CPT1基因对大口黑鲈脂肪沉积的影响及其调控机制的研究

The breaking of lipid homeostasis will lead to ectopic lipid deposition, which seriously affects the growth and health of fish. As a key regulatory enzyme in fatty acid oxidation, carnitine palmitoyltransferase (CPT) 1 plays a crucial role in regulating the body fat metabolism.At present, it is still obscure how the CPT1 gene regulates the fat metabolism in aquatic animals. Micropterus salmoides, is an important economic species of freshwater fish in China. The aim of this project is to clone, express and characterize the CPT1 of M. salmoides; analyze the gene structure, amino acid sequences and the important functional sites of CPT1; detect the tissue distribution and differential expression of CPT1 gene during different development stages of M. salmoides and in mature fish, as well as the dynamic changes of CPT1 expression and fat metabolism related genes after stimulations with different dietary or nutrition levels, shedding light on the relationship between them and revealing its possible roles in fat metabolism. The project will also use AMPK agonists, AMPK inhibitors and CPT1 inhibitors to explore the molecular mechanism of AMPK regulating CPT1 and the effect of AMPK regulating fat metabolism related genes and establish the signaling pathways of CPT1 regulating the fat metabolism in fish. The implementation of this project will be conducive to illuminating the mechanism of CPT1 gene regulating the fat deposition in fish body and provide a theoretical basis for solving the problem of abnormal fat deposition.

脂肪代谢的平衡状态被打破，将会导致机体脂肪异常沉积，严重影响鱼类生长和健康。作为脂肪酸氧化的关键酶—CPT1，在脂肪代谢的调控中至关重要。目前，CPT1影响水产动物机体脂质代谢机理尚不明确。本项目以我国重要淡水经济鱼类—大口黑鲈为研究对象，利用同源克隆和RACE技术得到大口黑鲈CPT1基因全序列，分析其分子结构与功能特性；研究CPT1在大口黑鲈各发育期及成鱼组织中的差异表达与定位，不同日粮或不同营养水平刺激后组织中CPT1及脂肪代谢相关基因的相对表达量，弄清CPT1的应答表达规律及其与脂肪沉积的关系，探讨其在脂肪代谢中的作用；采用AMPK的激动剂和抑制剂及CPT1的抑制剂探讨AMPK对CPT1调控的分子机理及对脂肪代谢相关基因的调控作用，构建CPT1在鱼类上影响机体脂肪代谢的信号通路。本项目旨在阐明CPT1基因对鱼体脂肪沉积的影响机理及其调控机制，为解决脂肪异常沉积问题提供理论基础。

摄入过量或水平不适宜的物质会导致动物出现代谢紊乱，进而诱发一系列的病理变化。作为代谢关键基因——CPT1，是如何参与调节机体的物质代谢？为此，本项目以大口黑鲈为实验对象，探索了CPT1基因参与鱼类物质代谢的调节机制。研究发现（1）利用RT-PCR和SMART RACE方法从大口黑鲈（Micropterus Salmoides）肝脏中，克隆了CPT1基因的全长序列，该序列全长2359 bp，编码一个由433个氨基酸组成的蛋白质，分子量49 kDa，等电点5.71。氨基酸序列分析，大口黑鲈CPT1具有较高的保守性。（2）探明了CPT1基因在9个组织中均有表达，以肝脏中表达最高，明确了CPT1表达受鱼类的生长、饲料脂肪水平和淀粉水平的调控，构建了AMPK-CPT1调控机体代谢的信号通路。（3）大口黑鲈通过调节机体糖代谢酶和脂代谢酶来适应外源性淀粉水平的变化。高淀粉水平会诱使肝脏发炎、细胞调亡、抗氧化能力降低，进而增加肝糖原和肝脂肪含量，从而危害大口黑鲈的健康生长。（4）大口黑鲈能够有效利用饲料适宜水平的脂肪，促进生长。对于高脂肪水平而言，大口黑鲈一方面通过调节摄食量来限制脂肪的摄取，另一方面通过加强脂肪的氧化分解以及脂肪向肝外的转运来适应和调整，避免脂肪异常或过量沉积。（5）确定了大口黑鲈饲料中脂肪的适宜添加水平为18.42%，纤维素添加水平为4.07%，饲料淀粉不适宜作为大口黑鲈的能量来源。（6）明确了饲料中补充适宜水平的纤维、桑叶、多糖、亚麻籽油或胆汁酸，可在一定程度上改善大口黑鲈的健康。综合上述结果，本研究为大口黑鲈的绿色健康养殖奠定了坚实的工作基础。同时对CPT1功能的深入认识为鱼类代谢相关疾病的预防或治疗提供了重要的启示性线索。