Molecular Cloning Of FTH1gene And Its Functional Analysis In Jinding Duck

Ferritin occurs as a soluble protein which can store iron in eukaryotic. It is composed of two subunits:heavy chain and light chain, encoded by two distinct genes. The heavy chain contains a ferroxidase center and is thought to be responsible for the rapid detoxification of iron. It performs the primary function of ferritin. Ferritin heavy chain (FHC) is encoded by ferritin heavy chain polypeptide1(FTH1) gene, the gene expression is regulated transcriptionally and post-transcriptionally, and iron response proteins mediate the post-transcriptional regulation. Although ferritin possesses many iron-related functions, recent studies have shown that it also has various functions that are might be unrelated to iron. More and more studies are indicating that ferritin is associated with disease pathogenesis, such as hepatitis, diabetes, tumour and leukemia. So the major function of FHC is protecting the cell from oxidative damage. The report also indicates that ferritins are known to be involved in innate immunity in vertebrates. Our group screened out differentially expressed genes after duck hepatitis virus (DHV) infection by suppression subtractive hybridization technique, and found that FTH1was one of the key genes in duck virus hepatitis.In this study, the JinDing duck FTH1coding region sequence was cloned, and then FTH1mRNA expression of different tissues was detected by qRT-PCR technique. Furthermore, the change of FTH1and apoptosis-related genes expression were analyzed after DHV-1infection to find the internal relation between FTH1and duck virus hepatitis. In order to further study the function of FTH1in duck, we used the genetic engineering and transfection technique to construct the FTH1eukaryotic expression vector and RNA interference expression vector and identify them. And the mRNA expression of apoptosis-related genes was analyzed by qRT-PCR after vector transfection. This study aimed to explore the regulation mechanism of FTH1and its function in duck virus hepatitis. It provided new evidence for the study of the relationship between FTH1and diseases, and supplied new genetic resources for the disease resistance breeding. Specific findings are as follows: 1. In this research, the FTH1coding region sequence was obtained by RT-PCR and TA cloning technique. The length of FTH1CDS is546bp, which encodes181amino acids. Afterwards, the FTH1mRNA expression in different tissues was analyzed by qRT-PCR. The result showed that FTH1expressed in all detective tissues, the expression was relatively high in liver, spleen and lung, and relatively low in heart and kidney.2. The FTH1expression in liver and spleen was detected after DHV-1infection, and found that the mRNA expression of FTH1in infection group down-regulated significantly (P<0.05). It means that the FTH1expression of liver and spleen is inhibited by DHV-1infection. After infection, apoptosis inhibitory factor Bcl-2down-regulated significantly (P<0.05) and apoptosis promoting factor Caspase-3, Caspase-8expression increased significantly (P<0.05). Therefore, DHV-1may induce apoptosis.3. FTH1eukaryotic expression vector and RNA interference expression vector were constructed using genetic engineering technique and identified by transfecting into the duck embryo fibroblasts. Furthermore, we found that overexpressed FTH1could promote the expression of Bcl-2and inhibit the expression of Caspase-3, Caspase-8. On the contrary, the suppression of FTH1expression could promote apoptosis. The results indicated that the change of FTH1expression may affect apoptosis.