Isolation,Identification And Functional Analysis Of Ferritin And Subunits Of Eri-silkworm,Samia Cynthia Ricini

Iron is an important nutrient and is ubiquitous in various tissues of living organisms.Ferritin is an iron-binding protein that is widely distributed and plays a vital role in regulating iron storage and transport,antioxidant stress,cell cycle,and resistance to pathogens.In recent years,insect ferritin has attracted much attention in the regulation of immune responses.In this study,functional analysis of ScFerritin and subunits is expected to complement the study of insect Ferritin to some extent.This study cloned the cDNA sequence encoding the eri-silkworm Ferritin heavy chain subunit(ScFerHCH)and light chain subunit(ScFerLCH)genes.The eri-silkworm complex and its subunits were isolated.The tissues distribution of the heay chain subunit(ScFerHCH)and light chain subunit(ScFerLCH)of ScFerritin and the expression mode of ScFerHCH and ScFerLCH response to iron ions and pathogens were obtained.A recombinant ScFerritin subunit can self-assembly form complex in vitro.The complex and subunits can bind to the pathogen.The specific research results are as follows:1.Bioinformatics analysis of the ScFerHCH and ScFerLCHBased on the transcriptome data of eri-silkworm,the cDNA nucleotide sequence and encoded amino acid sequence of ScFerHCH and ScFerLCH were identified and analyzed,and the signal peptides and the ORF reading frame of ScFerHCH and ScFerLCH were determined.Homology analysis show that the ScFerritin subunits have high homology with the Bombyx mori Ferritin subunits and is also very similar in three-dimensional structure,suggesting that the two have similar functions.Both ScFerHCH and ScFerLCH have an iron responsive element(IRE),but only ScFerHCH contains a ferroxidase center.2.Extraction and identification of ScFerritin and subunitsScFerritin was isolated from hemolymph of the larvae by Native-PAGE,and two subunits(ScFerHCH and ScFerLCH)were identified by Western-blot and MALDI-TOF/TOF MS.3.Tissue distribution of ScFerHCH and ScFerLCHThe tissue distribution of ScFerHCH and ScFerLCH was analyzed by RT-qPCR and Western-blot from the transcriptional level and protein level.The results showed that ScFerHCH and ScFerLCH were widely present in various tissues of eri-silkworm,and have a high expression in the midgut,hemolymph,silk gland and fat body.4.Analysis of iron ion chelation ability,antioxidant capacity and expression pattern of iron ion stimulation of ScFerHCH and ScFerLCHThe recombinant ScFerHCH protein can chelate free iron ions and also exhibit the ability of the antioxidant H2O2,However,recombinant ScFerLCH protein does not chelate free iron ions and has no antioxidant capacity.After iron ion challenged,the expression levels of ScFerHCH and ScFerLCH were significantly up-regulated in the midgut,hemolymph and fat body.5.Self-assembly of recombinant ScFerHCH and ScFerLCH proteins in vitro and the ability to respond to pathogenic bacteriaThe refolded recombinant ferritin subunits can self-assemble to form a complex in vitro,and the ability of the complex to chelate iron ions is significantly higher than ScFerHCH.Both the complex and the two subunits can bind to four pathogens,but complex don’t directly inhibit the growth of pathogens.When S.aureus and P.aeruginosa stimulated eri-silkworm,the expression levels of ScFerHCH and ScFerLCH were significantly up-regulated.In summary,this study extracted the Ferritin from the hemolymph of larvae of the eri-silkworm for the first time and isolated two Ferritin subunit,and proved that ScFerHCH has the ability to chelate iron ions and resist oxidation.when stimulated by iron ions,the expression levels of ScFerHCH and ScFerLCH in tissues were significantly up-regulated.Studies have also demonstrated that Ferritin subunits can be involved in the immune stimulation of pathogenic bacteria.In addition,recombinant Ferritin subunits can self-assembled form complex in vitro.Both the complex and subunits can bind to the pathogen,but it does not directly inhibit the pathogen.The above results provide a certain reference value for further study of the biological function of ScFerritin and the molecular mechanism of related immune responses.