花鲈味觉受体T1Rs参与摄食调控的机制研究

In our previous studies, it has been shown that when dietary fishmeal was totally replaced with plant protein, the Japanese seabass, a typical carnivorous fish, showed appetite shifts of anorexia followed by adaptation even compensation. Taste 1 receptor family members (T1Rs), which include T1R1, T1R2 and T1R3 subtypes, are not conserved among species and play vital roles in fish feeding preferences, swallowing and digestion. The aims of the project are to clone full lengths and carry out transmembrane domain analysis of T1R1, T1R2 and T1R3 of Japanese seabass, combine with establishment of overexpression and RNAi vectors to transfect into the primary tongue bud cells of Japanese seabass to clarify the roles of T1Rs in taste perception. Expressions of T1Rs and signal pathway genes PLC-β2, TRPM5 in tongues and intestines of Japanese seabass at anorexia, adaptation and compensation feeding response periods are examined to screen the key subtypes of T1Rs involved in appetite shifts. The key subtypes of T1Rs are further mutated and transfected into the tongue buds and intestinal epithelial cells of Japanese seabass, then Ca2+ cell imaging and hormones involved in feeding regulation are detected after stimulation with umami amino acids and anti-nutrient factors, which are marker compounds of fishmeal and plant protein diet respectively. This project will be helpful to clarify the mechanism of T1Rs involved in feed intake regulation and nutrient sensing of carnivorous fish and can provide important theoretical guidance in the efficient utilization of plant protein.

肉食性鱼类花鲈摄食植物蛋白完全替代鱼粉的饲料后出现厌食到适应再到补偿摄食的食欲转变。味觉受体第一家族T1Rs存在T1R1，T1R2和T1R3亚型，具有物种特异性，在食物的选择、吞咽和消化中起重要作用。本项目拟克隆花鲈T1Rs基因，开展跨膜结构域分析，并在花鲈舌味蕾细胞中构建过表达和RNA干扰体系阐明T1Rs作用方式；检测花鲈摄食植物蛋白饲料后在厌食期、适应期、补偿期不同摄食反应阶段舌和肠道T1Rs及信号传导通路重要蛋白PLC-β2和TRPM5表达水平，筛选参与食欲转变的关键T1Rs亚型；进一步对该亚型的关键作用位点进行突变后转染花鲈舌味蕾和肠上皮细胞，分别采用鱼粉和植物蛋白标志性化合物‒鲜味氨基酸和抗营养因子刺激，结合Ca2+细胞成像和摄食调控相关激素检测进行体外机制验证。本项目有助于阐明肉食性鱼类T1Rs参与摄食调控和营养感知的作用机制，对实现植物蛋白高效利用具有重要的理论指导意义。

肉食性鱼类花鲈摄食植物蛋白完全替代鱼粉的饲料后出现厌食到适应再到补偿摄食的食欲转变。味觉受体第一家族T1R1和T1R3异源二聚体在食物的选择、吞咽和消化中起重要作用。本项目首先克隆花鲈T1R1和T1R3基因，开展组织表达和细胞共定位；通过在HEK293T细胞中体外构建花鲈T1R1和T1R3单表达体系和T1R1/T1R3共表达体系，阐明花鲈T1R1和T1R3异源二聚体的作用形式；通过检测花鲈摄食植物蛋白饲料后在厌食期、适应期、补偿期不同摄食反应阶段味蕾电镜结构以及舌和肠道T1R1和T1R3在蛋白和mRNA表达水平表明花鲈味觉系统长期应对植物蛋白饲料发生适应性改变，随着对植物蛋白适应能力的增加，对鲜味的感知能力下降；结合Ca2+细胞成像和分子对接进一步发现摄食抑制的主要原因是T1R1和T1R3均与植物蛋白中含量高的精氨酸结合活性最强。本研究对阐明鱼类味觉感知和摄食调控机制奠定基础，为实现植物蛋白饲料的高效利用提供科学依据。