| Biorhythm is a kind of periodic activity that is formed during the long-term evolution of natural things,such as circadian rhythm,annual rhythm,(migration,migration,etc.).The biological clock system regulates the physiological,behavioral and biochemical processes of the organism.Accumulating studies have shown that the biological clock system not only regulates biological rhythms,but also regulates the body’s immune system.In the present study,the research on the biological clock system is mainly focused on the biological rhythm behavior and the study on the influence of the immune system.Given Eriocheir sinensis belongs nocturnal and migratory species,the protein cryptochrome and iron sulfur cluster assembly protein were chosed to preliminaryly explore their functions in circadian rhythm and immune regulation utilizing the means such as bioinformatics,cell biology and immunology.Cryptochromes,as one of important flavoproteins,play important roles in regulating circadian clock.Numerous studies have shown that biorhythm is closely associated with other important life events.Circadian clock system could interact with the immune system by cryptochromes.In the present study,the full length of EsCry cDNA was cloned and was 1662 bp in length including a 5’ untranslated region(UTR)of 132 bp,a 3’ UTR of 132 bp with a poly(A)tail and an open reading frame(ORF)of 1518 bp encoding a polypeptide of 505 amino acids with an estimated molecular mass of 58.14 kDa.The typical DNA photolyase domain,FAD binding domain and a low complexity region were identified in EsCry by SMARTER program analysis.BLASTp homology analysis revealed that EsCry was homologous to Crys from other known model organisms with above 60% identities.EsCry was clustered with other types of Crys of vertebrates,such as Cry4 and Cry5 from D.rerio,Cry4 from G.gallus and C.livia.The mRNA transcripts of EsCry showed the circadian expression at different time points in one day.The highest mRNA expression level of EsCry was detected in hepatopancreas,eyestalk and brain,which was approximate 100.00-fold(p < 0.05),110.00-fold(p < 0.05)and 120.00-fold(p < 0.05)of that in muscle,respectively.The mRNA expression level of EsCry was relatively higher in gill,hemocytes,HPT and heart.EsCry protein was mainly located in the cytoplasm of hemocytes.The mRNA expression of EsCry in hemocytes of crabs from A.hydrophila and LPS stimulation group both was sharply down-regulated from 3 h,reached the minimum level at 24 h,which was 0.13-fold(p < 0.05)and 0.19-fold(p < 0.05)of that in control group,respectively.The expression of EsCry mRNA displayed the similarly inhibition tendency in hepatopancreas.After A.hydrophila stimulation,the mRNA transcripts decreased sharply at 6 h(0.04-fold compared to control group,p < 0.05),and kept at low level until 72 h post stimulation(p < 0.05).And in LPS stimulation group,the mRNA transcripts were obvious inhibited at 3 h(0.38-fold compared to control group,p < 0.05)and dropped to the minimum level at 48 h,which was 0.02-fold(p < 0.05)of that in control group and maintained at the low level until 72 h post stimulation(p < 0.05).The expression level of EsLITAF and EsILF mRNA were up-regulated after EsCry interference,which were 1.61-fold(p < 0.05)and 2.21-fold(p < 0.05)of dsEGFP group.Iron-sulphur clusters(ISCs),one of the oldest and most versatile cofactors of proteins,are involved in catalysis reactions,electron transport reactions,regulation processes as well as sensing of ambient conditions.Iron-sulphur cluster assembly protein(IscA)is a scaffold protein member of ISC formation system,which plays a significant role in the assembly and maturation process of ISC proteins.In the present study,the cDNA sequence of iron-sulphur cluster assembly protein 2(designated as EsIscA2)was cloned from Eriocheir sinensis.The open reading frame(ORF)of EsIscA2 was of 507 bp,encoding a peptide of 168 amino acids with a typically conserved Fe-S domain.A tetrameric form was predicated by the SWISS-MODEL prediction algorithm,and three conserved cysteine residues(Cys-93,Cys-158,Cys-160)from each IscA monomer were predicted to form a ‘cysteine pocket’.The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs.EsIscA2 was clustered with IscA2 proteins from other animals including invertebrates and vertebrates.rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM.EsIscA2 transcripts were detected in all the tested tissues including gonad,hemocytes,gill,muscle,heart,hepatopancreas and eyestalk,and EsIscA2 protein was detected in the mitochondria of hemocytes.The highest mRNA expression level of Es IscA2 was detected in muscle and hepatopancreas,which was about 34.66-fold(p < 0.05)and 27.07-fold(p < 0.05)of that in hemocytes,respectively.After Aeromonas hydrophila and lipopolysaccharide(LPS)stimulations,the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h(0.31-fold,p < 0.05)and 48 h(0.29-fold,p < 0.05)compared to control group,respectively.And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation(p < 0.05).When the primary cultured crab hemocytes were incubated with different concentrations of H2O2 for 15 minutes,the expression level of EsIscA2 mRNA was significantly repressed to the 0.34~0.44-fold of that in the control group.After A.hydrophila stimulation,the mRNA expression of Es Grx2 was up-regulated at 3 h(3.22-fold compared to control group,p < 0.05)and reached the peak at 12 h(4.88-fold,p < 0.05).The above results indicated that cryptochrome,as a biological clock protein,showed circadian oscillation in Chinese mitten crab.The decreased expression after immune stimulation and the up-regulation of the immune effect factors after the intervention of the disturbance,indicated that EsCry could activate the immune system of Chinese mitten crab.Meanwhile,EsIscA2 has iron-binding capabilities as observed in IscA proteins from other organisms,supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab.Its differential mRNA expression after immune and oxidative stress challenges suggests adaptations of ISC synthesis rates to these stress conditions.In a word,this study verified that EsCry and EsIscA2 can regulate the biological rhythm and immune response in Eriocheir sinensis,providing the basic theory basis.for researching the behaviour physiology and immune defense. |
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