Study On Genetic Variation And Its Effects And Expression Of Visfatin And PRDM16 Gene In Chickens

With the rapid development of molecular biology, many works on searching major genes or closely linked molecular markers of growth and production traits from DNA level and on studying the gene functions have been carried out in recent years. The approaches of marker-assisted selection and transgenic breeding have become the future trend in poultry breeding. Visfatin and PRDM16 are the newly discovered genes and they play an important role in determination of fat traits, no information on genetic variation and functional studies of chicken visfatin and PRDM16 genes is available. In this study, the genetic variations of visfatin and PRDM16 genes were detected by single stranded conformation polymorphism and DNA pool sequencing. SNPs of visfatin and PRDM16 genes were genotyped in an F2 resource population of Gushi chickens crossing with Anka broilers using forced-PCR-RFLP. Haplotypes were constructed based on 3 SNPs with linkage disequilibrium of visfatin gene. The associations of 9 SNPs polymorphisms with 84 parameter in 849 F2 chickens were analyzed. The coding sequence of Silky Chicken's visfatin gene was cloned and analyzed by bioinformatics methods. Using real-time PCR techique, the mRNA of visfatin and PRDM16 gene in heart, liver, kidney, spleen, breast muscle, subcutaneous fat, viscera fat and bone marrowfrom broiler and silky fowl were analyzed. Expressions of heart, liver, bone morrow, sebum and leg muscle from silky fowl at 1-day, 4, 8, 12, 16 weeks old were also investigated. The goal of this study is to identify the sequence variations of the visfatin and PRDM16 gene, to analyze the relationship between gene variation and chicken performance, and to find molecular markers for chicken genetics and breeding programs. In addition, it would provide theoretical basis for studying visfatin and PRDM16 functions and diagnosing and treating the obesity. The main results were summarized as below:1.Genetic variations of visfatin gene and their associations with chicken production traitsTwelve SNPs were found in visfatin gene with the location of intron 3 (14360C>T), exon 4 (14404A>T), exon 6 (15526C>T), intron 6 (15673C>G), exon 7 (17873C>T), intron 7 (17928T>C), intron 8 (24019A>G), intron 9 (4238T>C, 24262G>T, 24285A>G, 24294A>G) and intron 10 (9 bp Indel), respectively. Except for 14404A>T, 17873C>T and 24262G>T, other 9 SNPs were newly discoverd, of which eight SNPs were genotyped. Three were main haplotypes including H1(ACG), H2(ACT) and H8(TTT) and 5 main diplotypes based on three SNPs with strong linkage disequilibrium.14404A>T polymorphisms was significantly associated with body weight and body size traits at different age (P<0.05). For the traits body weight and carcass weight with genotype TT were significantly higher than those of genotype AA. 15526C>T polymorphisms was associated with body weight at birth and at 4 weeks, and with breast angle at 8 weeks. 17873 C>T polymorphisms was associated with the early growth traits. The growth traits at 4 weeks of the individual with the genotype TT was higher than those with genotype CC (P<0.05). 24238 T>C polymorphisms was also associated with growth traits and genotype CC is a favorable genotype for chicken growth and development. The genotype TT of 24262G>T polymorphisms was a favorable gene for chicken early growth traits and breast muscle weight. 24285A>G polymorphism was associated with growth traits at 8 weeks. 24294A>G polymorphisms was associated with chest depth at 8 weeks, pelvis breadth at 4 weeks and12 weeks, skin fat thicknessand and low density lipoprotein cholesterol (P<0.05). 9 bp Indel polymorphisms was associated with growth traits and pancreas weight, allele D (9 bp deletion) of the vistafin gene had a negative effect on skeletal growth. The five main diplotypes was associated with breast angle at 8 weeks, breast muscle fiber density and low density lipoprotein cholesterol (P<0.05). The genotype combination TTTTTTIICCTTGGAA had a positivie effects on chicken growth and development. The results has affirmed that there is a close relationship between visfatin gene and chicken growth and development..2.Genetic variations of PRDM16 gene and associations with production traitsSix novel SNPs of the PRDM16 gene were found and 92188G>A and 115305C>T was located at non-cording region, 102872C>T, 102944C>T, 103144G>A and 115512G>A located at cording region. The polymorphism of the 115512G>A was significantly associated with the fatness and carcass traits and the genotype AA is an optional genotype for decreasing abdominal fat weight and triglyceride (P<0.05). On the other hand, the genotype GG is an optional genotype for enhancing evisceration weight and meat quality. The results indicated that the polymorphism of PRDM16 gene in chicken may influence the carcass and fat traits, but no association with the growth traits.3. Clone and bioinformatics analysis of visfatin gene of Silky fowlThe 1482 bp full coding region sequence of visfatin gene in Silky fowl were obtained and it coded 493 amino acid sequence. It has 26 phosphorylation sites and a conserved domain of NAPRTase family but no signal peptide sequence. There is a hydrophobic region in 140-160aa of the visfatin. The amino acid sequence contains four kinds of secondary structure:α-helix,β-fold,β-angle and random coil. It has six function motifs including an amidation site. The visfatin is a highly conserved protein.4. Study of the expression of chicken visfatin gene in different tissue and developmental stagesThe highest expression of visfatin was found in breast muscle and the lowest in bone marrow. There was no difference of the expression in visceral fat and subcutaneous fat. However, the expressions of visfatin in bone marrow, liver, kidney, subcutaneous and visceral fat in broiler were significantly higher than in silky fowl (P< 0.05). Visfatin mRNA levels decreased in the bone marrow with the body development (P< 0.05) but increased in the liver and leg muscle.4. Study of the expression of chicken PRDM16 gene in different tissue and developmental stagesThe highest expression of PRDM16 was found in heart and the lowest in liver. There was no difference of the expression in visceral fat and abdominal fat. The expressions of PRDM16 in breast muscle, abdominal fat, subcutaneous fat and visceral fat in broiler were significantly higher than in silky fowl (P<0.05).The expressions of PRDM16 decreased in the bone marrow with the growth and development (P<0.05). The highest expression in the bone marrow was found at the age of 1 day, 8 weeks and 12 weeks, and the highest expression in the liver was found at the age of 4 weeks, the highest expression in the liver was found at the age of 16 weeks. The mRNA levels of ten tissues were much lower in PRDM16 than in visfatin (P<0.05). The visfatin and PRDM16 gene expression in the liver, heart and bone marrow has no gender difference in silky fowl.