The Genetic Variation And Expression Of Lpin2 And Lpin3 Genes And Their Associations With Traits In Sheep

Lipin family proteins act as lipid phosphatase enzymes in triglyceride synthesis and catalyze the dephosphorylation of phosphatidic acid to form diacylglycerol, they can also act as the transcriptional co-activator to regulate the gene expression related to the lipid metabolism. Mammalian lipin proteins consist of lipin 1, lipin2 and lipin3 and are encoded by their respective genes Lpin1, Lpin2 and Lpin3. To date, most studies focus on Lpinl, only a few have addressed Lpin2 and Lpin3. No studies concerned with Lpin2 and Lpin3 are found in sheep. Studies on the structures, genetic variation, expression of Lpin2 and Lpin3 and their associations with traits would help to explore their genetic characteristics and function mechanisms in lipid or other possible metabolic pathways in sheep.In this study, the RT-PCR,5'-RACE and sequencing technologies were used to amplify coding sequence (CDS) of Lpin2 gene in sheep, the CDS and corresponding amino acid sequences profiles were analyzed by bioinformatics methods. Genetic polymorphism of 5'-regulatory region in Lpin2 and Lpin3 in 96 animals from two sheep breeds with phenotypic differences in tail fat phenotype (Guangling Large Tailed and Small Tailed Han Sheep) was detected using the direct sequencing methods, haplotypes were constructed across SNPs loci and associations between genotypes (haplotypes) and tail and slaughter traits were analyzed. Meanwhile, two sheep breeds totaling 96 were chosen to study the ontogenetic expression of Lpin2 and Lpin3 mRNA in seven different tissues (liver, kidney, small intestines, femoral biceps, testis and ovary, perirenal fat, tail fat) by quantitative real-time PCR, associations between gene expression and tail and slaughter traits were also analysed.10 months old GLT were chosen to examine the protein expression of lipin2 and lipin3 in seven different tissues (liver, kidney, small intestines, femoral biceps, testis, perirenal fat, tail fat) by western blotting technique. Main research results were as follows:(1)The full length of CDS of Lpin2 in sheep was 2670bp, encoding protein lipin2 was an unstable, neutral and hydrophilic fat-soluble protein that 889 amino acid residues composed. Lipin2 may not have signal peptide but have a bidirectional alpha helical transmembrane structure, and may reside in the cytosol and the nucleus. Proteins disulfides might be formed by the reactive oxygen species (ROS) produced by mitochondria in cytoplasm by the mechanism of cell redox status regulation. Lipin2 protein may not have potential N-glycosylation sites but have the potential O-glycosylation sites which might be glycosylated in the cytoplasm. Lipin2 protein may have 89 phosphorylation sites, conserved site of Ser106 was predicted to be phosphorylated. Secondary structures such as alpha helix and extended strand and the buried status of amino acids solvent accessibility were found to locate in the conserved domains of Lipin-N and LNS2. A peptide sequence located in the LNS2 domain was found to form tertiary structures. The similarities of Lpin2 CDS and amino acid sequences between sheep and other 14 vertebrates were high, however, which was low between lipin 1 and lipin2 in sheep.(2) Three novel SNPs were found within 5'-regulatory region of Lpin2 gene about 1200bp upstream sequence from start codon ATG, namely duplication mutation of SNP1 (-663 dup ATT), transtions of SNP2 (-388 T>C) and transversions of SNP3 (-330 T>G), sequencing data revealed that the trinucleotide ATT repeat expansion at SNP 1 locus was cope number variation (CNV) of short tandem repeats (STR), which caused the significant decreasing in carcass weight and dressing percentage in GLT (P<0.05).13 novel SNPs were discovered within 5'-regulatory region of Lpin3 about 1200bp upstream sequence from start codon ATG, they are SNP1 (-815 T>G), SNP2 (-788 C>G), SNP3 (-753 G>A), SNP4 (-725 C>T), SNP5 (-699 T>G), SNP6 (-613 A>G), SNP7 (-604 A>G), SNP8 (-443 T>C), SNP9 (-334--332 ins TAA), SNP10 (-189 OT), SNP11 (-136 G>A), SNP12 (-95 A>G) and SNP13 (-88 G>A), which include 9 transtions,3 transversions and one trinucleotide insert mutation. SNP 1-7 could be constructed to the haplotype block in GLT and STH according to the Linkage disequilibrium (LD) tests, respectively. However, SNP 10-13 could be constructed to the haplotpye block only in GLT. Diplotypes from haplotypes didn't exhibit significant genetic effects on tail and slaughter traits in sheep (.P>0.05). SNP9 insertion mutation significantly decreased the tail width in STH (P<0.05). SNP8 point mutation didn't significantly alter the tail and slaughter traits in GLT and STH, and SNP 10-13 didn't significantly affect the tail and slaughter traits in STH sheep breed (P>0.05).(3) Sheep Lpin2 mRNA was highly expressed in perirenal and tail fats, and was also substantially expressed in liver, kidney, reproductive organs (testis and ovary), with the lowest levels in small intestine and femoral biceps (P<0.001). Sheep Lpin3 mRNA was prominently expressed in liver and small intestine, and was also expressed at high levels in kidney, perirenal and tail fats as well as reproductive organs (testis and ovary), with the lowest level in femoral biceps (P<0.001). Global expression of Lpin2 and Lpin3 in GLT both were significantly higher than those in STH (P<0.05). Spatiotemporal expression showed that the highest levels of Lpin2 expression occurred at 10 months of age in two breeds of sheep, with the lowest expression at 2 months of age in STH and at 8 months of age in GLT (P<0.001). The greatest levels of Lpin3 expression occurred at 4 months of age in STH and at 10 months of age in GLT, with the lowest expression at 12 months of age in STH and at 8 months of age in GLT (P<0.01). Breed and age significantly influenced the tissue expression patterns of Lpin2 and Lpin3, respectively (.P<0.001), and sex significantly influenced the spatiotemporal expression patterns of Lpin3 (P<0.01). Meanwhile, Lpin2 and Lpin3 mRNA expression both showed significant correlations with tail and slaughter traits (P<0.01, P<0.05), and the associations appear to be related with the potential functions of lipin2 and lipin3 in sheep.(4) The expression of lipin2 protein in seven different tissues in 10 months old GLT was similar to that of mRNA in the corresponding tissues. Lipin3 was highly expressed in these seven tissues and the protein expression abundance was not significant different among these tissues, the expression of lipin3 protein was not consistent with that of mRNA.Studies of Lpin2 and Lpin3 based on DNA, mRNA and protein levels would lay the foundation for further exploring the potential function mechanism of the two genes in sheep.