CRISPR/Cas9-mediated knockout of the Vanin-1 gene in the Leghorn Male Hepatoma cell line and its effects on lipid metabolism.

OBJECTIVE: Vanin-1 (VNN1) is a pantetheinase that catalyses the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies have shown that the VNN1 is specifically expressed in chicken liver which negatively regulated by microRNA-122. However, the functions of the VNN1 in lipid metabolism in chicken liver haven't been elucidated. METHODS: First, we detected the VNN1 mRNA expression in 4-week chickens which were fasted 24 hours. Next, knocked out VNN1 via CRISPR/Cas9 system in the chicken Leghorn Male Hepatoma cell line. Detected the lipid deposition via oil red staining and analysis the content of triglycerides (TG), low-density lipoprotein-C (LDL-C), and highdensity lipoprotein-C (HDL-C) after VNN1 knockout in Leghorn Male Hepatoma cell line. Then we captured various differentially expressed genes (DEGs) between VNN1-modified LMH cells and original LMH cells by RNA-seq. RESULTS: Firstly, fasting-induced expression of VNN1. Meanwhile, we successfully used the CRISPR/Cas9 system to achieve targeted mutations of the VNN1 in the chicken LMH cell line. Moreover, the expression level of VNN1 mRNA in LMH-KO-VNN1 cells decreased compared with that in the wild-type LMH cells (p<0.0001). Compared with control, lipid deposition was decreased after knockout VNN1 via oil red staining, meanwhile, the contents of TG and LDL-C were significantly reduced, and the content of HDL-C was increased in LMH-KO-VNN1 cells. Transcriptome sequencing showed that there were 1,335 DEGs between LMH-KO-VNN1 cells and original LMH cells. Of these DEGs, 431 were upregulated, and 904 were downregulated. Gene ontology analyses of all DEGs showed that the lipid metabolism-related pathways, such as fatty acid biosynthesis and long-chain fatty acid biosynthesis, were enriched. KEGG pathway analyses showed that "lipid metabolism pathway", "energy metabolism", and "carbohydrate metabolism" were enriched. A total of 76 DEGs were involved in these pathways, of which 29 genes were upregulated (such as cytochrome P450 family 7 subfamily A member 1, ELOVL fatty acid elongase 2, and apolipoprotein A4) and 47 genes were downregulated (such as phosphoenolpyruvate carboxykinase 1) by VNN1 knockout in the LMH cells. CONCLUSION: These results suggest that VNN1 plays an important role in coordinating lipid metabolism in the chicken liver.

Phase wavefront perturbation calculation model for spectroscopic refractive index matching of hybrid materials.

A low-cost flexible spectroscopic refractive index matching (SRIM) material with bandpass filtering properties without incidence angle and polarization dependence by randomly dispersing inorganic C a F 2 particles in organic polydimethylsiloxane (PDMS) materials was proposed in our previous study. Since the micron size of the dispersed particles is much larger than the visible wavelength, the calculation based on the commonly used finite-difference time-domain (FDTD) method to simulate light propagation through the SRIM material is too bulky; however, on the other hand, the light tracing method based on Monte Carlo theory in our previous study cannot adequately explain the process. Therefore, a novel approximate calculation model, to the best of our knowledge, based on phase wavefront perturbation is proposed that can well explain the propagation of light through this SRIM sample material and can also be used to approximate the soft scattering of light through composite materials with small refractive index differences, such as translucent ceramics. The model simplifies the complex superposition of wavefront phase disturbances and the calculation of scattered light propagation in space. The scattered and nonscattered light ratios; the light intensity distribution after transmission through the spectroscopic material; and the influence of absorption attenuation of the PDMS organic material on the spectroscopic performance are also considered. The simulation results based on the model are in great agreement with the experimental results. This work is important to further improve the performance of SRIM materials.

Regulation of chicken vanin1 gene expression by peroxisome proliferators activated receptor α and miRNA-181a-5p.

