The CD8α gene in duck (Anatidae): cloning, characterization, and expression during viral infection.

Cluster of differentiation 8 alpha (CD8α) is critical for cell-mediated immune defense and T-cell development. Although CD8α sequences have been reported for several species, very little is known about CD8α in ducks. To elucidate the mechanisms involved in the innate and adaptive immune responses of ducks, we cloned CD8α coding sequences from domestic, Muscovy, Mallard, and Spotbill ducks using reverse transcription polymerase chain reaction (RT-PCR). Each sequence consisted of 714 nucleotides and encoded a signal peptide, an IgV-like domain, a stalk region, a transmembrane region, and a cytoplasmic tail. We identified 58 nucleotide differences and 37 amino acid differences among the four types of duck; of these, 53 nucleotide and 33 amino acid differences were between Muscovy ducks and the other duck species. The CD8α cDNA sequence from domestic duck consisted of a 61-nucleotide 5' untranslated region (UTR), a 714-nucleotide open reading frame, and an 849-nucleotide 3' UTR. Multiple sequence alignments showed that the amino acid sequence of CD8α is conserved in vertebrates. RT-PCR revealed that expression of CD8α mRNA of domestic ducks was highest in the thymus and very low in the kidney, cerebrum, cerebellum, and muscle. Immunohistochemical analyses detected CD8α on the splenic corpuscle and periarterial lymphatic sheath of the spleen. CD8α mRNA in domestic ducklings was initially up-regulated, and then down-regulated, in the thymus, spleen, and liver after treatment with duck hepatitis virus type I (DHV-1) or the immunostimulant polyriboinosinic polyribocytidylic acid (poly I:C).

Molecular cloning and expression analysis of ferritin, heavy polypeptide 1 gene from duck (Anas platyrhynchos).

H-ferritin is a core subunit of the iron storage protein ferritin, and is related to the pathogenesis of malignant diseases. A differential expressed sequence tag of the ferritin, heavy polypeptide 1 gene (FTH1) was obtained from our previously constructed suppression subtractive cDNA library from 3-day-old ducklings challenged with duck hepatitis virus type I (DHV-1). The expression and function of FTH1 in immune defense against infection remains largely unknown in ducks. In this study, the full-length duFTH1 cDNA was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. It consisted of 153 basepairs (bp) 5'untranslated region (UTR), 183 bp 3'UTR, and 546 bp open reading frame that encodes a single protein of 181 amino acid residues. duFTH1 shares high similarity with FTH1 genes from other vertebrates. The amino acid sequence possesses the conserved domain of typical ferritin H subunits, including seven metal ligands in the ferroxidase center, one iron binding region signature, and a potential bio-mineralization residue (Thy(29)). Moreover, in agreement with a previously reported ferritin H subunit, we identified an iron response element in the 5'UTR. RT-PCR analyses revealed duFTH1 mRNA is widely expressed in various tissues. Real-time quantitative polymerase chain reaction analyses suggested that duFTH1 mRNA is significantly up-regulated in the liver after DHV-1 injection or polyriboinosinic polyribocytidylic acid (polyI:C) treatment, reaching a peak 4 h post-infection, and dropping progressively and returning to normal after 24 h. Our findings suggest that duFTH1 functions as an iron chelating protein subunit in duck and contributes to the innate immune responses against viral infections.

Identification and differential expression of microRNAs in ovaries of laying and Broody geese (Anser cygnoides) by Solexa sequencing.

BACKGROUND: Recent functional studies have demonstrated that the microRNAs (miRNAs) play critical roles in ovarian gonadal development, steroidogenesis, apoptosis, and ovulation in mammals. However, little is known about the involvement of miRNAs in the ovarian function of fowl. The goose (Anas cygnoides) is a commercially important food that is cultivated widely in China but the goose industry has been hampered by high broodiness and poor egg laying performance, which are influenced by ovarian function. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the miRNA transcriptomes of ovaries from laying and broody geese were profiled using Solexa deep sequencing and bioinformatics was used to determine differential expression of the miRNAs. As a result, 11,350,396 and 9,890,887 clean reads were obtained in laying and broodiness goose, respectively, and 1,328 conserved known miRNAs and 22 novel potential miRNA candidates were identified. A total of 353 conserved microRNAs were significantly differentially expressed between laying and broody ovaries. Compared with miRNA expression in the laying ovary, 127 miRNAs were up-regulated and 126 miRNAs were down-regulated in the ovary of broody birds. A subset of the differentially expressed miRNAs (G-miR-320, G-miR-202, G-miR-146, and G-miR-143*) were validated using real-time quantitative PCR. In addition, 130,458 annotated mRNA transcripts were identified as putative target genes. Gene ontology annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that the differentially expressed miRNAs are involved in ovarian function, including hormone secretion, reproduction processes and so on. CONCLUSIONS: The present study provides the first global miRNA transcriptome data in A. cygnoides and identifies novel and known miRNAs that are differentially expressed between the ovaries of laying and broody geese. These findings contribute to our understanding of the functional involvement of miRNAs in the broody period of goose.

DNA methylation and regulation of the CD8A after duck hepatitis virus type 1 infection.

