The Cloning, Polymorphism, Expression Of Lpin1Gene And Their Associations With Traits In Sheep

Lpinl gene plays an important role in fat metabolism of the body. Lpinl not only serves as an enzyme direct participating glycerolipid and phospholipids biosynthesis, it also as a transcriptional coactivator indirectly regulates expression of the genes related with lipid metabolism. Studies on the structures, genetic variation, expression of Lpinl gene and their associations with traits can be useful for understanding their complex fat metabolic mechanism in the body. The fat metabolism pathway may be distinct in different fat-tailed sheep. However, at present, no report is found about its nucleotide sequences, molecular structures, polymorphism and expression.In this study, the gene sequences characteristics, genetic variation, mRNA and protein expression of sheep Lpinl gene were studied at DNA, mRNA and protein levels, respectively. First, we used RT-PCR techniques to amplify coding sequence (CDS) of Lpinl gene and sequencing. Second, the CDS and corresponding amino acid sequences profiles was analyzed by bioinformatics methods. Third, the genie polymorphism of the5’regulatory region in96animals from two sheep breeds (Guangling Large Tailed Sheep and Small Tailed Han Sheep) was detected using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. Associations between genotype and tail type and slaughter traits were analyzed. Fourth, two sheep breeds was used to study the ontogenetic expression of Lpinl mRNA and protein in seven different adipose tissues by quantitative real-time PCR and western blotting technique. Associations between mRNA and traits were analyzed. The main results of studies were as follows:(1)The full length of CDS sequences of sheep was2688bp and encoding895amino acids. Lpinl gene encoding protein was named Lipinl. Lipinl was an unstable hydrophilic fat-soluble protein and had no signal peptide and transmembrane domain but with5potential O-glycosylation sites and5N-glycosylation sites and88predicted phosphorylation sites. Lipinl protein amino and carboxyl terminal contain a highly conserved region was Lipin-N and LNS2, respectively. The similarities of CDS and amino sequences between sheep and most species were higher. (2)9SNPs loci were detected in the5’ regulatory region, ie-2484G>A,-217OC>T,-1782A>G,-1717A>C,-1237C<A,-1218C>T,-534C>T,-474X>G, and-227C>T. Among these had six transition and3transversion. Correlation analysis about genotype and traits showed different genotypes of P1primers amplified fragments had a significant influence on absolute tail fat weight and relative tail fat weight (P<0.05). Different genotype of P4primers and P9primers amplified fragments had a significant influence on long tail and tail respectively (P<0.05). These results indicated Lpinl genes have certain influence on the sheep tail type and tail fat deposition, it provides a theoretical basis for marker assisted selection of the sheep meat quality traits.(3)The Lpinl mRNA expressed in all seven tissues of sheep and had a significant difference (P<0.001). The perirenal fat Lpinl mRNA expression was the highest, followed by the tail fat. Expression was lowest in mesenteric fats and significantly lower than other tissue. The expression of Guang Large Tailed Sheep was significantly (P<0.001) higher than the Small Tailed Han Sheep. Significant difference between the months of age (P<0.01) with the highest expression in10months of age and the lowest expression in8months of age had a significant impact on the Lpinl mRNA expression. The breed by sex and breed by tissue interactions significantly affected the Lpinl mRNA expression. The age by breed and age by tissue interactions had a significant impact on the Lpinl mRNA expression, too. Associates analysis between mRNA and traits indicated that different association degrees existed for differern tissues in two breed. These results suggest that genetic expression of Lpinl genes may play different roles in fat metabolic pathways in different fat-tailed sheep.(4)Breed and sex had a significant impact on Lipinl protein expression. The age by sex and age by breed interactions significantly affected the Lipinl protein expression. Although Lipinl protein expression on the whole consistent with the expression of mRNA, but not linear.In summary, this study lay the scientific foundation for revealing the genetic regulation mechanism of Lpinl gene in fat metabolic pathways of different fat-tailed sheep through the systematic study on the sheep Lpinl gene at different levels.