Expression Regularity And Polymorphism Analysis Of Chicken SIRT1 Gene

SIRT1,a NAD~+-dependent histone deacetylase,is the mounting studies member of the mammalian sirtuin family.SIRT1 regulates physiological processes in mammalian,especially fat metabolism,glucose Metabolism,apoptosis and insulin secretion.The liver is important critical tissues that regulate fat metabolism,which was closely related to egg nutrition and laying performance.In mammals,SIRT1 was found to repress lipid synthesis and promote lipolysis.As a star gene in mammal,What is the expression pattern in poultry? We found that expression of SIRT1 is up-regulation after visfatin treatment in chicken embryonic primary hepatocytes.To further investigate the biological characteristics on lipid metabolism,q PCR was performed to explore its expression levels in different tissues.Meanwhile,We analyse the SNP polymorphism and economic trait in F2 population of Gushi chickens and Anka chickens.To explore the effect of different genotypes on SIRT1 expression in breast muscle,leg muscle,adominal fat and sebum.Finally Dual-luciferase reports was performed to inspect the SIRT1 transcriptional activity of different haplotype.We get results as follows:1.We found that expression of SIRT1 was up-regulation after visfatin treatment in chicken embryonic primary hepatocytes(P <0.05).The expression of SIRT1,ap2,PPARγ were low and no significant changes in 0-4d.But they were significantly increased at 6d and no significant changes in 6d and 8d.SIRT1 was generalized expression in different tissues in 75 week chickens,the highest expression levels of the heart,liver and luns were significantly different with other tissues(P<0.05).The second were breast muscle,leg muscle,kidney,hypothalamus,the lowest expression is sebum and abdominal fat.2.By means of DNA mixed pool sequencing,two mutations in the 5 ’UTR region of SIRT1 gene were identified,the g.-1552_-1553 ins was a 2bp indel that CG insertion,the g.-450_-451 del was 2bp indel that GC deletion.Analyse with F2 population of Gushi chickens and Anka chickens,g.-1552_-1553 ins was significantly correlated with breast muscle fiber length and DD individuals are significantly longer than II individuals(P <0.05).g.-450_-451 del was significantly correlated with leg muscle fiber length,leg muscle fiber short diameter,leg muscle fiber circumference,leg muscle fiber area ratio and II individuals was significantly longer than ID individuals(P <0.05).It is suggested that SIRT1 participates in the growth process of muscle fiber.In addition,g.-1552_-1553 ins was significantly correlated with cholesterol and low density lipoprotein(P <0.05).g.-450_-451 del was significantly correlated with sebum weight,subcutaneous fat thickness and alkaline phosphatase.DD individuals were higher than II and ID in two indels.It is suggested DD genotypes was dominant genotypes in fat-related traits,It is speculated that SIRT1 is involved on the transport of fat in the blood and the metabolism of sebum.3.At g.-1552_-1553 ins site,the expression of SIRT1 in genotype II and ID was significantly higher than DD in breast muscle(P <0.05);The expression of SIRT1 in ID and II genotype was higher than DD in the leg muscle(P <0.05);ID and II genotype were higher than DD in sebum tissue and abdominal fat(P <0.05).At g.-450_-451 del site,the expression of SIRT1 in genotype ID was significantly higher than DD and II in breast muscle(P <0.05);The level of SIRT1 in genotype ID and DD was higher than II in the leg muscle(P <0.05);The expression of SIRT1 in genotype ID and II was higher than DD in the sebum tissue(P <0.05);The level of SIRT1 in genotype ID was significantly higer than II and DD in abdominal fat(P <0.05).These results show that the different genotype can influence the expression of SIRT1 and DD type SIRT1 expression in adipose tissue volume is the lowest.So we hypothesized that SIRT1 could reduce the weight and thickness of sebum,reducing the amount of cholesterol and LDL in the blood.4.Dual-luciferase reports was performed to inspect the SIRT1 transcriptional activity of different heplotypes and transfected 293 T cell lines.We found the activity of SIRT1 was highest in heplotype IIII that was 1.5 fold than other heplotypes(P <0.05);The transcription activity of SIRT1 was no significant difference in heplotypes DDDD,DDII and IIDD(P >0.05).Bioinformatics analysis found that Indels mutations which in SIRT1 gene 5 ’UTR region that affect the change of transcription factor binding sites and leads to a change in transcription activity of SIRT1.Meanwhile,double fluorescent carrier report result also verifies the different Indels genotype influence SIRT1 expression.From the above results,SIRT1 involved in former adipocyte differentiation process.Not only reduce the weight and thickness of the leather fat,but also reduce the content of cholesterol and low density lipoprotein in the blood.Moreover,the two Indels of the 5 ’UTR that by affecting the binding site of transcription factors and lead to the change of the activity of SIRT1,and its role in fat metabolism in chicken is worth further exploration.