Functional Study On The Involvment Of Beclin-1,AMPK? And Akirin In Antibacterial Immunity In Chinese Mitten Crab Eriocheir Sinensis

Chinese mitten crab Eriocheir sinensis is one of the most importantly aquacultural species with prominently economic and scientific value in China.However,the increasing diseases caused by bacteria have resulted in considerably economic losses.In the present study,the molecular characteristics of Beclin-1(designated as EsBeclin-1),AMP-activated protein kinase ? subunit(designated as EsAMPK?)and Akirin(designated as EsAkirin),as well as their functions in regulating the expression of AMPs in E.sinensis were explored by molecular biological methods including immunofluorescence,quantitative real-time PCR(qRT-PCR),western blot analysis and double strands RNA interference(dsRNAi).The open reading frame(ORF)of EsBeclin-1,EsAMPK? and EsAkirin were of 1 275 bp,888 bp and 615 bp,encoding a polypeptide of 424,295 and 204 amino acids,respectively.EsBeclin-1 contained a typical APG6 domain,as well as CCD and ECD domains.Es AMPK? possessed a typical AMPK? domain and a CBM domain.There were no definite domains in EsAkirin,while the C-and N-terminal regions were relatively conserved with two nuclear localization signals(NLSs).The mRNA expression of EsBeclin-1,EsAMPK? and EsAkirin could be detected in all the examined tissues with relatively higher expression in hemocytes.Further,the immunofluorescence was carried out to explore the subcellular localization of the three proteins.EsBeclin-1 and EsAMPK? protein mainly located in the cytoplasm of hemocytes,while the EsAkirin protein located in the nucleus.The mRNA expression patterns of EsBeclin-1,EsAMPK? and EsAkirin in E.sinensis hemocytes were determined by qRT-PCR after lipopolysaccharide(LPS)and Aeromonas hydrophila stimulations.Post stimulations with LPS and A.hydrophila,the mRNA expression of EsBeclin-1,EsAMPK? and EsAkirin in hemocytes were obviously up-regulated and reached the highest level at 6 h(4.70 ± 0.70-fold,p < 0.01,and 2.91 ± 0.46-fold,p < 0.01),12 h(3.19 ± 0.58-fold,p < 0.01,and 2.69 ± 0.30-fold,p < 0.01)and 24 h(5.04 ± 1.01-fold,p < 0.01,and 3.01 ± 0.84-fold,p < 0.01),respectively.Additionally,EsBeclin-1 protein in crab hemocytes gathered together and displayed as punctuate in the cytoplasm at 2 h after LPS stimulation.For exploring the function of EsBeclin-1,EsAMPK? and EsAkirin in control of antimicrobial peptides(AMPs)expression,dsRNAi was used to inhibit the expression of EsBeclin-1,EsAMPK? and EsAkirin(the interfering efficiency all over 60% at 24 h post dsRNA injections),and the mRNA transcripts of some AMPs in hemocytes were examined.The results showed,the transcripts of EsALF2(0.55 ± 0.08-fold,p < 0.05),EsLYZ(0.21 ± 0.04-fold,p < 0.01),EsCrus(0.49 ± 0.08-fold,p < 0.01)and EsCrus2(0.46 ± 0.11-fold,p < 0.01)were all significantly down-regulated at 6 h after LPS stimulation in EsBeclin-1-interfered crabs compared to the control group.After EsAMPK? was interfered,the mRNA expression of EsALF2(0.44 ± 0.10-fold,p < 0.01)and EsLYZ(0.69 ± 0.20-fold,p < 0.05)were significantly decreased.After EsAkirin was interfered,the transcripts of EsALF2(0.26 ± 0.07-fold,p < 0.01),EsLYZ(0.54 ± 0.10-fold,p < 0.01),EsCrus2(0.54 ± 0.17-fold,p < 0.01)and EsDWD1(0.56 ± 0.10-fold,p < 0.05)were all obviously decreased at 12 h after LPS stimulation.Further,the dual-luciferase reporter assay showed that EsAkirin could significantly enhance the luciferase activity of NF-?B promoter(2.72 ± 0.40-fold,p < 0.01).The above results have collectively indicated that EsBeclin-1,EsAMPK? and EsAkirin were highly conserved with functional regions.EsBeclin-1,EsAMPK? and EsAkirin were all constituvly expressed and involved in the immune responses aroused by LPS and A.hydrophila in E.sinensis.EsBeclin-1 and EsAMPK? protein mainly located in the cytoplasm of hemocytes,EsAkirin protein located in the nucleus.EsBeclin-1 could affect the mRNA transcripts of EsALF2,EsLYZ,EsCrus and EsCrus2,EsAMPK? might participate in regulating the expression of EsALF2 and EsLYZ,EsAkirin might play an essential role in regulating EsALF2,EsLYZ,Es Crus2 and EsDWD1.These imply that some relations might exist among EsBeclin-1,EsAMPK? and EsAkirin in coregulating of EsALF2 and Es LYZ,and participating in the antibacterial immunity in E.sinensis.These studies not only laid an important theoretical foundation for further studies on the antibacterial immunity of crustaceans,but also provided a reference for studying the invertebrate immune system.