Vol 49(2015) N 1 p. 120-129; DOI 10.1134/S0026893315010021
R.A. Kajungiro, L. Xue*, M. Aynealem
Molecular cloning and expression patterns of two tumor necrosis factor alpha genes in Crucian carp (Carassius carassius)
College of Marine Sciences, Laboratory of Molecular Biology, Ningbo University, Ningbo, Zhejiang, 315211, P.R. China
Tumor necrosis factor (TNF) is a potent inflammatory cytokine produced during inflammation. In this study, two crucian carp TNF-α genes, TNFα-1 and TNFα-2, were cloned and sequenced. The TNFα-1 is 720 bp long and consists of a 699 bp opening reading frame (ORF) encoding 232 amino acids and TNFα-2 is 793 bp long and contains an ORF of 687 bp, which encodes 228 amino acids. The genomic structure of both genes consists of 4 exons and 3 introns similar to the other known TNF-α genes. The amino acid sequences of the crucian carp TNFα-1 and TNFα-2 share the highest identity with common carp, goldfish and the lowest with flounder and humans. The phylogenetic analysis grouped crucian carp TNFα-1 and TNFα-2 with other cypriniformes, which are more closely related to goldfish and common carp TNFα-1 and TNFα-2. Real time PCR analysis showed a constitutive expression of crucian carp TNFα-1 and TNFα-2 in all seven tissues examined. The TNFα-1 mRNA was expressed significantly higher in the liver and kidney than those of TNFα-2, while TNFα-2 in turn was expressed significantly higher in the muscle. The Aeromonas hydrophila BSK-10 strain upregulated the expression level of both TNFα-1 and TNFα-2 in all of the tested tissues. At 6 h, the expression levels of TNFα-1 were increased significantly higher in the muscles, skin, and liver, while the expression levels of TNFα-2 were increased significantly higher in the muscles and gills. TNFα-1 was expressed much stronger than TNFα-2. At 12 h the expression levels started to decline and were even lower at 24 h. These results imply that both TNFα-1 and TNFα-2 mRNA are distributed differently in tissues and are implicated in the immune response to bacterial infection.
Aeromonas hydrophila BSK-10, Crucian carp Carassius carassius, mRNA, expression patterns, real time PCR, tumor necrosis factor α