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.

[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.

[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.

[Single nucleotide polymorphism analysis of the chicken Myostatin gene in different chicken lines].

Myostatin is a new member of the TGF-beta superfamily. It is specifically expressed in skeletal muscle cells and functions as a negative regulator of skeletal muscle growth. This study was aimed to identify the single nucleotide polymorphisms (SNPs) of Myostatin gene in various breeds including Beijing Youji, Baierji, Shiqiza, Dwarf Yellow Chickens, Mini Yellow Chickens, Huiyang Huxuji, Recessive White Chickens, AA and Hyline Layer. The 17 pairs of primers for Myostatin gene were designed based on the chicken genomic sequence. The total 5 SNPs were identified in the chicken Myostatin gene by PCR-SSCP of which 3 were in the 5'-regulatory region and 2 were in the 3'-regulatory region of the Myostatin gene in different chicken lines. The sequence data showed that one of the three SNPs localized in the 5'-regulatory region was due to 3 single point mutations (G304A, A322G, C334T, respectively), other two were single point mutation of G167A and T177C, respectively. The other two SNPs in the 3'-regulatory region of the Myostatin gene are A to T (7263) and A to G (6935). Population genetic analysis indicated that genotype frequencies of the Myostatin locus (P60/P61) in Beijing youji were quite different from those of other tested chicken lines. Frequency of the BB genotype was very high (0.70) while the AA genotype was only 0.033 in 5'-regulatory region (P60/P61) in Beijing youji population. The genotype frequencies (P93/P94) were significantly different among the lines (P < 0.01): frequencies of the EE genotype in Beijing Youji were lower than those in the other lines, while frequencies of the EE genotype in Baierji and Hyline Layer were higher than the rest. Frequency of allele C (P80/P81) in 9 breeds was higher than that of allele D. Frequency of genotype MM (P76/P77) was low, and that of MN was high in 7 breeds.

[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.

[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.