[Characterization of chicken PPARγ expression and its impact on adipocyte proliferation and differentiation].

To characterize the chicken PPARγ gene expression and its impact on chicken adipocyte proliferation and differentiation, western blotting approach was conducted to investigate the expression of PPARγ in various chicken tissues and the difference of expression level in abdominal adipose tissues between the NEAU broiler lines divergently selected for abdominal fat content. The expression of PPARγ gene was suppressed in chicken adipocytes using RNAi technology, and the roles of PPARγ gene in the adipocytes proliferation and differentiation were investigated by MTT assay and Oil Red O staining extraction assay, respectively. After PPARγ gene was downregulated, the expression level of other transcript factors and marker genes related to the adipocyte differentiation was detected by Real-time PCR and Western blotting analyses. The results showed that PPARγ highly expressed in abdominal adipose tissue, gizzard, spleen, kidney, lowly expressed in heart, and not expressed in liver, breast muscle, leg muscle, and duodenum. Meanwhile, PPARγ expressed much higher in fat birds than in lean ones in abdominal adipose tissue at 5 and 7 weeks of age (P<0.05). RNAi analysis showed that knockdown of PPARγ gene increased chicken adipocyte proliferation and decreased cell differentiation and significantly decreased the expression levels of C/EBPα, SREBP1, A-FABP, Perilipin1, LPL, and IGFBP-2 (P<0.05). In summary, PPARγ gene may be related to the broiler abdominal fat deposition, and be probably a key regulator of chicken adipocyte proliferation and differentiation.

[Association between chicken A-FABP gene polymorphisms and growth and body composition traits].

This experiment was designed to study the effects of polymorphism of A-FABP gene on growth and body composition traits in chicken. The 10th generation broiler population, derived from the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) was used. Polymorphism among individuals was detected by DNA sequencing, PCR-RFLP, PCR-LP, and DHPLC. Linkage disequilibrium analysis for eight SNPs was performed, and five htSNPs were selected to construct haplotypes. The association analysis between the individual SNPs and haplotypes and growth and body composition traits were investigated, respectively. The results showed that there were consistently significant effects on muscle stomach weight (MSW) and percentage of muscle stomach (MSW/BW) (Pamp;0.05) in the seven SNPs (except for SNP 5) and haplotypes, but no significant effect on any other trait (P>0.05). Due to no evidence on effects of A-FABP for digestion system in other species, future experiments need to be developed to confirm whether A-FABP could be a major gene of MSW and MSW/BW traits in broiler chicken.

Profiling of chicken adipose tissue gene expression by genome array.

BACKGROUND: Excessive accumulation of lipids in the adipose tissue is a major problem in the present-day broiler industry. However, few studies have analyzed the expression of adipose tissue genes that are involved in pathways and mechanisms leading to adiposity in chickens. Gene expression profiling of chicken adipose tissue could provide key information about the ontogenesis of fatness and clarify the molecular mechanisms underlying obesity. In this study, Chicken Genome Arrays were used to construct an adipose tissue gene expression profile of 7-week-old broilers, and to screen adipose tissue genes that are differentially expressed in lean and fat lines divergently selected over eight generations for high and low abdominal fat weight. RESULTS: The gene expression profiles detected 13,234-16,858 probe sets in chicken adipose tissue at 7 weeks, and genes involved in lipid metabolism and immunity such as fatty acid binding protein (FABP), thyroid hormone-responsive protein (Spot14), lipoprotein lipase(LPL), insulin-like growth factor binding protein 7(IGFBP7) and major histocompatibility complex (MHC), were highly expressed. In contrast, some genes related to lipogenesis, such as leptin receptor, sterol regulatory element binding proteins1 (SREBP1), apolipoprotein B(ApoB) and insulin-like growth factor 2(IGF2), were not detected. Moreover, 230 genes that were differentially expressed between the two lines were screened out; these were mainly involved in lipid metabolism, signal transduction, energy metabolism, tumorigenesis and immunity. Subsequently, real-time RT-PCR was performed to validate fifteen differentially expressed genes screened out by the microarray approach and high consistency was observed between the two methods. CONCLUSION: Our results establish the groundwork for further studies of the basic genetic control of growth and development of chicken adipose tissue, and will be beneficial in clarifying the molecular mechanism of obesity in chickens.

Goose FMO3 gene cloning, tissue expression profiling, polymorphism detection and association analysis with trimethylamine level in the egg yolk.

