Cloning, Expression And Snps Analysis Of Two Genes Encoding Lipoprotein Lipases From Micropterus Salmoides

Largemouth bass（Micropterus salmoides） is a eurythermal carnivorous species of fish andnative to the North America. Owing to its broad adaptablity, fast-growing and delicious taste, itwas introduced into China, and the breeding area and yield have reached high levels. However,largemouth bass are susceptible tofatty liver, especially when they were fed on artificial diets.The cDNA of two lipoprotein lipase (LPL) genes from largemouth bass are cloned by PCRand RACE technology. The sequence analysis indicates that LPLtype1cDNA is2156bp inlength, encoding516amino acids; and LPLtype2cDNA is1710bp in length, encoding346amino acids. The amino acid sequence of LPLtype1is43.5%identified to that of LPLtype2.Phylogenetic analysis indicates that LPLtype1of M. salmoides and LPL of Sinipercachuatsigather to one branch. LPLtype2of M. salmoides and LPLtype2of Oncorhynchus clarkii gatherto the other branch. The predictive analysis of the protein encoded by LPLtype1and LPLtype2genes in M. salmoide shows that the main functional regions are conservative compared with thebony fishes and other vertebrates, such as active site residue, N-glycosylation site, conservedhydrophobic interaction site, heparin binding region. Speculating that LPL type1and LPL type2play an important role in lipid metabolism.The tissue expression levels of LPL mRNA are detected by real time PCR, and the resultsshow that expression levels of LPLtype1and LPLtype2are the most in liver, which suggests thatfish liver plays essential roles in storing lipids induced by nutrition.The study also detected the effects of different fat levels in compound diets on theexpression levels of two lipoprotein lipase genes mRNA in largemouth bass liver. The fat levelsof the ice fresh fish, A compound diets, B compound diets are4.4%,8.2%,14.5%. The resultsshow that the expression levels of LPLtype1mRNA in group which were fed on A and Bcompound diets are1.3times and1.8times of those in group which were fed on ice fresh fish,respectively. And the expression levels of LPLtype2mRNA in group A and B feeding on mixedfeed are2.7times and3.1times of that in group which were fed on ice fresh fish. These dataindicate that the expression levels of LPLtype1and LPLtype2mRNA in livers of largemouth bass fed on compound diets are all higher than those fed on ice fresh fish, and the expressionlevels were highest in group B fed on compound diets with the highest concentration of fat.When the largemouth bass were dissected to collect their liver tissue, varying degree liverpathological changes were detected in the fish fed on compound diets. Speculating that LPLplays an important role in lipid metabolism of liver and has association with the formation of thefatty liver.In order to analyze the differences between LPLtype1and LPLtype2, all gene sequences oftwo lipoprotein lipases from largemouth bass were cloned using directly sequencing method toscreen SNP sites. Thirteen SNP s were detected in LPLtype1and six SNP sites were detected inLPLtype2. The19SNP sites locate in introns and exons. These SNPs were genotyped in acultured largemouth bass population, and the correlation analysis between growth traits (bodyweight, full length, body length, body height, caudal peduncle length, caudal peduncle height)and SNP sites was performed.