草鱼AdipoR1-B基因克隆、组织分布及其表达的营养调控

Molecular cloning, tissue distribution and nutritional regulation of AdipoR1-B in grass carp(Ctenopharyngodon idella)

为探讨Adipo R1-B在鱼类糖类和脂质代谢中的作用,本实验采用RACE方法获得了草鱼Adipo R1-B的全长c DNA序列(登录号:KP733846),利用生物信息学技术对该基因及其所编码蛋白的结构特征进行了分析;采用实时定量PCR技术,检测了Adipo R1-B在19个不同组织中的表达特性以及高糖(45%)、高脂(8%)和高糖高脂饲喂对草鱼肝脏中该基因表达的影响。结果显示,草鱼Adipo R1-B c DNA全长2186 bp,其中开放读码框为1122 bp,编码373个氨基酸;跨膜结构分析表明草鱼Adipo R1-B为典型的7次跨膜蛋白;同源性分析结果显示,草鱼Adipo R1-B与其他物种的Adipo R1-B高度同源(氨基酸相似度78%以上),并与斑马鱼Adipo R1-B的进化关系最近;组织分布结果显示,Adipo R1-B在草鱼肝脏中表达量最高,中枢神经系统和红肌次之;此外,高脂和高糖高脂饲喂均能够显著提高草鱼肝脏中Adipo R1-B的表达水平。因此,草鱼肝脏中Adipo R1-B的表达水平受到日粮中脂类水平的调控,推测该受体可能在调节鱼类脂质代谢中发挥重要作用。

Adiponectin is an abundantly secreted adipokine from the adipose tissue of mammals, and plays central roles in the regulation of glucose and lipid metabolism. The biological function of adiponectin is mediated by at least two putative receptors（AdipoR1 and AdipoR2）. In order to investigate the molecular regulatory mechanisms of lipid metabolism and liver lipid accumulation in fish, the full-length cDNA sequences of adiponectin receptor1-B（AdipoR1-B） from grass carp has been identified by rapid amplification cDNA ends（RACE）, and the structural features of the gene and its encoded protein have been analysed through bioinformatics methods. Using the method of real-time PCR, the expression pattern of AdipoR1-B in nineteen different tissues of grass carp has been studied,and the expression levels of AdipoR1-B in the liver of grass carp under different nutrient conditions with high carbohydrate（H-CHO）, high lipid（H-LIP） and high carbohydrate high lipid（H-CHO H-LIP） have been studied. The result revealed that the obtained c DNA of AdipoR1-B in grass carp was 2186 bp in length, which consisted of a 1122 bp open reading frame（ORF）, encoding 373 amino acids. Alignment based on amino acid sequences showed that grass carp AdipoR1-B had a homology of 81.0%–94.1% to its counterparts in other fishes.Phylogenetic analysis showed that grass carp AdipoR1-B clustered with that of zebrafish. The AdipoR1-B m RNA could be detected in all the examined tissues of grass carp. But the expression level in liver was the highest,followed by central nervous system and red muscle. Compared with the control group, the expression levels of AdipoR1-B in the H-LIP and H-CHO H-LIP groups had increased significantly. This result suggested that adiponectin may improve the utilization of lipid in grass carp and it may be involved in the lipid metabolism process in fish. The research data can enrich the knowledge of lipid metabolism regulation mechanism of fish, and lay a foundation for the further functional research of the adiponectin/AdipoRs system in fish.