Analysis Of The Role Of Ferritin Gene In The Resistance To Bacterial Infection Of Rana Dybowskii

Ferritin（Fer）,a factor closely related to the antioxidant response and immune response that occurs in living organisms,is mainly composed of two subunits,a light chain（Ferritin light chain,Fer-L）and a heavy chain（Ferritin heavy chain,Fer-H）,of which the Fer-H gene is mainly involved in binding intracellular free Fe²⁺,which is closely related to disease resistance against microbial infection.In recent years,the number of wild populations of Rana dybowskii has declined year by year due to factors such as habitat destruction and excessive hunting capture,and the high density of artificial farming has in turn made microbial infection gradually an important cause of R.dybowskii mortality.To explore the role of the Fer-H gene in amphibian bacterial infection.In this study,we first cloned the full-length coding region of Fer-H gene and analyzed its gene characteristics using bioinformatics software,based on the data obtained from R.dybowskii;Secondly,the infection stress of R.dybowskii by Aeromonas hydrophila（Ah）and bacterial Lipopolysaccharide（LPS）was used to construct an inflammation model,the tissue lesions were observed by HE staining,and the activities of tissue antioxidant enzymes were measured;Finally,we analyzed the changes of gene transcript levels and protein levels of R.dybowskii Fer-H after infection using quantitative real-time PCR（q RT-PCR）,immunofluorescence detection,and Western blot,respectively.The main findings are as follows:（1）In this study,534 bp of the full-length coding region of the R.dybowskii Fer-H gene encoding 177 amino acids was obtained.This sequence presents 13 potential sites of phosphorylation,no N-glycosylation sites;Contains a Pfam ferritin conserved domain;The Fer-H genes of R.dybowskii are most homologous to Rana temporaria,with the closest relative,followed by other amphibians and fish,and more distantly related to mammals.（2）Pathological changes and tissue damage were observed in the liver,skin and muscle tissues of R.dybowskii under Ah and LPS stress;Within 6-120h of stress,there were changes in the activities of antioxidant enzymes in liver、spleen、kidney、skin and muscle,and the activities of SOD and CAT in each tissue basically showed a tendency to increase first and then to reach,and were generally higher than those in the control group;GSH activity almost all showed a decreasing trend and was lower than that of the control group.（3）Using q RT-PCR,we found that under normal physiological conditions,Fer-H gene was expressed in all tissues of R.dybowskii,with the highest expression in spleen and the lowest expression in lung.Upon ah stress,upregulated expression of Fer-H genes was observed in all tissues of R.dybowskii,with kidney tissues peaking at 24h of infection and heart,liver,spleen,lung,stomach,skin and muscle tissues peaking at 48h of infection;Upon LPS stress,Fer-H genes also showed up-regulated expression,with heart tissue peaking at 72h of infection,liver and kidney peaking at 48h of infection,and spleen,lung,stomach,skin and muscle tissues peaking at 24h of infection.（4）The results of immunofluorescence assay showed that the trends of positive granules in the liver and muscle tissues of R.dybowskii after stressed by Ah and LPS were largely consistent with the results of q RT-PCR;The results of Western blot showed that the Fer-H protein in both liver and muscle tissues of R.dybowskii peaked after 48h of Ah stress,which was the same as that detected by immunofluorescence.In this study,the Fer-H gene of R.dybowskii was cloned to construct the model of bacterial infection of R.dybowskii by Ah and LPS,and the altered regulation of the Fer-H gene at the transcriptional and translational levels under both stresses was examined and analyzed,which may provide a basis for analyzing the function of the Fer-H gene in bacterial infection of amphibians.