草鱼脂肪代谢关键酶基因的分子特性及营养调控研究

Lipid is one of the most important energy sources and necessary nutrients for cultured animals. Lipid metabolism has close relation with growth and health of cultured animals. Excess fat deposition in animals was reported not only leading to poor meat quality and low feed conversion, but also causing some lipid metabolism associated diseases such as fatty liver syndrome. In recent years, reducing excess fat deposition in animals, revealing regulation mechanisms of lipid metabolism and developing its regulation technology have become main tasks in animal nutrition research. In this project, we select grass carp (Ctenopharyngodon idella), an economically important aquaculture species in China, as a model organism and analyze 8 genes coding key enzymes related to lipid synthesis, catabolism and β-oxidation pathway. First, the RT-PCR technology will be used for cloning the full-length cDNA sequences of above-mentioned genes. Second, the real-time PCR method will be applied to detecting the temporal expression of these genes in different tissues and at various developmental stages. Third, mRNA expression changes of fish intravenously injected with glucose or hypolipidemic drugs, or intraperitoneally injected with siRNA, will be determined with the real-time PCR to reveal the functions of these genes. Finally, experimental fish will be fed for 8 weeks with diets containing three different lipid sources or starch sources, and then the real-time PCR method will be applied to evaluating the effects of dietary lipid or starch sources on the expression of these genes. Meanwhile, growth parameters including specific growth rate (SGR), feed conversion ratio (FCR), hepatopancreas somatic indices (HSI) and viscera somatic indices (VSI), and physiological- biochemical indices such as blood glucose levels, serum lipid levels, muscle fatty acid composition and lipase and amylase activities will also be investigated. This research will reveal the mechanism of lipid metabolism in herbivorous fish at the molecular levels, and to provide the theoretical basis for lipid metabolism regulation in fish nutrition.

脂肪代谢与养殖动物的生长和健康关系密切，脂肪过度沉积不仅影响肉质、降低饲料利用率，还会引发营养性脂肪肝等疾病。因此，通过调控脂肪代谢来降低机体脂肪沉积已成为动物营养学研究一个重要课题。本项目以我国重要养殖鱼类草鱼为研究对象，对其脂肪合成、转运和分解代谢中8个关键酶基因进行研究。首先，采用RT-PCR技术克隆这些基因全长cDNA；然后，采用实时定量PCR技术检测这些基因在草鱼不同发育阶段和不同组织中的时空表达；接着，静脉注射葡萄糖、降脂药物或腹腔注射siRNA，定量检测处理后这些基因mRNA表达量的变化，解析基因功能；最后，用3种不同淀粉源及脂肪源日粮喂养草鱼8周，采用实时定量PCR技术，研究淀粉和脂肪种类对8个基因表达的影响；同时，测定鱼体增重率、饲料系数、肝体指数、血脂血糖水平、脂肪酶活力等生理生化指标。从分子水平上揭示营养素调控脂肪代谢规律，为养殖鱼类脂肪代谢营养调控提供理论依据。

采用RT-PCR和RACE技术从草鱼肝胰脏中克隆了乙酰辅酶A羧化酶（ACC1）、葡萄糖激酶（GK）、脂蛋白脂肪酶（LPL）、6-磷酸果糖激酶（PFK）、丙酮酸激酶（PK）、丙酮酸脱氢酶（PDH）、长链脂酰辅酶A合成酶（ACSL1）和肉碱脂酰转移酶Ⅰ（CPTⅠ）等8个脂肪代谢关键酶基因全长cDNA。结果表明，草鱼ACC1基因cDNA全长7515bp，开放阅读框（ORF）7173bp，主要在肝胰脏中表达。ACSL1a基因全长3129 bp，ORF 2094bp，主要在心脏、肠系膜脂肪和全脑中表达；ACSL1b基因全长2468bp，ORF 2094bp，主要在心脏和肌肉中表达。CPT-I基因cDNA全长2513bp，ORF 2322bp，主要在全脑中表达。GK基因cDNA全长2039bp，ORF 1422bp，主要在肝胰脏中表达。LPL基因cDNA全长2118bp，ORF 1542bp，主要在肠系膜脂肪组织和肝胰脏中表达。PDH基因cDNA全长1662bp，ORF框1182bp，主要在全脑和心脏中表达。PFK基因cDNA的全长2609bp，ORF 2343bp，主要在全脑中表达。PK基因cDNA全长1806bp，ORF 1584bp，主要在肝胰脏中相对表达。设计4组日粮，分别添加5%的鱼油、豆油、猪油为脂肪源，羧甲基纤维素为对照，饲喂76.7±1.2g/尾的草鱼12周，探讨不同脂肪源对草鱼生长及肝胰脏脂肪代谢相关基因表达的影响。结果表明：鱼油组和豆油组增重率显著高于猪油组和对照组；不同脂肪源对GK、PDH基因表达没有影响，对CPTⅠ、PFK和PK基因表达有一定影响，但差别不大，日粮中添加鱼油、猪油和豆油均显著抑制ACC1、ACSL1a、LPL基因表达。设计4组日粮，分别添加25%的玉米淀粉、马铃薯淀粉和小麦淀粉为淀粉源，羧甲基纤维素为对照，饲喂规格39.4±1.6g/尾的草鱼12周，探讨不同淀粉源对草鱼生长、肝胰脏脂肪代谢相关基因表达的影响。结果表明：马铃薯淀粉组和小麦淀粉组的增重率显著高于玉米淀粉组和对照组；不同淀粉源对ACC、CPT、LPL和PK等基因表达没有影响，对ACSL1a、PDH和PFK基因表达有一定影响，但差别不大，日粮中添加淀粉极显著诱导GK基因表达，小麦淀粉组GK基因表达量是对照的3546倍。