OBJECTIVE: Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that VNN1 is specifically expressed in chicken liver. In this study, we aimed to investigate the roles of peroxisome proliferators activated receptor α (PPARα) and miRNA-181a-5p in regulating VNN1 gene expression in chicken liver. METHODS: 5'-RACE was performed to identify the transcription start site of chicken VNN1. JASPAR and TFSEARCH were used to analyze the potential transcription factor binding sites in the promoter region of chicken VNN1 and miRanda was used to search miRNA binding sites in 3' untranslated region (3'UTR) of chicken VNN1. We used a knock-down strategy to manipulate PPARα (or miRNA-181a-5p) expression levels in vitro to further investigate its effect on VNN1 gene transcription. Luciferase reporter assays were used to explore the specific regions of VNN1 targeted by PPARα and miRNA-181a-5p. RESULTS: Sequence analysis of the VNN1 promoter region revealed several transcription factor-binding sites, including hepatocyte nuclear factor 1α (HNF1α), PPARα, and CCAAT/enhancer binding protein α. GW7647 (a specific agonist of PPARα) increased the expression level of VNN1 mRNA in chicken primary hepatocytes, whereas knockdown of PPARα with siRNA increased VNN1 mRNA expression. Moreover, the predicted PPARα-binding site was confirmed to be necessary for PPARα regulation of VNN1 gene expression. In addition, the VNN1 3'UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression level of VNN1 mRNA. CONCLUSION: This study demonstrates that PPARα is an important transcriptional activator of VNN1 gene expression and that miRNA-181a-5p acts as a negative regulator of VNN1 expression in chicken hepatocytes.

MiR-122 targets the vanin 1 gene to regulate its expression in chickens.

As the most abundant microRNA (miRNA) in the liver, miR-122 plays important roles in the growth and development of liver, lipid metabolism, and liver diseases. Vanin 1 (VNN1) plays an important role in hepatic lipid metabolism, and VNN1 may serve as a potential therapeutic target for the treatment of metabolic diseases caused by overactivated gluconeogenesis. In our previous RNA-seq study, we found the expression of VNN1 increased significantly when the expression of miR-122 (gga-miR-122-5p) was knocked down in primary chicken hepatocytes. In this study, we verified this result by real-time qRT-PCR, and we also found that the chicken VNN1 was highly expressed in the liver. By bioinformatics analyses, we found the 3'UTR of VNN1 contained sequences completely complementary to the nucleotides 1 to 8 of miR-122. Co-transfection and dual-luciferase reporter assays showed that overexpression of miR-122 decreased the expression of luciferase reporter gene linked to the 3'UTR of chicken VNN1 in the Chinese hamster ovary cells (P<0.01), and the decrease was further demonstrated to be dependent on the predicted miR-122 binding sites by site mutation analyses. These results further support miR-122 as a negative regulator of VNN1 expression in chicken hepatocytes. Overall, this study suggests that miR-122 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the VNN1 gene.

MicroRNA-122 targets genes related to liver metabolism in chickens.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting mRNAs. MicroRNA-122 (miR-122) has important functions in mammalian and fish livers, but its functions in the poultry liver are largely unknown. In this study, we determined the expression patterns of miR-122 in the chicken and identified its target genes in the chicken liver. We found that chicken miR-122 was highly expressed in the liver and that its expression in the liver was up-regulated during the early posthatch life. By bioinformatics and reporter gene analyses, we identified PKM2, TGFB3, FABP5 and ARCN1 as miR-122 target genes in the chicken liver. miR-122 knockdown in primary chicken hepatocytes and expression analysis of miR-122 and predicted target mRNAs in the chicken liver suggested that the expression of PKM2 and FABP5 in the chicken liver is regulated by miR-122. Knockdown of miR-122 affected the expression of 123 genes in cultured chicken hepatocytes. Among these genes, the largest cluster, which consisted of 21 genes, was involved in liver metabolism. These findings suggest that miR-122 plays a role in liver metabolism in the chicken by directly or indirectly regulating the expression of genes involved in liver metabolism.

