Cloning And Functional Characteristics Of Some Immune-related Genes From Mud Crab, Scylla Paramamosain

Scylla paramamosain is one of the most important cultivated marine crabs in Southern China.However,the knowledge on its immune responses against bacterial infection is still poor.Eleven novel genes belonging to five families have cloned and their genomic organizations have been clarified in this study.In addition,their expression profiles in different developmental stages of embryo and larvae,and multiple tissues from na?ve and bacterial challenged adult crab were investigated.The results will enrich the base immunologic knowledge of crab and will be helpful for controlling and preventing the bacterial diseases of mud crab.1.Two novel C-type lectins,named Sp-lectin3 and Sp-lectin4,were cloned from Scylla paramamosain.The genomic organizations of Sp-lectin3 and Sp-lectin4 were 5-exon/4-intron and 4-exon/3-intron,respectively.The putative peptides possessed several conserved structure of C-type lectins,such as a signal peptide and a single Carbohydrate Recognition Domain（CRD）.In na?ve adult crabs,both genes were expressed highest in hepatopancreas,and the expression level of Sp-lectin4 was higher than that of Sp-lectin3 in almost all the tested tissues with exception in brain and ejaculatory ducts,where Sp-lectin3 expressed significantly higher than Sp-lectin4（p<0.01）.Furthermore,with the development of embryo and larvae,the expression levels of both genes increased gradually and reached a peak at the stage of zoea.In vivo,artificial challenge with Vibrio parahaemolyticus significantly up-regulated the expression of Sp-lectin3in reproductive system and Sp-lectin4 in hepatopancreas.The results suggested that the two C-type lectins might be involved in the immune response of Scylla paramamosain against bacterial infection and Sp-lectin3 functioned bias in reproductive system whilst Sp-lectin4 in hepatopancreas.2.Three Macrophage migration Inhibitory Factor（MIF）genes,named Sp-MIF,Sp-MIF1and Sp-MIF2,were cloned.All of their genomic organization was 3-exon/2-intron.Their precursor peptides possessed a conservative catalytic site of isomerase.The expression of Sp-MIF was focused in hepatopancreas.Contrarily,the expressions of both Sp-MIF1 and Sp-MIF2 were detected in all the tested development stages and tissues,where Sp-MIF2expressed higher than Sp-MIF1 with exceptions in heart and crablet.In vivo,artificial challenge of Vibrio parahaemolyticus significantly up-regulated the expression of Sp-MIF in hepatopancreas and down-regulated the expression of Sp-MIF1 in gill.The results indicated that both Sp-MIF and Sp-MIF1 might play a role in the innate immunity of crab against bacterial infection.3.Three Gamma-interferon Induced Lysosomal Thiol reductase genes（GILT）were firstly cloned in crab,named Sp-GILT1、Sp-GILT2 and Sp-GILT3 and their precursor peptides were encoded by four exons,one exon and 5 exons,respectively.The putative peptides possessed conservative characterizes of GILT family,such as a functional unknown signature“CQHGX₂ECX₂NX₄C”,an active site motif of thiol reductase activity“CXXS（expect Sp-GILT2）”and conserved 10 to 11 cysteines.Sp-GILT1 was expressed concentrated in hepatopancreas,Sp-GILT2 was mainly expressed in reproductive system and digestive system and Sp-GILT3 was highly expressed in both hepatopancreas and hemocyte.SpGILT3 was expressed highest in early developmental stages of embryo while SpGILT1 in the other stages.In vivo,artificial challenge of Vibrio parahaemolyticus significantly up-regulated their expression in hepatopancreas and Sp-GILT2 also in reproductive system.The results indicated that Scylla GILT had multiple variants and all of them might participate in the innate immune response against bacterial infection.4.A novel fatty acid binding protein was cloned from Scylla paramamosain,named Sp-FABP1.In na?ve crabs,it was highly expressed in hepatopancreas and widely distributed in the development stages.In vivo,artificial challenge with mixed bacteria significantly down-regulated its expression in hepatopancreas and gill,which suggest that Sp-FABP1 might involve in the immune response of crab to mixed bacterial infection.Furthermore,two metallothionein genes,Sp-MT1 and Sp-MT2,were cloned.They were expressed highest in hepatopancreas,and showed a basal expression in embryonic and larval stages with high expression at megalopa.In vivo,artificial challenge of Vibrio parahaemolyticus could significantly regulated their expressions in multiple tissues.The results indicated that metallothionein might also involve in the immune response against bacterial infection.