUOXA)/p22phox/NOX isoform signaling pathway. These results will give an important theoretical foundation for the mechanism of glycinin-damaged the structural integrity of the intestine.