鞍带石斑鱼仔稚鱼对糖的利用能力及代谢调控机制研究

This project aims to study carbohydrate utilization and glycometabolic regulations' mechanism in grouper Epinephelus lanceolatus frys. Growth performance, stomach and intestine structure, digestive ability of carbohydrate, blood glucose levels, glucose transport capcity of liver and muscle tissue cells for carbohydrates fed Epinephelus lanceolatus frys will be focused on. Research methods such as fry culture, biochemical analysis, histological observation, RT-PCR, Realtime-PCR, mathematics statistics and so on, will be applied in this project. Carbohydrate digestive enzymes activities in the degestive system in carbohydrates fed frys will be measured, and also, activities of key enzymes in glycometabolism, including glycolysis, gluconeogenesis,pentose phosphate pathway,tricarboxylic acid cycle, glycogenolysis and glycogenesis, will be analyzed.These key enzymes contain glucokinase, 6－phosphofructokinase, pyruvate kinase, glucose-6-phosphatase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, Fructose-1，6-diphosphatase, glycogen synthase, glycogen phosphorylase, glucose-6-phosphate dehydrogenase and citrate synthase. Gene express patterns and levels of glucose transporters (GLUTs) in liver and muscle tissue cells will be identified or measured by using molecular tools. Through the research works above, kinds of GLUTs existed in liver and muscle tissue cells will be ascertained, and relativity between blood glucose levels and carbohydrate digestive enzymes activities, gene express levels of GLUTs, activities of key enzymes in glucometabolism will be explored, and thus, capacities of carbohydrate digesting, transporting and utilizing as well as partial glycometabolic regulations' mechanismin of Epinephelus lanceolatus frys will be understood. It would be expected that results from this project will lay a theoretical foundation for technique exploitation of high-effeciency carbohydrate utilization for Epinephelus lanceolatus frys in future. Implementation of this project will enrich nutriology theory in marine fish frys and have scientific significance.

本项目以鞍带石斑鱼仔稚鱼为研究对象，采用仔稚鱼培育、生化分析、组织学观察、RT-PCR、Realtime-PCR和数理统计等综合研究方法，从糖在鱼体内的消化、转运及代谢入手，重点研究仔稚鱼摄食糖后生长性能的变化、胃肠道结构的变化、消化系统对糖的消化分解能力（糖消化酶活性）、血液葡萄糖水平的变化、肝脏和肌肉组织细胞葡萄糖转运能力（葡萄糖转运蛋白GLUTs基因表达类型及表达水平）及糖代谢（糖酵解、糖异生、磷酸戊糖途径、柠檬酸循环、糖原合成、糖原分解）能力（代谢关键酶活性），鉴定其肌肉和肝脏组织中GLUTs类型，探索血糖水平与消化系统糖消化酶活性、肝脏和肌肉组织细胞GLUTs基因表达水平及糖代谢关键酶活性之间的关联，阐明鞍带石斑鱼仔稚鱼糖消化、转运及利用能力，揭示其糖代谢部分调控机制，为开发鞍带石斑鱼仔稚鱼高效糖利用技术奠定基础。项目的实施可丰富海水鱼类仔稚鱼营养学理论，会有一定的科学意义。

本项目以鞍带石斑鱼仔稚鱼为研究对象，采用仔稚鱼培育、生化分析、组织学观察、RT-PCR、Realtime-PCR和数理统计等综合研究方法，从糖在鱼体内的消化、转运及代谢入手，主要研究仔稚鱼摄食糖后生长性能的变化、消化系统对糖的消化分解能力（糖消化酶活性）、肝脏组织细胞葡萄糖转运能力及糖代谢能力。相对于葡萄糖、麦芽糖和纤维素，淀粉更适合作为仔稚鱼糖源，投喂淀粉饲料仔稚鱼糖分解代谢关键酶基因表达水平更高。饲料中糖的添加可提高肝脏细胞GLUT4蛋白基因表达水平。仔稚鱼发育过程中，胰蛋白酶和肠脂肪酶活性升高，胰淀粉酶活性下降。饲料1.30糖/脂比（14.79%粗脂肪、19.23淀粉）适合仔稚鱼生长，糖/脂比过高（1.88），生长显著下降，饲料脂肪含量过高会导致机体脂肪沉积。饲喂CHO14.4/P50、CHO10.4/P54、CHO6.4/P58、CHO2.4/P62饲料组仔稚鱼肝细胞丙酮酸激酶/PK及己糖激酶/HK基因mRNA表达水平低于饲喂CHO26.4/P38、CHO22.4/P42、CHO18.4/P46饲料组仔稚鱼。肌细胞丙酮酸激酶/PK基因mRNA表达水平随饲料糖/蛋比下降而下降。肝细胞丙酮酸羧化酶/ PC及1,6-二磷酸果糖酶/FBP基因mRNA表达水平随饲料糖/蛋比降低而升高。摄食高糖蛋比饲料后，仔稚鱼肝细胞脂肪合成代谢相关酶（脂肪酸合酶/ FAS、ATP-柠檬酸裂解酶/ ACLY）基因mRNA表达水平降低。仔稚鱼肝细胞脂肪氧化代谢关键酶（肉碱棕榈酰转移酶1A/CPT1A）基因mRNA表达水平表现出随饲料糖/蛋比降低而升高的趋势。摄食高糖蛋比饲料后，仔稚鱼肝细胞蛋白质合成相关酶（核糖体蛋白S6 /RPS6）基因mRNA表达水平降低。肝脏和肌肉细胞蛋白质代谢也受饲料糖蛋比影响。本项目实施阐明了鞍带石斑鱼仔稚鱼对糖的利用能力，初步揭示其糖代谢调控部分机制，项目成果为开发鞍带石斑鱼仔稚鱼人工饵料提供了理论依据，学术价值和应用价值较高。