Growth hormone-regulated mRNAs and miRNAs in chicken hepatocytes.

Growth hormone (GH) is a key regulatory factor in animal growth, development and metabolism. Based on the expression level of the GH receptor, the chicken liver is a major target organ of GH, but the biological effects of GH on the chicken liver are not fully understood. In this work we identified mRNAs and miRNAs that are regulated by GH in primary hepatocytes from female chickens through RNA-seq, and analyzed the functional relevance of these mRNAs and miRNAs through GO enrichment analysis and miRNA target prediction. A total of 164 mRNAs were found to be differentially expressed between GH-treated and control chicken hepatocytes, of which 112 were up-regulated and 52 were down-regulated by GH. A total of 225 chicken miRNAs were identified by the RNA-Seq analysis. Among these miRNAs 16 were up-regulated and 1 miRNA was down-regulated by GH. The GH-regulated mRNAs were mainly involved in growth and metabolism. Most of the GH-upregulated or GH-downregulated miRNAs were predicted to target the GH-downregulated or GH-upregulated mRNAs, respectively, involved in lipid metabolism. This study reveals that GH regulates the expression of many mRNAs involved in metabolism in female chicken hepatocytes, which suggests that GH plays an important role in regulating liver metabolism in female chickens. The results of this study also support the hypothesis that GH regulates lipid metabolism in chicken liver in part by regulating the expression of miRNAs that target the mRNAs involved in lipid metabolism.

Expression of miR-33 from an SREBF2 intron targets the FTO gene in the chicken.

The sterol regulatory element binding transcription factor 2 (SREBF2) gene encodes a transcription factor that activates the expression of many genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. Through bioinformatics, we found that intron 16 of the chicken SREBF2 gene might encode the chicken miR-33. Using quantitative RT-PCR, we detected the expression of miR-33 in a variety of chicken tissues including skeletal muscle, adipose tissue, and liver. Three hundred and seventy eight genes were predicted to be potential targets of miR-33 in chickens via miRNA target prediction programs "miRanda" and "TargetScan". Among these targets, the gene FTO (fat mass and obesity associated) encodes a Fe(II)- and 2-oxoglutarate-dependent nucleic acid demethylase that regulates lipid metabolism, and the possibility that its expression is negatively regulated by miR-33 in the chicken liver was therefore further studied. Co-transfection and dual-luciferase reporter assays showed that the expression of luciferase reporter gene linked to the 3'-untranslated region (3'UTR) of the chicken FTO mRNA was down-regulated by overexpression of the chicken miR-33 in the C2C12 cells (P<0.05). Furthermore, this down-regulation was completely abolished when the predicted miR-33 target site in the FTO 3'UTR was mutated. In contrast, the expression of FTO mRNA in the primary chicken hepatocytes was up-regulated after transfection with the miR-33 inhibitor LNA-anti-miR-33. Using quantitative RT-PCR, we also found that the expression of miR-33 was increased in the chicken liver from day 0 to day 49 of age, whereas that of the FTO mRNA was decreased during the same age period. These data together suggest that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the FTO gene.

MicroRNA-126 expression is decreased in cultured primary chicken hepatocytes and targets the sprouty-related EVH1 domain containing 1 mRNA.

The microRNA-126 (miR-126) is a miRNA expressed in highly vascularized tissues, and it is believed to play a role in angiogenesis by repressing sprouty-related EVH1 domain containing 1 (Spred1). In the current study, we determined the expression pattern of chicken miR-126 (gga-miR-126) and predicted and validated its target genes. The quantitative reverse-transcription (qRT) PCR analysis showed that miR-126 was expressed in various chicken tissues with the highest level in lung. In liver, the expression level of miR-126 increased from 0 to 7 wk of age. The expression of miR-126 in primary chicken hepatocytes decreased with culturing. A miR-126 binding site was predicted in the 3' UTR (untranslated region) of chicken Spred1. Dual-luciferase reporter assays indicated that miR-126 could bind to the predicted site to repress the expression of Spred1. These data validate Spred1 as a target gene of chicken miR-126. These results will help further understand the function and regulation of miR-126 and Spred1 in chickens.

