大口黑鲈两种脂蛋白脂肪酶基因cDNA的克隆及表达特征分析

Cloning and expression analysis of two cDNAs encoding lipoprotein lipase of Micropterus salmoides

为促进肉食性鱼类人工配合饲料开发的理论基础研究,分析鱼类脂肪代谢的机制,实验克隆了大口黑鲈2个脂蛋白脂肪酶基因LPLtype1和LPLtype2的cDNA。序列分析表明,LPLtype1基因cDNA序列全长2 156 bp,编码516个氨基酸;LPLtype2基因cDNA序列全长1 710 bp,编码346个氨基酸。大口黑鲈LPLtype2与LPLtype1氨基酸序列之间的同源性为43.5%。系统进化分析表明,大口黑鲈LPLtype1和鳜LPL聚为一支,大口黑鲈LPLtype2和大麻哈鱼LPLtype2紧密聚为一支。预测分析发现,大口黑鲈LPLtype1和LPLtype2基因编码蛋白的活性中心位点、N-糖基化位点、二聚体形成的保守疏水残基位点、肝素结合域等主要功能域与硬骨鱼类和其他脊椎动物对比都比较保守。运用实时定量PCR方法检测脂蛋白脂肪酶mRNA的组织分布,发现LPLtype1和LPLtype2都在肝脏中表达量最高,推测这与肝脏是最主要的营养诱导性储脂部位有关。

To promote the theoretical basic research on developing artificial feed for carnivorous fishes, and the analysis of fat metabolizing mechanism in fish, the cDNA of LPLtypel and LPLtype2 genes from largemouth bass（Micropterus salmoides）are cloned. The sequence analysis indicates that LPLtypel cDNA is 2 156 bp in length, encoding 516 amino acids; and LPLtype2 cDNA is 1 710 bp in length, encoding 346 amino acids. The amino acid sequence of LPLtypel is 43.5% identified to that of LPLtype2. Phylogenetic analysis indicates that LPLtypel of M. salrnoides and LPL of Siniperca chuatsi gather to one branch. LPLtype2 of M. salmoides and LPLtype2 of Oncorhynchus clarkii gather to the other branch. The predictive analysis of the protein encoded by LPLtypel and LPLtype2 genes in M. salmoides shows that the main functional regions are conservative compared with the bony fishes and other vertebrates, such as active site residue, N-glycosylation site, conserved hydrophobic interaction site, heparin binding region. The tissue expression levels of LPL mRNA are detected by real time PCR, and the results show that expression levels of LPLtypel and LPLtype2 are the highese in liver, which suggests that fish liver plays an essential role in storing lipids induced by nutrition.