乙二醛酶Ⅰ基因对刀鲚应激猝死的调控功能及作用机制

In this study, basing on the research foundations, biochemistry, molecular biology and genetics research methods are used to study the function and regulated mechanism of GLO1 gene for stress-induced sudden death in Coilia nasus. In vitro, activity of GLO1 promoter is analysised to determine the core promoter region. In vivo for C. nasus, studies are carried out for the three groups of materials. The First one, the stress induced death rates of larval and adult fish are significantly different, methylation levels of core promoter region are measured, then clarifying the relationgship with the transcriptional regulation of GLO1, and try to revealing its law on the regulation of stress-induced sudden death. The second one, agonist and inhibitor of GLO1 are applicated to treat C. nasus, after which studies are carried out to show how are stress-induced sudden death regulated under the conditions of GLO1 protein overexpression and inhibition. The third one, Juvenile transport experiment are carried out and then the correlation between survival rate and haplotypes of GLO1 using,the survival and death samples, the purpose of which is to explore its potential as an anti-stress breeding target genes. In vivo for Danio rerio, GLO1 is knocked out, and which is used to study how the indicators of stress induced sudden death response. In this study, the regulation of GLO1 to sudden death which stress induced are studied comprehensively from the DNA level, transcriptional regulation level and protein level, this would provid a new target gene for anti-stress breeding in C.naus and new ideas for resolving anti-stress sudden death in human and mammalian.

本项目针对应激性猝死的科学问题，以刀鲚为主要研究对象，运用生物化学、分子生物学和遗传学的多种手段，系统地研究乙二醛酶Ⅰ(Glyoxalase 1，GLO1) 对应激性猝死的调控作用。细胞水平上，分析GLO1的启动子活性，确定核心启动子区。刀鲚个体水平上，针对三组材料分别展开研究：①仔鱼和成鱼应激猝死率差异明显，研究核心启动子区的甲基化水平对GLO1的转录调控作用，明确其对应激性猝死的调控规律；②应用GLO1激动剂和抑制剂分别增强和抑制其蛋白表达，研究其对应激性猝死的调控规律；③开展幼鱼运输应激实验，研究GLO1不同单倍型与猝死率的相关性。斑马鱼个体水平上，构建GLO1敲除模型，研究其对应激性猝死相关指标的调控规律。本项目在GLO1的DNA层面、转录调控层面和蛋白层面系统地研究其对应激性猝死的调控作用，探讨其作为刀鲚抗应激选育靶标基因的潜力，也为人和哺乳类的抗应激猝死研究提供新思路。

本项目针对鱼类应激性猝死的科学问题，以刀鲚、斑马鱼、团头鲂等为主要研究对象，运用生物化学、分子生物学和遗传学的多种手段，系统地研究了乙二醛酶Ⅰ(Glyoxalase 1，Glo1) 对应激性猝死的调控作用。项目完成各项考核指标。. 细胞水平上，获得了Glo1基因的DNA全长序列、cDNA序列。分析Glo1的启动子活性，确定核心启动子区位于转录起始点上游800-1200 bp。刀鲚个体水平上，针对三组材料分别开展了研究：①项目研究发现幼鱼运输过程中，Glo1的mRNA和蛋白的表达与应激死亡率存在显著的相关性。②仔鱼和成鱼应激猝死率差异明显，研究了核心启动子区的甲基化水平对Glo1的转录调控作用，但两组的甲基化水平无显著差异。我们检测了Glo1基因组，发现了2个SNP标记，发现这两个标记与刀鲚应激猝死显著相关，该成果为刀鲚抗应激育种提供了靶标基因；③项目研究发现NaCl和山梨醇是Glo1的激动剂，Ca2+是抑制剂，应用Glo1激动剂和抑制剂分别增强和抑制其蛋白表达，发现其可以显著提高和降低刀鲚的存活率；其次，研究发现鱼类应激通过HPI轴提升糖类和脂类的分解代谢，产生过量氧自由基，引发氧化应激，对机体造成损伤。斑马鱼个体水平上，构建了Glo1敲除模型，发现敲除品系斑马鱼的氧化应激指标和死亡率显著上升，表明Glo1在调控鱼类应激中发挥重要功能。另外，本项目的研究还发现除了上述神经内分泌对应激的调控，还发现了肠道微生物通过磷酸戊糖途径调控了鱼类的应激应答。. 本项目在细胞水平、刀鲚个体和斑马鱼个体水平，从Glo1的DNA层面、转录调控层面和蛋白层面系统地研究其对应激性猝死的调控作用，探讨了其作为刀鲚抗应激选育靶标基因的潜力，也为人和哺乳类的抗应激猝死研究提供新思路。.