泥鳅去饱和酶基因（fads）在高度不饱和脂肪酸合成中的作用机制

Investigating the molecular mechanism of highly unsaturated fatty acids (HUFA) biosynthesis in fish is the basis for regulating the ability of HUFA biosynthesis, which is very meaningful for increasing nutritional quality of seafood. Fatty acid desaturase (fad) genes play an important role in HUFA biosynthesis. Our previous works detected that loach (Misgurnus anguillicaudatus) has an ability of HUFA biosynthesis with three fad genes. However, their regulation functions on HUFA biosynthesis are unknown. In this project based on our previous works, first, we plan to confirm the functional characterizations of three fads genes using transgenic yeast expressing method. And then, we will construct three fads-deficiency loach and fads-overexpression loach, respectively, to identify the key genes and metabolites of three fads by the comparative sequencing analysis and metabonomics profiles, respectively. Moreover, we will study the key genes and interacting proteins of three fads genes in cell line using biochemical analysis, including Co-IP, western blot and immunofluorescence technique, to uncover the molecular mechanism of fads genes for regulating HUFA biosynthesis. Last, we will reveal the functional mechanism of fad on HUFA biosynthesis in fish fed with stable isotope labeling fatty acids substrates diets. The functional characterization of the three fad genes will not only make great contributions to comprehensively understanding the mechanism of regulating HUFA biosynthesis, but also provide a novel strategy for improving the nutritional quality of freshwater fish, which is significance in both theory and application.

了解鱼类高度不饱和脂肪酸（HUFA）合成的分子机理是调控鱼类自身合成HUFA能力的前提，对提高水产品的营养品质有重要的意义。去饱和酶（fads）基因在HUFA合成中起着重要作用。申请人在前期研究中已经证实泥鳅具有很强的HUFA合成能力，其具有三条fads基因，但它们的作用机制尚不清楚。本项目在前期研究基础上，拟先用体外酵母表达明确这三条fads的生物学功能；再分别构建三条fad敲除泥鳅和fads过表达泥鳅，基于转录组和代谢组学手段，确定与fads作用的关键基因和代谢产物，再在细胞水平采用免疫共沉淀、Western blot等筛选出互作蛋白并验证，来剖析三条fads基因在HUFA合成中的作用机制；最后通过投喂稳定同位素标记饲料，揭示fads基因在不同底物下的作用机制。本研究不仅将丰富人们对鱼类fads在HUFA中作用机制的认识，还将为提高淡水鱼营养品质提供新线索，在理论和应用均具有重要意义。