BACKGROUND: Cluster of differentiation 8 (CD8) is expressed in cytotoxic T cells, where it functions as a co-receptor for the T-cell receptor by binding to major histocompatibility complex class I (MHCI) proteins, which present peptides on the cell surface. CD8A is critical for cell-mediated immune defense and T-cell development. CD8A transcription is controlled by several cis-acting elements and trans-acting elements and is also regulated by DNA methylation. However, the epigenetic regulation of CD8A in the duck and its relationship with virus infection are still unclear. RESULTS: We investigated the epigenetic transcriptional regulatory mechanisms, such as DNA methylation, for the expression of the CD8A and further evaluated the contribution of such epigenetic regulatory mechanisms to DHV-I infection in the duck. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed the highest level of CD8A expression to be in the thymus, followed by the lungs, spleen, and liver, and the levels of CD8A expression were very low in the kidney, cerebrum, cerebellum, and muscle in the duck. RT-qPCR also demonstrated that the CD8A mRNA was down-regulated significantly in morbid ducklings treated with DHV-1 and up-regulated significantly in non-morbid ducklings in all the tissues tested. In addition, hypermethylation of CD8A was detected in the morbid ducklings, whereas relatively low methylation of CD8A was evident in the non-morbid ducklings. The CD8A mRNA level was negatively associated with the CpG methylation level of CD8A and global methylation status. CONCLUSIONS: We concluded that the mRNA level of the CD8A was negatively associated with the CpG methylation level of CD8A and global methylation status in the duck, suggesting that the hypermethylation of CD8A may be associated with DHV-1 infection. The first two CpG sites of the CD8A promoter region could be considered as epigenetic biomarkers for resistance breeding against duckling hepatitis disease in the duck.

Identification and expression analysis of the leukocyte cell-derived chemotaxin-2 (LECT2) gene in duck (Anas platyrhynchos).

Leukocyte cell-derived chemotaxin 2 (LECT2), first identified as a chemotactic factor, is involved in the regulation of liver regeneration, carcinogenesis, and natural killer T-cell homeostasis in mammals. The function of LECT2 in the duck remains unclear, however. A suppression subtractive cDNA library was constructed from the livers of 3-day-old ducklings treated with duck hepatitis virus type I (DHV-1). A total of 66 expressed sequence tags (ESTs) were identified in the libraries. Among the novel gene fragments identified was the LECT2 gene. Full-length duck LECT2 (duLECT2) complementary DNA (cDNA) was obtained using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). The cDNA consisted of a 50 nucleotide 5' untranslated region (UTR), an 84 nucleotide 3' UTR, and a 1020 nucleotide open reading frame encoding a single protein of 339 amino acids. In agreement with a previously reported LECT2 sequence, the predicted amino acid sequence contains characteristic phosphorylation and N-glycosylation sites. DuLECT2 is highly similar to LECT2 genes from other vertebrates. Phylogenetic analysis demonstrated that the LECT2 gene has been highly conserved throughout vertebrate evolution. RT-PCR analyses revealed that duLECT2 mRNA is widely expressed in healthy tissues. They also showed that duLECT2 mRNA is significantly up-regulated in the liver and spleen following injection with DHV-1 or polyriboinosinic polyribocytidylic acid (poly I:C), peaking 4 or 12h post-challenge in the liver and spleen, respectively, and afterwards gradually returning to normal. Our findings suggest that duLECT2 contributes to the innate immune response against viral infections.

[Study on genetic diversity of wild quail in China with microsatellite DNA markers].

Genetic diversity of domestic quail and two wild quail species distributed in China, wild Japanese quail and wild common quail,was studied by using microsatellite DNA markers. According to the comparison of corresponding genetic index in the three quail populations, such as polymorphism information content (PIC), mean heterozygosity (H) and fixation index etc, wild common quail possessed rich genetic diversity of 4.67 alleles per locus. Its value of PIC and H were the highest, 0.5732 and 0.6621, respectively. Meanwhile, domestic quail had the lowest value, 0.5467 and 0.5933, respectively. Wild Japanese quail had little difference in genetic diversity with domestic quail. In addition,from analyses of fuzzy cluster based on standard genetic distance,the similarity relation matrix coefficients between wild Japanese quail and domestic quail was 0.937, and that between wild common quail and domestic quail was 0.783. All these results showed that wild Japanese quail was closer to the domestic quail in phylogenetic relationship than wild common quail. These results at the molecular level further proved the thesis that domestic quail is originated from wild Japanese quail.

[Determination of major and minor components in standard talc samples by ICP-AES].

The standard talc sample was fused with Na2CO3 and Na2B4O7 and then the fused disc was dissolved with HCl solution. SiO2, MgO, Al2O3, Fe3O2 and CaO in talc samples were determined simultaneously by ICP-AES. The optimum analytical line with high sensitivity and low spectral interference were carefully chosen. The sources and properties of the interference were discussed. The recoveries for these elements were 98.8%-104.4%, with precision of 0.12%-2.4% RSD (n = 6). The results of major and minor components in talc samples by this method were in agreement with those provided by the standard method.