Cloning And Expression Analysis Of C Type Lectin Genes Of Scylla Paramamosain

C type lectins are animal lectins with one or more calcium-dependent carbohydrate recognition domains(CRD),which exist in almost all metazoan animals and play an important role in innate immunity and adaptive immunity.In vertebrates,C type lectins are conservative,most contains only a carbohydrate recognition domain,while C type lectins’ types of invertebrates are more diverse,and functions are also more complex.Researches of C type lectins in invertebrates help clarify innate immune mechanisms,and enrich the researches of C type lectin family.Scylla paramamosain is an important crab species in China,mainly distributed in the southeast coastal areas of the Yangtze river.But the survival rate of crabs in artificial culture is low,and bacterials or virals infection will aggravate this situation.As a kind of invertebrate animal with only innate immune system,the study on the innate immune mechanism of Scylla paramamosain to improve its own immunity is of great significance for the production.In this study,three C type lectin genes of Scylla paramamosain were obtained,and the pathogen stress experiment was carried out.The expression model was analyzed by real-time fluorescence quantitative technology,and the following results were obtained:1.A C type lectin gene containing CTLD domain was successfully cloned by RACE method and named SpCTLD.The cDNA sequence of the gene was 1237 bp,containing a 468 bp open reading frame,which encoding 155 amino acids.And the predicted molecular weight is 18.32 KDa and isoelectric point is 7.56.The gene contained two SNP sites at 390(A390G)and 1020(T1020C)sites.The expression pattern of SpCTLD in different stages of the larval and tissues of Scylla paramamosain showed that SpCTLD was expressed in all developmental stages of larval,and the highest expression was observed in the fifith stage of the larval,followed by the early juvenile crab.And the second juvenile crab is the lowest.The tissue distribution analysis of SpCTLD showed that the highest expression level was found in the gill,followed by the ejaculatory duct.The expression level of the two tissues was much higher than that of other tissues,and it was also found in hemolymph,hepatopancreas and other immune-related tissues.The stress experiment of Vibrio parahaemolyticus showed that the mRNA expression levels in the hepatopancreas,hemolymph and gill of the live bacteria group were significantly up-regulated,and reached the highest at the 12 th hour.Based on the results of this study,it is speculated that SpCTLD is a novel gene containing the CTLD domain,which may be involved in the antibacterial response of Scylla paramamosain.The discovery of this gene enriches the study of C type lectin genes in Scylla paramamosain,and is conducive to the improvement of the innate immune mechanism of crustaceans.2.In this study,two other C type lectin genes were obtained from Scylla paramamosain: SpMR and SpCollectin-12-like.The SpMR gene has a full-length cDNA of 1762 bp and contains a 936 bp open reading frame encoding 311 amino acids.It also includes a 258 bp 5’ UTR and a 568 bp 3’ UTR.It was predicted 35.4KDa of molecular weight and 4.12 of isoelectric point,containing a signal peptide of 18 amino acids.There are two C-type lectin-like domains(CTLDs)in the coding region: The 30 th to 157 th amino acids and the 180 th to 307 th amino acids,and each of them contains a conserved recognized motif of EPN and WND.The cDNA of Spcollectin-12-like is 2293 bp,and contains an 819 bp open reading frame,which encoding 272 amino acids.The gene also contains a 346 bp 5 ’UTR and a 1128 bp 3’ UTR.Its molecular weight was predicted to be 29.8KDa,and its isoelectric point was 6.09,containing a signal peptide with 24 amino acids.There is a CTLD domain,the 143rd-266 th amino acid,which contains a conserved recognized motif of WND.Through the stress experiment of Vibrio parahaemolyticus,it was preliminarily proved that both of two genes were involved in the anti-bacterial immune response of Scylla paramamosain.This study enriches the researches of C type lectins in the innate immunity of Scylla paramamosain,and helps to promote the development of relevant immune enhancers.And also has positive significance for the improvement of the study on the innate immunity mechanism of crustaceans,which is conducive to the healthy development of the aquaculture industry.