草鱼SREBP-1基因的克隆及糖对其在肝脏中表达的影响

Molecular cloning of SREBP-1 gene and effects of carbohydrates on its expression in liver of Ctenopharyngodon idella

固醇调节元件结合蛋白（SREBPs）是调控糖脂代谢相关基因表达的关键核转录因子。为获知草鱼SREBP-1基因的序列及其在肝脏中的表达规律,本实验采用同源克隆和RACE方法获得了草鱼SREBP-1基因的部分cDNA序列,并通过生物信息学方法对该基因及所编码蛋白的结构特征进行了分析;采用实时荧光定量PCR技术,对SREBP-1基因在8种不同组织的表达规律及低糖（糖含量24%）和高糖（糖含量42%）投喂条件下肝脏中的表达水平进行了研究。结果显示,所克隆到的草鱼SREBP-1基因cDNA长4 760 bp,其中包括开放读码框3 426bp,编码1 141个氨基酸;草鱼SREBP-1具有一个典型的碱性螺旋-环-螺旋亮氨酸拉链结构（bHLH-zip）;氨基酸序列比对结果显示,草鱼SREBP-1与其他鱼类的同源性在76%~88%之间,与斑马鱼的进化关系最近;草鱼SREBP-1基因在脑中的表达量最高,肝脏和肠次之,在肾脏、脾脏、肌肉、脂肪和性腺中均有少量表达;与对照组相比,SREBP-1基因在高糖诱导下表达量显著提高（P〈0.05）,低糖诱导下没有显著性差异。研究表明,在高糖负荷条件下,草鱼肝脏中SREBP-1可能会促进糖的利用和转化,从而参与糖代谢调节过程,为丰富鱼类糖代谢调控机理提供研究资料,并有望为提高鱼类对饲料糖的利用效率提供理论依据。

Sterol regulatory element binding proteins（ SREBPs） are nuclear transcription factors critical for regulating the expression of genes related to glucose and lipid metabolism. In order to investigate the molecular regulatory mechanisms of lipid metabolism and liver lipid accumulation in fish,the partial cDNA sequence of sterol regulatory element binding protein 1（ SREBP-1） from grass carp has been identified by homology cloning and rapid amplification cDNA ends（ RACE）,and the structural features of the gene and its encoded protein have been analysed by bioinformatics methods. Using the method of real-time PCR,the expression pattern of SREBP-1 in eight different tissues of grass carp has been studied,and the expression levels of SREBP-1 in liver of grass carp under different feeding conditions with low-carbohydrates（ carbohydrates content 24%） and high carbohydrates（ carbohydrates content of 42%） have been studied. The result revealed that the obtained cDNA of SREBP-1 in grass carp is 4 760 bp in length,which consisted of a3 426 bp open reading frame（ ORF）,encoding 1 141 amino acids. The amino acid sequence has a typical basic helix-loop-helix leucine zipper（ bHLH-zip）. Alignment based on amino acid sequence showed that grass carp SREBP-1 had a homology of 76%- 88% to its counterparts of other fishes. Phylogenetic analysis showed that grass carp SREBP-1 clustered with that of zebrafish. The SREBP-1 mRNA could be detected in all the examined tissues of grass carp. But the expression level in brain was the highest,followed by liver and intestines,and with a little expression in the kidney,spleen,muscle,fat and gonad tissues. Compared with the control,the expression level of SREBP-1 in high carbohydrates（ 42%） induced group increased significantly（ P 〈 0. 05）. The results showed that SREBP-1 gene may promote the utilization and in version of carbohydrates in grass carp under high carbohydrates loading conditions,which suggested that it may improve the utilization of carbohydrates in grass carp and be involved in its carbohydrates metabolism process. The research data can enrich the glucose metabolism regulation mechanism of fish,and are expected to provide a theoretical basis for improving the utilization efficiency of sugar in fish feed.