[Cloning and characterization of Caveolin-1 gene in pigeon, Columba livia domestica].

Caveolins, a class of principal proteins forming the structure of caveolae in plasmalemma, were encoded by caveolins gene family. Caveolin-1 gene is a member of caveolins gene family. In the present study, a full-length of 2605 bp caveolin-1 cDNA sequence in Columba livia domestica, which included a 537 bp complete ORF encoding a 178 amino acids long putative peptide, were obtained by using RT-PCR and RACE technique. The Columba livia domestica caveolin-1 CDS shared 80.1% - 93.4% homology with Bos taurus, Canis lupus familiaris, Gallus gallus and Rattus norvegicus. Meanwhile, the putative amino acid sequence of Columba livia domestica caveolin-1 shared 85.4% - 97.2% homology with the above species. The semi-quantity RT-PCR revealed that Caveolin-1 expressions were detectable in all the Columba livia domestica tissues and the expressional level of caveolin-1 gene was high in adipose, medium in various muscles, low in liver. These results demonstrated that Caveolin-1 gene was potentially involved in some metabolic pathways in adipose and muscle.

[Identification and characterization of myostatin gene in rough-skinned sculp, Trachidermus fasciatus].

Myostatin is a member of the TGF-beta superfamily and acts as a negative regulator of skeletal muscle growth. The characterization of the myostatin gene and its expression in Trachidermus fasciatus was reported in the current study. A full-length of 2 568 bp myostatin cDNA sequence in T. fasciats was cloned by 5' and 3' RACE, which included a 1 131 bp complete ORF encoding a 376 amino acid peptide, a 106 bp long 5'-UTR and a 1331 bp long 3'UTR. As other MSTN, the putative peptide contains a 22 amino acids long signal peptide, a conserved RARR proteolytic processing site, and 10 conserved cysteine residues in the C terminal of the protein. The Trachidermus fasciatus MSTN has high homology with Umbrina cirrosa, Morone saxatilis, Morone americana, Morone chrysops myostatin while has low homology with mammalian and birds myostatin. The phylogenetic analysis showed that the T. fasciatus myostatin had the closest relationship with U. cirrosa. In the four examined tissues, the myostatin gene was highly expressed in muscle and intestine and weakly expressed in brain and liver. These results suggested that the fish myostatin gene might not only play roles in muscle development but also contribute to other biological functions.

Patterns of East Asian pig domestication, migration, and turnover revealed by modern and ancient DNA.

The establishment of agricultural economies based upon domestic animals began independently in many parts of the world and led to both increases in human population size and the migration of people carrying domestic plants and animals. The precise circumstances of the earliest phases of these events remain mysterious given their antiquity and the fact that subsequent waves of migrants have often replaced the first. Through the use of more than 1,500 modern (including 151 previously uncharacterized specimens) and 18 ancient (representing six East Asian archeological sites) pig (Sus scrofa) DNA sequences sampled across East Asia, we provide evidence for the long-term genetic continuity between modern and ancient Chinese domestic pigs. Although the Chinese case for independent pig domestication is supported by both genetic and archaeological evidence, we discuss five additional (and possibly) independent domestications of indigenous wild boar populations: one in India, three in peninsular Southeast Asia, and one off the coast of Taiwan. Collectively, we refer to these instances as "cryptic domestication," given the current lack of corroborating archaeological evidence. In addition, we demonstrate the existence of numerous populations of genetically distinct and widespread wild boar populations that have not contributed maternal genetic material to modern domestic stocks. The overall findings provide the most complete picture yet of pig evolution and domestication in East Asia, and generate testable hypotheses regarding the development and spread of early farmers in the Far East.

