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Cloning and expression analysis of IRF3 in Gadus macrocephalus us-ing absolute quantitative PCR
Pages: 887-895
Year: Issue:  5
Journal: Journal of Fishery Sciences of China

Keyword:  Gadus macrocephalusinterferonabsolute quantitative PCR;
Abstract: Gadus macrocephalus is a marine teleost that is economically important throughout the world. The species has a high nutritional value and is processed into a variety of products. As a result of the increase in fisheries targeting this species, the abundance of wild populations has declined. Our laboratory has successfully carried out artificial breeding and rearing of G. macrocephalus. However, we observed significant mortality as a result of immune system dysfunction. To improve artificial breeding techniques for G. macrocephalus, the molecular mechanisms and expression characteristics of immune-related genes needs to be understood. Interferon regulatory factor 3 (IRF3) is a member of the interferon regulatory factor family. It is an important transcription factor for the expression of the interferon α/βgene and plays an important role in the host antiviral response mechanism. We obtained a cDNA sequence of G. mac-rocephalus IRF3 gene for the first time using gene cloning. The sequence was 1878 bp in length with a whole open reading frame (ORF) of 1377 bp that encoded a 459 amino acid protein. The molecular weight of the encoded protein was~51 kDa. Amino acid sequence alignment revealed that the protein included a DNA binding domain (DBD) con-taining five conserved tryptophan residues and a conserved C-terminal IRF association domain (IAD). The phylogenetic tree revealed that the IRF3 of G. macrocephalus was clustered with the IRF3 of other species, distant from IRF1 and IRF2. The tissue and age-specific expression of IRF3 was detected using absolute quantitative PCR. Additionally, the effect of interferon during the larval stages was analyzed. The expression levels were highest in the gonad, liver, and thymus. The copy number of IRF3 in the different tissues was: 231.244 copies/ng, 516.649 copies/ng, 695.158 cop-ies/ng, 2128.273 copies/ng, 198.548 copies/ng, 101.758 copies/ng, 419.927 copies/ng, 13.016 copies/ng, 1102.775 cop-ies/ng, and 13.016 copies/ng in the intestine, heart, thymus, gonad, gill, muscle, spleen, brain, liver, and kidney, respec-tively. The expression of IRF3 was detected in fertilized eggs and remained relatively constant to 5 d post-hatching (dph) but increased by 25 dph. The copy number of IRF3 at different ages was:6.189 copies/ng, 6.809 copies/ng, 8.066 cop-ies/ng, 5.009 copies/ng, 5.009 copies/ng, 10.390 copies/ng, and 10.390 copies/ng in the fertilized egg, 1 dph, 5 dph, 9 dph, 17 dph, 25 dph, or 33 dph, respectively. Our results suggest that the gonad, liver, and thymus are the primary or-gans for IRF3 expression. Additionally, our results suggest that interferon plays an antiviral role at 25 dph. The results of this study lay the foundation for further study of the mechanism of interferon action during the early development of G. macrocephalus and provide basic data for studies of the immune system function in G. Macrocephalus.
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