Molecular Cloning And Transcriptional Regulation Of FTH1 Gene In Duck

Numerous studies confirmed that the change in the expression of ferritin (FTH1) was closely related to hepatitis.It was found that FTH1 gene was one of the main genes which expression in infection group was down-regulated by suppression subtractive hybridization technique. By mean of the CDS of duFTHl gene analysis among susceptible group, resistant group and control group that treated with duckling virus hepatitis by sequencing, only a silent mutation was found and there were no any other sense mutations, deletions or rearrangements, In order to reveal more of the transcription regulatory mechanism of FTH1 gene in duck, this research was mainly carried out as follows:1.The duFTH1 gene upstream-1110 bp of the ATG was successfully obtained by genome walking, the sequence of 5’flanking was analyzed by online software, a typical CpG island was found, meanwhile, some transcriptional factor binding sites such as Sp-1 were detected and these trans-acting element is also located on CG dinucleotide sites, however, level of duFTH1 gene detected by bisulfite sequencing PCR remained hypomethylation in blood and spleen, and found the same result in susceptible group, resistance group and control group.2. The promoter missing mutants were constructed according to the sequence of FTH1 gene promoter:pGL3-Basic-Ml (+145～765)、pGL3-Basic-M2 (+145～486)、pGL3-Basic-M3 (+145～265)、pGL3-Basic-M4 (+145～144)、pGL3-Basic-M5 (+145～35)、pGL3-Basic-M6 (+145～11)and pGL3-Basic-M7(+145～+50), Dual Luciferase reporter gene showed that, there were important action elements in the region -144 bp/-35 bp which had an effect on promoter activity. Then with screening of the SNP of-144 bp/-35 bp in the susceptible group and the resistance group, only one mutation (-54 C>T) was found among different treatment groups, this mutation was predicted to affect the binding of NRF1 by the software of alibaba2 and jasper online.3. In order to confirm the action mechanism of the SNP(-54 C>T), a point mutagenesis to the possibly existed transcription factor binding sites of NRF1 in the core promoter region, and dual luciferase reporter gene was conducted and it showed that, the luciferase activity of mutant recombinant plasmid(-54 T) was significantly higher than the non-mutant recombinant plasmids (-54 C) (P<0.01). Furthermore, the results of EMS A trail showed that:transcription factor NRFl could bind to the promoter sequence of duFTHl gene, the mutation of site (-54 C>T) significantly reduced the binding bands, competition and super EMSA experiment further showed it could bind with the target DNA fragment specially.From all of above, it can be concluded that the transcription of duFTHl gene was not regulated by its promoter methylation modification, but by the affect of -54 C>T mutation which in -144 bp/-35 bp of core promoter region.