Growth hormone regulation of insulin-like growth factor-I gene expression may be mediated by multiple distal signal transducer and activator of transcription 5 binding sites.

The transcription factor signal transducer and activator of transcription (STAT)-5 mediates GH stimulation of IGF-I gene expression in the liver. Previous studies suggested that STAT5 might exert this effect by binding to an IGF-I intron 2 region and a distal 5'-flanking region each containing two STAT5 binding sites. Here we report the identification of three additional chromosomal regions containing a total of five putative STAT5 binding sites that may mediate GH-induced STAT5 activation of IGF-I gene expression in the mouse liver. By comparing an 170-kb mouse genomic DNA containing the IGF-I gene with the corresponding human sequence, we identified 19 putative STAT5 binding sites that bear the consensus sequence of STAT5 binding site and are conserved across the two species. Chromatin immunoprecipitation assays indicated that five chromosomal regions containing a total of nine of the 19 putative STAT5 binding sites were bound by STAT5 in the mouse liver in response to GH administration and that these bindings preceded or coincided with GH-increased IGF-I gene transcription. Two of the five chromosomal regions correspond to those previously identified in other species, and the three new chromosomal regions that contain a total of five putative STAT5 binding sites are IGF-I intron 3 regions located at least 26 kb from the transcription start site. Gel-shift assays confirmed the binding of the five new putative STAT5 binding sites as well as three of the four previously identified STAT5 binding sites to GH-activated STAT5 from the mouse liver. Cotransfection analyses indicated that, although each of the five chromosomal regions was able to mediate STAT5 activation of reporter gene expression, together they mediated greater STAT5 activation of reporter gene expression in response to GH. Overall, these results suggest that GH-induced STAT5 activation of IGF-I gene expression in the mouse liver might be collectively mediated by at least eight STAT5 binding sites located in distal intronic and 5'-flanking regions of the IGF-I gene.

[Identification and genetic analysis of SNPs in duck adiponectin gene].

Single nucleotide polymorphisms (SNPs) within the duck adiponectin gene were detected by single strand conformation polymorphism (SSCP) using 5 pairs of primers to amplify an area spanning the open reading frame. Eight duck breeds, including Kunshan Sheldrake, Cherry Valley Meat duck, Gaoyou duck, Shanma duck, Jinding duck, Longbai duck, Jingjiang Sheldrake and White feather Muscovy duck, were used. Seven nucleotide variations were found, of which G430A, A457G, and T523C resulted in amino acid changes of A144T, I153V, and Y175H, respectively. The remaining 4 SNPs were C507T, T540C, C576T and C597T. Eight genotypes (AA, AB, AC, BB, BC, CC, DD, and DE) were detected in the 8 breeds. Chi(2) analysis showed that the distribution of the eight genotypes was very different among the different breeds (P < 0.01). Ex-cept for the Jingding duck, all breeds were in accordance with the Hardy-Weinberg equilibrium. Genetic analysis indicated that homozygosity was highest in the Jinding duck, lowest in the Gaoyou duck and similar in other breeds. Polymorphism information content (PIC) was low in the Jinding, high in the Gaoyou and intermediate in other breeds. These results showed that the adiponectin gene had a high level of polymorphism in different duck breeds, and could be used as a candidate gene to analyze the correlation between its polymorphism and fat traits in duck.

Molecular cloning and characterization of Rab6 gene in duck.

Rab (ras-like in rat brain) proteins are small GTP-binding proteins that belong to largest subfamily in the small G protein, which are important for molecular modulation of membrane in the vesicular trafficking pathways. We have cloned and sequenced full length cDNA of Rab6 gene in duck. The cDNA sequence consists of 761 nucleotides and contains a complete open reading frame (ORF) of 627 nucleotides; the putative protein includes 208 amino acids. The CDS of duck Rab6 gene shares 86.1-90.0% homology with house mouse, silurana tropicalis, dog, human and orangutan, which indicates the Rab6 gene is high evolutional conservation in above animals.

