| Ferritin, as an important immune factor, is involved in the immune response of the freshwater pearl shellfish-Hyriopsis schlegelii. In our study, when Hyriopsis schlegelii was challenged by the Gram-positive bacteria Staphylococcus aureus, the expression level of ferrritin(designated as HsFer-1 and HsFer-2) was detected by qRT-PCR. The results showed that the expression level of HsFer-1 increased 1.80-fold, 7.69-fold and 2.82-fold in the haemocyte, gonad and hepatopancreas, respectively. Comparatively, the expression of HsFer-2 increased 1.80-fold, 2.25-fold and 2.34-fold, respectively. Following stimulation with the Gram-negative bacteria Vibrio anguillarum, HsFer-1/2 mRNAs also experienced a different degree of increase in three tissues. After Cu2+ stimulation, the peak levels of Hs Fer-1 were reached to 9.37-fold and 9.61-fold in the tissues of haemocyte and gonad, respectively. The expression level of HsFer-2 was similar to HsFer-1, the peak value were 3.65-fold and 12.64-fold in two tissues. After Pb2+ stimulation, the highest expression levels of HsFer-1 were 1.76-fold and 4.16-fold in the tissues of hepatopancreas and gonad, and HsFer-2 were 2.61-fold and 4.02-fold. The expression of HsFer-1 which exposure to Fe3+ increased 1.53-fold, 6.14-fold and 2.74-fold, and that of HsFer-2 increased 3.59-fold, 4.29-fold and 10.1-fold in three tissues, respectively. After pearl-nucleus-inserting operation, the expression levels of HsFer-1/-2 were down-regulated in haemocyte. However, the expression levels of HsFer-1/-2 reached to 1.80-fold and 6.46-fold in gonad.Antibacterial tests showed that the two subunits of recombinant ferritin had a significant inhibitory effect on Gram-negative bacteria and Gram-positive bacteria. And the antibacterial effect was enhanced with the increase of protein. By analyzing rHsFer-1 and Fe3+-chelating experiments, we could infer that rHsFer-1 could combine with Fe3+ to form a type of chelate and the chelating activity was 39.35% when the concentration of rHsFer-1 was 6ug/ml. In the subsequent inhibition experiments with the chelate, we preliminarily determined that this chelate might be involved in certain types of detoxification. In this study, RNA interference was applied to the freshwater pearl oyster to explore the relationship between the expression of HsFer-1 and HsFer-2. The observation results showed that the haemocyte could stabilize the survival of 24 h. And it confirmed that the existence of the relationship between the expression of HsFer-1 and HsFer-2. When the HsFer-1 was silenced, its expression level was down-regulated and the expression level of HsFer-2 was up-regulated. We inferred conjecture that there was functional complementarity between the two subtypes of HsFer.The location of HsFer in hepatopancreas was researched by immunofluorescence. The results showed that two subtypes of HsFer were all found on the surface of the hepatopancreas cell. In our study, the eukaryotic vectors of PDSred2-N1-Ferritin-1 and PEGFP-C1-Ferritin-2 were successfully constructed. After the vectors were transfected into the haemocyte, we found that ferritin was distributed in the nucleus. The technique of eukaryotic transfection was applied in the freshwater mussel. We found that there was no difference of the position about two subtypes ferritin. However, ferritin was distributed in different types of cell. We hypothesize that there was the functional complementarity relationship between two subtypes of HsFer, so the location of distribution was consistent. However, in different cells, the different functions of ferritin led to the different distribution. |
PDF Full Text Request