Flavin-containing monooxygenase 3 (FMO3) plays a critical role in catalyzing the conversion of trimethylamine (TMA) to trimethylamine-N-oxide (TMAO) in vivo. Despite the well-documented association between FMO3 mutations and a 'fishy' off-flavor eggs in chicken and quail, little information is available regarding the molecular characteristic of goose (Anser cygnoides) FMO3 and its relationship with the yolk TMA content. To fill these gaps, we cloned the full-length cDNA sequence of goose FMO3, which comprised 1851bp encoding 531 amino acids. FMO3 mRNA was dramatically expressed in liver than in other tissues in the geese. Eight single nucleotide polymorphisms (SNPs) were detected in the entire coding region. The CC genotype at the T669C site, GG at the A723G site, and AA at the G734A site of FMO3 were highly significantly associated with elevated TMA content in goose egg yolk (P<0.001). Carriers of the A allele of G734A or C allele of T885C had yolk TMA content that had a high probability of being elevated after feeding with additional choline chloride (P=0.0429, OR=4.1300, 95%CI=1.0390-16.4270, and P=0.0251, OR=4.6060, 95%CI=1.1620-18.2620, respectively). This work lays a foundation for studying the function of FMO3 and yolk TMA content in goose. However, studies using larger sample sizes and more goose breeds are required to determine whether the fishy off-flavor trait exists in goose.

[Detection of major gene on litter size in sheep].

The current study was designed to detect SNPs within BMP15 and BMPR-IB gene and investigate the effect of the genes on sheep litter size. Four sheep lines, HU-Yang, Chinese Merino monotocous, Chinese Merino multiparous for wool production and Chinese Merino multiparous for mutton production, were used in this study. Litter sizes were recorded for each ewe in the four lines. Primers for BMP15 and BMPR-IB gene were designed from database sheep sequence and polymorphisms were detected by PCR-RFLP method. The results showed that there was no polymorphism with BMP15 gene among the four lines, and there was an A / G SNP with BMPR-IB gene at base 746 among the four lines. Three types of genotype (BB, B+ and ++), based on A / G locus, were found within each line. The frequencies of genotypes were significantly different among the lines (P<0.001), with BB genotype primarily existing in HU-Yang, ++ genotype in Chinese Merino monotocous line, and B+ genotype in Chinnese Merino multiparous lines. The A / G mutation influence significantly the sheep litter sizes, and the BB and B+ ewes had significant higher litter sizes than ++ ewes. The results of present study showed simultaneously that the genotype of BMPR-IB was a perfect predictor of the sheep litter sizes. These results intensively indicated that BMPR-IB is a major gene to affect litter size in sheep, and could be used as the molecular genetic marker to select litter size in sheep.

[Correlation analysis between single nucleotide polymorphism of the leptin receptor intron 8 and fatness traits in chickens].

Leptin receptor plays an important role in leptin functioning signal transduction and it may have direct effects on the deposition of adipose tissues and the body weight, the leptin receptor (OBR) gene, therefore, can be considered as a candidate gene in the study of fat deposition of the chicken. The function of OBR gene has been intensively studied in mammals, but study of OBR gene in the chicken is still rare. In this paper, the NEAU divergent selection broiler lines for abdominal fat were used. Body weight and fatness traits were measured in the sixth generation broiler population of the two lines at 7 week of age. Two pairs of primers for intron 8 of OBR gene were designed according to the database of chicken genomic sequence (Accession No. AF222783). The SNP was detected by DNA sequencing, and PCR-SSCP method was then developed to screen the population. The correlation analysis between the polymorphisms of the intron 8 of OBR gene and growth and fatness traits in the population was carried out using the appropriate statistical model. Two SNPs were found in the population. Those were T500C and G659A. The least square analysis showed that BB genotype birds had significant higher (P < 0.05) abdominal fat weight and percentage of abdominal fat than AA and AB genotype birds, and AA genotype birds had significant lower (P < 0.05) weights of livers than AB and BB genotype birds at the same time. From these results we can putatively drew the conclusion that OBR gene may be a major gene to affect the fatness traits or linked to the major gene, and the two polymorphisms found in OBR gene intron 8 region could be used to select the chicken for low abdominal fat in molecular marker-assisted selection programs.

[The study of the uncoulping protein gene as the candidate gene for fatness traits in chicken].

