Preliminary Study On The Immunomodulatory Role Of Apoptosis-related Gene In Eriocheir Sinensis

The immune defense of organisms is mainly achieved through the specific cells?such as phagocytes?and specific immune processes?such as apoptosis?.Apoptosis plays an important role in embryo development and immune defense.In vertebrates,apoptosis-related genes and their roles in the immune defense have been well studied,but the studies of that in invertebrates,especially in crustaceans are still unclear.In the present study,three apoptosis-related genes including EsCaspase-3/7-1,EsIAP1 and EsSOCS6 were cloned and identified from Eriocheir sinensis.The function and immune regulation mechanism were conducted by using the method of molecular biology,cell biology and bioinformatics.The full length open reading frames of EsCaspase-3/7-1,EsIAP1 and EsSOCS6 were 927 bp,1356 bp and 1266 bp,respectively,which encoded a polypeptide of 323,451 and 421 amino acids,respectively.EsCaspase-3/7-1 contained a CASc domain,which had the conserved Caspase active site motif?QACRG?.EsIAP1 was consisted of two BIR domains.A SH₂ domain and a SOCS box were identified in EsSOCS6.The mRNA of EsCaspase-3/7-1,EsIAP1and EsSOCS6 was constitutively expressed in all the examined tissues with higher expression levels in hepatopancreas and hemocytes,and the immunofluorescence results indicated that the EsCaspase-3/7-1 and EsIAP1 mainly located in the cytoplasm of the hemocytes.The mRNA expression levels of EsCaspase-3/7-1 in hemocytes were obviously up-regulated after challenged with lipopolysaccharide?LPS?and Aeromonas hydrophila.The mRNA expression of EsCaspase-3/7-1 in hemocytes was significantly upregulated at 24 h post challenged with LPS?14.71-fold,p<0.01?,and 12 h post challenged with A.hydrophila?4.93-fold,p<0.01?,respectively.Furthermore,the mRNA expression of EsCaspase-3/7-1 was gradually up-regulated in 0-30 min after the challenge of H₂O₂ in the primary hemocytes of crabs.The recombinant protein of EsCaspase-3/7-1 had hydrolytic activity on the substrate of Caspase-3.In addition,EsCaspase-3/7-1 recombinant protein could directly combine with LPS.The mRNA expression levels of the EsIAP1 in hemocytes were increased after challenged with LPS and A.hydrophila.The mRNA expression of EsIAP1 in hemocytes was significantly upregulated and peaked at 24 h post challenged with LPS?13.86-fold,p<0.01?,and peaked at 48 h post challenged with A.hydrophila?2.71-fold,p<0.01?,compared with that in PBS group.Furthermore,in the challenge of H2O2in the primary hemocytes of crabs,the mRNA expression of EsIAP1 was decreased in0-30 min.The rEsIAP1 could inhibit the hydrolytic activity of rEsCaspase-3/7-1.Pull-down experiment revealed that rEsIAP1 could bind with rEsCaspase-3/7-1 directly.After EsIAP1 was interfered by dsRNA,the activity of Caspase-3 in total protein was up-regulated in hemocytes,and the mRNA expression of EsCaspase-3/7-1 was also increased.The EsSOCS6 could response to the challenge of LPS,A.hydrophila and Poly?I:C?.The mRNA expression of EsSOCS6 in hemocytes peaked at 24 h post challenged with LPS?2.11-fold,p<0.05?compared with that in PBS group.And after the challenge of A.hydrophila,the mRNA expression of EsSOCS6 peaked at 48 h?4.93-fold,p<0.01?compared with that in PBS group.In addition,the mRNA expression of EsSOCS6peaked at 12 h?3.97-fold,p<0.01?post the injection of Poly?I:C?compared with that in PBS group.Furthermore,the mRNA expression of EsSOCS6 was gradually up-regulated in 0-30 min after the challenge of H2O2 in the primary hemocytes of crabs.After EsSOCS6 was interfered by dsRNA,the expression of Caspase-3 was up-regulated in hemocytes,while the expression of EsIAP1 was decreased.The results above have collectively indicated that the apoptosis-related genes,EsCaspase-3/7-1,EsIAP1 and EsSOCS6,were highly conserved in E.sinensis.EsIAP1could decrease the activity of EsCaspase-3/7-1 through combing with it,and EsSOCS6played an essential role in regulating EsCaspas-3/7-1 and EsIAP1.The efficient combination of those three genes was involved in the immune response in Chinese mitten crab.It could play an important foundation in the immune regulation mechanism of crustaceans.