Genetic Polymorphism Analysis And Effect On Lipid Metabolism Of SIRT1 And SIRT3 Genes In Guizhou White Goat

SIRT1 and SIRT3 are the members of the family of silencing information regulators,and they have similar structures.They are highly conserved class nicotinamide adenine dinucleotides(NAD+)-dependent deacetylatases,which are widely distributed and active in animals.As a local goat breed in Guizhou,Guizhou white goat plays an important role in the local meat goat industry.In this experiment,Guizhou white goat was used as the research object,and SIRT1 and SIRT3 genes were the target genes.Firstly,we screened the SNPs of SIRT1 and SIRT3 genes by direct sequencing,analyzed genetic polymorphisms,and performed association analysis with serum levels of triglycerides,cholesterol,and fatty acids,three lipid metabolism indicators.Then,we detected the expression levels of SIRT1 and SIRT3 genes in different tissues by real-time fluorescence quantitative PCR technology.In addition,we isolated and cultured primary goat adipocytes,constructed eukaryotic expression vectors of SIRT1 and SIRT3 genes,and transfected the cells.Cell proliferation was detected by CCK-8 method,cell scratching,and proliferation-related gene methods.Finally,we used the expression levels of differentiation-related genes to detect the effects of SIRT1 and SIRT3 genes on cell differentiation.The findings are as follows:(1)We detected 11 SNPs of the SIRT1 gene in Guizhou white goats.Among them,the genetic balance of g.21207042C>T and g.21217404T>C mutation sites deviates from the Hardy-Weinberg equilibrium state,and the m RNA secondary structure and minimum free energy of the mutation sites are changed.There are strong linkage disequilibrium effects between g.21206621T>C,g.21206920T>G,g.21207071A>C,g.21215871G>A,g.21216960T>C,g.21217717T>C and g.21217777A>G,and different genotypes were associated with three lipid metabolism indexes.We detected 6 SNPs of the SIRT3 gene in Guizhou white goats.The genetic balance of the g.42501C>A locus population deviates from the Hardy-Weinberg equilibrium state,and the m RNA secondary structure,morphology and minimum free energy of all loci are changed.g.42500T>C and g.42501C>A site,g.58062G>A,g.58066A>G,g.58166G>A site have a strong chain imbalance effect.However,there was no association with lipid metabolism indicators.(2)The SIRT1 and SIRT3 genes of Guizhou white goat were expressed in different tissues,and the expression in the liver was significantly higher than that of other tissues(P<0.01);no base mutations were found after cloning of SIRT1 and SIRT3 genes in Guizhou white goat.Bioinformatics analysis shows that SIRT1 and SIRT3 proteins are mainly composed of irregular curls and α-helixes.The SIRT1 is an unstable non-transmembrane protein,and the SIRT3 latter is a stable non-transmembrane protein.They all have the closest genetic distance to sheep,indicating that SIRT1 and SIRT3 mainly regulate fat metabolism in the liver.(3)After transfection of eukaryotic expression vectors p EGFP-N3-SIRT1 and p EGFP-N3-SIRT3 to primary adipocytes,it could significantly upregulate the expression of SIRT1 and SIRT3 transcriptional levels in primary adipocytes(P<0.01),and CCK-8 and cell scratches showed that SIRT1 and SIRT3 genes could inhibit the proliferation of primary adipocytes,and significantly inhibited the expression of PCNA,Cyclin E,Cyclin D1,PPARγ,AP2,C/EBPα gene(P<0.01).In summary,SIRT1 and SIRT3 genes may inhibit the growth and development of goat adipocytes by inhibiting fat proliferation and differentiation-related genes,and affect the level of serum fat metabolism,thereby becoming candidate genes that affect goat fat metabolism and meat quality traits.In summary,SIRT1 and SIRT3 genes may hinder the growth and development of goat primary adipocytes by inhibiting fat proliferation and differentiation-related genes,affecting the level of serum lipid metabolism,and then becoming candidate genes affecting goat fat metabolism and meat quality traits.