The UCPs are integral membrane proteins of the mitochondrial respiration from oxidative phosphorylation, diminishing the resulting production of ATP and instead yielding dissipative heat. The action of those proteins creates a futile cycle that decreases the metabolic efficiency of the organism. Thus UCPs provide new clue to obesity's causes. This study was designed to investigate the effect of UCP gene on chicken fatness traits. The fifth generation population of divergent selection broiler line, Hyline Brown layer and three native breeds (shiqiza, Beijing You, baier) were used in this research. Body weight and body composition traits were measured in broiler lines at 7 weeks of age. Primers for the 3'-untranslator region in UCP were designed from database of chicken genomic sequence. Polymorphisms were detected by PCR-SSCP and DNA sequencing. The results showed that there was significant difference (P < 0.01) in the frequency of genotype among breeds except broiler vs Beijing You and Baier vs Hyline Brown layer in mutation sites detected by the two pairs of primers. The distribution of genotype in Beijing You and broiler had no difference. It deduced that Beijing You belongs to the native breed that has dominant meat type traits and has the same genetic background with broiler. Baier and Hyline Brown Layer have no difference in the genotype, it can be viewed as they have same genetic background. A A/C mutation at base position 1197 was found among individuals in broiler line and the least square analysis showed that BB birds had significant lower (P < 0.01) abdominal fat weight and percentage of abdominal fat than AB or AA birds. From the results we can putatively draw the conclusion that UCP gene is the major gene to affect the fatness traits or it links with the major gene.

[Cloning and sequencing of fatty acid binding proteins gene in chicken].

A pair of primers was designed according to the sequence of mammal fatty acid binding protein (FABP) gene, then PCR amplified to chicken genome. After the product of PCR was cloned and sequenced, homologous comparison was done among porcine heart fatty acid binding protein gene and porcine adipocyte fatty acid binding protein gene. The result showed that the sequence of chicken FABP gene had 68% and 75% homology with porcine H-FABP and A-FABP gene respectively, and had 75% homology with porcine AFABP on amino acid level. The result of Northern showed that the gene only expressed in fat tissues.

[Studies of single nucleotide polymorphism of PPAR gene and its associations with fattiness trait in chicken].

In this experiment, the AA broiler fat traits and three Chinese special breeds (Shiqiza, Beijing youji and Baier) were used to study the effect of PPAR-alpha gene on fat trait. Coding region of the gene was amplified by seven pairs of primers, and then single nucleotide polymorphisms (SNPs) were detected by the technique of single strand conformation polymorphism (SSCP) and finally confirmed by sequencing. One nucleotide variation was found, the result of chi 2 analysis showed that the distribution of three genotypes was very different among different breeds(P < 0.01). The result of variance analysis showed that the birds with BB genotype had a higher abdominal fat weight than the birds with other genotypes (AA and AB). It implied that PPAR-alpha gene could be a candidate locus or linked to a major gene to significantly affect abdominal fat traits in chicken.

[Cloning and sequencing of the introns of UCP gene and construction of molecular phylogenetic tree in chicken].

The UCP genes were the newly discovered genes that can increase the energy expenditure and involve in the metabolism of fat and regulation of energy. Four pairs of primers in chicken UCP exon region were designed to amplify the introns of chicken UCP gene according to the splice ways of the mouse UCP2 gene (Accession No.AF096288). The sequence results showed that the chicken UCP gene also had five GT-AG type introns. The molecular phylogenetic tree was constructed based on the sequence of cds, intron 2 and intron 3 region, respectively. The phylogenetic tree based on the UCP cds region was consistent with the species phylogenetic tree. This result implicated that UCP gene can be regarded as the useful gene for the study of animal phylogenesis. On the contrast, the phylogenetic tree based on the intron 2 and intron 3 region was different from the species phylogenetic tree, which showed that the evolution of intron and cds region is different.

[Single nucleotide polymorphism analysis in chicken leptin receptor exon 9].

Leptin receptor is a type I cytokine super family member and plays an important role in leptin functioning signal transduction. This study was designed to investigate the single nucleotide polymorphism (SNP) of OBR gene in various breeds, including Fatness Line (FL), Leanness Line (LL), Beijing Youji, Baierji, Shiqiza, Dwarf Yellow Chickens, Mini Yellow Chickens, Huiyang Huxuji, Recessive White Chickens and Hyline Layer. The primers for exon 9 in OBR gene were designed from the database of chicken genomic sequence and the SNPs were detected by PCR-SSCP method. One SNP (C/A at 1167 in cds) was found among individuals within all breeds. However, the amino acid was not changed because it was a silence mutation. The result of population genetics analyses showed that the frequency of AA genotype in Beijing Youji was significantly higher than that in other lines. Also, the frequency of A allele in FL was significantly higher than that in LL.

[CDNA microarray on differentially expressed genes of adipose tissue in two breeds chicken].

cDNA microarray containing 9024 cDNAs was used to construct gene expression profile in order to screen differentially expressed genes of adipose tissue between broiler and Bai' er and investigate the molecular mechanism related with body fatness traits between the two breeds. Sixty seven differentially expressed genes, being involved in fat metabolism, energy metabolism, cytoskeleton, transcription and splicing factor, protein synthesis and degradation, were screened out. Furthermore, some genes that had no annotation in GenBank were screened out, they were presumed to be unknown new genes. The roles that they may play in chicken fat metabolism need clarify later.