Dynamic Expression Pattern of SERPINA1 Gene from Duck (Anas platyrhynchos).

SERPINA1 is a member of serine protease inhibitors and is increasingly considered to be a regulator of innate immunity in human and animals. However, the expression and function of SERPINA1 gene in immune defense against viral infection remain unknown in ducks. The full-length du SERPINA1 cDNA sequence was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It contained 1457 nucleotide, including 47-bp 5' UTR, 135-bp 3' UTR, and 1275-bp open reading frame (ORF), and encodes a 424-amino acid protein. Then, the tissue expression profile of du SERPINA1 gene was determined. Real-time quantitative polymerase chain reaction (real-time qPCR) analysis revealed that du SERPINA1 mRNA is ubiquitous in various tissues, but higher expression levels were observed in lung and liver tissues. In addition, the expression pattern was investigated when the ducklings were challenged with duck hepatitis virus 1(DHV-1) and polyriboinosinic polyribocytidylic acid (poly I:C). After DHV-1 injection or poly I:C treatment, du SERPINA1 mRNA was up-regulated in the liver and kidney tissues. However, the peak time in two tissues was not consistent. In kidney, the expression lever of SERPINA1 increased immediately after the treatment while in liver tissue it kept steady until 12 h post-infection. Our results indicate that SERPINA1 has an active role in the antiviral response, and thus improve our understanding of the role of this protein.

Roles of miRNA-1 and miRNA-133 in the proliferation and differentiation of myoblasts in duck skeletal muscle.

MicroRNA (miRNA)-1 and miRNA-133 are derived from the same bicistronic pairs with roles in skeletal muscle development. Many investigations have focused on the role of miRNA-1 and miRNA-133 in the regulation of skeletal muscle development in mammals and fish. However, the mechanisms of miRNA-1 and miRNA-133 underlying the differences in skeletal muscle development between different breeds are not well known. Our study found that the weights of body and breast at 42 days of age were greater in Cherry Valley ducks than in Putian ducks and the areas of breast muscle fibers increased with age; the areas of muscle fibers of Cherry Valley ducks were always greater than those of Putian ducks. Besides, quantitative reverse-transcriptase polymerase chain reaction analysis revealed that relatively high levels of miRNA-1 and miRNA-133 were detected in heart, breast, and leg muscles compared with the liver, spleen, lung, kidney, and the expression levels of miRNA-1 and miRNA-133 remained stable in the embryo stage, and in the growth period, the fluctuation in miRNA expression levels in Putian ducks was considerably higher than that in Cherry Valley ducks, especially from 7 to 28 days. However, in the late growth period, the expression of miRNA-1 and miRNA-133 of Cherry Valley duck was higher than that of Putian duck, which may indicate that miRNA-1 and miRNA-133 play a more important role during the growth period. To determine the function of miRNA-1 and miRNA-133 in skeletal muscle development, we found that the overexpression of miRNA-1, but not miRNA-133, promoted fusion of adjacent myoblasts. By contrast, a repressor of miRNA-1 promoted, whereas a miRNA-133 inhibitor inhibited, myoblast proliferation. Accordingly, the expression levels of myocyte enhancer factor 2D (MEF2D) and myogenic differentiation ( MYOD) were significantly increased by an miRNA-1 mimic and the miRNA-133 inhibitor. In addition, we found that the expression levels of miRNA-1 significantly affected the expression of histone deacetylase 4 ( HDAC4), and miRNA-133 affected serum response factor ( SRF) and transforming growth factor ß receptor 1 ( TGFBR1) levels. However, dual-luciferase reporter assays revealed that only miRNA-1 directly inhibited pGL- HDAC4 luciferase reporter activity, whereas miRNA-133 did not affect pGL- SRF or pGL- TGFBR1 fluorescence activity. Taken together, these results suggest that miRNA-1 targets HDAC4 to promote the differentiation of duck myoblasts and miRNA-133 may affect SRF and TGFBR1 expression to promote proliferation, which indicates that miRNA-1 and miRNA-133 play different important roles in skeletal muscle development.

FTH1 expression is affected by promoter polymorphism and not DNA methylation in response to DHV-1 challenge in duck.

Ferritin heavy polypeptide 1 (FTH1) plays a pivotal role in response to viral infections. FTH1 expression is modulated by various pathogens, but the regulatory mechanisms are unknown. We firstly construct duck hepatitis virus 1 (DHV-1) infection model, including morbid ducklings, non-morbid ducklings and control ducklings. Then the mRNA expression of duck FTH1 (duFTH1) was measured mRNA expression of duck FTH1 (duFTH1) in the liver and spleen after duck hepatitis virus 1 (DHV-1) infection using quantitative polymerase chain reaction (qPCR) and found that duFTH1 mRNA was down-regulated significantly in morbid ducklings (liver, P < 0.01; spleen, P < 0.05) compared with the control ducklings. We also found that duFTH1 expression was significantly higher in the spleen (P < 0.01) and liver (P < 0.05) of non-morbid ducklings than in morbid ducklings. Moreover, DNA methylation of the duFTH1 promoter was examined by bisulfite sequencing (BSP) and we found that the duFTH1 promoter was hypomethylated, the relative methylation was only 5.9% and 2.0% in the morbid ducklings and non-morbid ducklings, respectively. The promoter contained a -55 C/T mutation in 75% of non-morbid ducklings, and this polymorphism affected promoter activity. Further analysis suggested that this mutation altered the binding site of the transcription factor NRF1. Binding of NRF1 to the FTH1 promoter was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the NRF1 was a negative regulator, and lossed of binding of NRF1 to duFTH1 promoter due to -55C/T mutation enhances duFTH1 expression in non-morbid ducks, which provided molecular insights into the effect of duFTH1 expression via promoter polymorphisms, but not DNA methylation, in response to DHV-1 challenge.