Growth hormone stimulates hepatic expression of bovine growth hormone receptor messenger ribonucleic acid through signal transducer and activator of transcription 5 activation of a major growth hormone receptor gene promoter.

The objective of this study was to determine whether and how GH regulates hepatic expression of GH receptor (GHR) mRNA in cattle. Ribonuclease protection assays revealed that injection of GH in a slow-release formula increased both hepatic GHR and IGF-I mRNAs 1 wk after the injection. The increases in GHR and IGF-I mRNAs were highly correlated. Western blot analysis showed that the injection also increased liver GHR protein level. In cattle and other mammals, hepatic GHR mRNA is expressed as variants that differ in the 5'-untranslated region due to the use of different promoters in transcription and/or alternative splicing. We found that GH increased the expression of the liver-specific GHR mRNA variant GHR1A without affecting the other two major GHR mRNA variants in the bovine liver, GHR1B and GHR1C. In transient transfection analyses, GH could robustly activate reporter gene expression from a 2.7-kb GHR1A promoter, suggesting that GH augmentation of GHR1A mRNA expression in the liver is at least partially mediated at the transcriptional level. Additional transfection analyses of serially 5'-truncated fragments of this promoter narrowed the GH-responsive sequence element down to a 210-bp region that contained a putative signal transducer and activator of transcription 5 (STAT5) binding site. EMSAs demonstrated that this putative STAT5 binding site was able to bind to STAT5b protein. In cotransfection assays, deletion of this putative STAT5 binding site abolished most of the GH response of the GHR1A promoter. Like 1-wk GH action, 6-h (i.e. short-term) GH action also increased liver expression of GHR1A and total GHR mRNAs in cattle. These observations together suggest that GH directly stimulates the expression of one GHR mRNA variant, GHR1A, through binding STAT5 to its promoter, thereby increasing GHR mRNA and protein expression in the bovine liver.

Identification and characterization of microRNAs from the bovine adipose tissue and mammary gland.

We report the identification of bovine miRNAs by cloning small RNAs from adipose tissue and the mammary gland. Fifty-nine distinct miRNAs were identified, five of them were not homologous to known mammalian miRNAs, and many of them had 3' and/or 5' end variants. Ribonuclease protection assays indicated that miR-23a and miR-24, whose genes are closely located on the same chromosome, were co-expressed in different tissues. The assays also suggested a role for several miRNAs in the mammary gland and a role for miR-133, a previously known skeletal and cardiac muscle-specific miRNA, in the rumen, an organ unique to the ruminant.

Complete sequence of the yak (Bos grunniens) mitochondrial genome and its evolutionary relationship with other ruminants.

The yak (Bos grunniens) is the most important domesticated species in the Qinhai-Tibetan Plateau. In present study, the complete sequence of the yak mitochondrial genome was determined. Sequence analysis revealed that there are no differences with cattle in the yak mitochondrial genome organization. Interestingly, within the D-loop, the conserved sequence blocks are less conserved than surrounding regions. Neighbor-Joining (NJ) trees based on single genes, gene sets and concatenated genes of mitochondrial genome were constructed. The analysis identified the yak as a sister group of a cattle/zebu clade. Based on substitutions in 22 tRNA genes, 12S rRNA gene and 16S rRNA gene, the dating of divergence between yak and cattle/zebu, and yak and water buffalo, was proposed to have occurred 4.38-5.32 and 10.54-13.85 million years before present, respectively. This is consistent with the paleontologyical data. Yak and sheep/goat divergent dating predicts that their divergence occurred at 13.14-27.99 million years before the present day.

Cloning and characterization of chicken adipocyte fatty acid binding protein gene.

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.

[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.