Molecular Cloning, Expression And Characterization Of Three Ferritins From The Housefly, Musca Domestica

The homeostasis of serum ferritin which is very important for human health, is often used as a detection indicator of certain diseases. As well as, in the housefly, the change of ferritin often reflects to external environmental stress, which provides new insights for research of housefly immunity. Musca domestica is one of the most important model organism for the study of insect immunology for their special living environment and shorter life cycle. In this study, the housefly as experimental materials, the main contents are as follows:Part 1: c DNA sequence of three Mdfers were obtained by searching housefly transcriptome data and using RT-PCR, RACE techniques. The nucleotide sequences and deduced amino acid sequences of the three Mdfers were analyzed by bioinformatic method.Part 2: Quantitative real-time PCR(q RT-PCR) and Western blot were employed to investigate the tissue distribution of Mdfer L and Mdfer H subunit, and temporal expression in housefly larvae challenged with bacteria and Fe2+.The results are as follows:1. The complete c DNA sequence of Mdfer L was 1105 bp, containing a open reading frame of 672 bp and encoding 223 amino acids. The predicted molecular weights of Mdfer L was 24.9 k Da. Mdfer H1 and Mdfer H2 were two alternative spliceosome of the Mdfer H gene.The full-length c DNA of them were 1417 bp and 1172 bp, they had exactly the same ORF sequence of 618 bp and 3’-untranslated region, encoding the same 206 amino acids and predicted protein molecular size of 23.1 k Da.2. The results of q RT-PCR showed that all of three Mdfers had the highest relative expression in gut. The relative expression level of three Mdfers were up-regulated post bacterial challenge. Together with the result that all of three Mdfers were significantly induced after iron challenge, and stability maintained at a higher level.3. Western blot analysis revealed that the highest expression was detected in gut.Mdfer L was clearly up-regulated at 3 h to12 h post pathogen stress, while Mdfer H was nosignificant difference. Meanwhile, a significant up-regulation of two Mdfers are found post iron challenge, the expression level of Mdfer L reached peak at 24 h to 36 h after stimulation,while Mdfer H reached peak at 3 h to 6 h. At last, Mdfer L and Mdfer H were found to be more abundant in the third-instar larvae followed by adult fly, while, only a very slight expression was found in the first-instar larvae. The relative expression levels of the Mdfers in response to different concentration of iron were slightly different, the relative expression level of Mdfer L was increasesd with the concentration of iron increased, which was in range of 0.15-0.2 M.while the relative expression levels of Mdfer H reached peak exposed to 0.05 M iron.