Cloning, Molecular Characterization And Functional Analysis Of A C-type And Lily-type Lectin In Turbot(Scophthalmus Maximus)

C-type lectin of Scophthalmus maximus(Sm Lec1)and Lily type lectin of Scophthalmus maximus(Sm LTL) play important roles in the non-self innate immune system with important physiological, biochemical and immune function. In this study, we analysed activity of these two lectins by gene cloning, recombinant tissue expression and recombinant protein. It is significant to explore potential function in innate immune response and the application in cultural environment of these two lectins of turbot.1 By cloning, molecular characterization and structure analysis of Sm LTL, we made theoretical basis for further study of Sm LTL. In this study we reported a full-length Lily type lectin(SmLTL) identified from turbot(Scophthalmus maximus). The full-length cDNA of SmLTL is composed of 569 bp with a 336 bp ORF that encodes 112 amino acid residues. The quantitative real-time PCR analysis of the tissues revealed that the SmLTL mRNA was abundantly expressed in skin, gill and intestine and rarely expressed other tissues. The SmLTL peptide contains mannose binding sites at 30-99 along with its specific motif of β-prism architecture of two repeats(QxDxNxVxY) and a special site( TxTxGxRxV)(x is one of any amino acid residues) in three-fold internal repeat. The primary, secondary and tertiary structure of Sm LTL was predicted and analyzed by biological engineering software, suggested that the Sm LTL protein was a hydrophilic protein, contained 5.36% of α- helix, 39.29% extended strand, 16.07% and 39.29% of β- fold random coil, had three β-folding, three protein binding sites. The overall results showed that SmLTL is a member of Lily type lectin family and the reaserch of SmLTL provided an important foundation to our study in the future.2 Through environmental stress experiments, recombinant protein and agglutination test for Sm LTL to expand deep research of function, lay the foundation for disease prevention and control in new ways of turbot. In the study, we examined the regulation of SmLTL by environment stress at transcription level(included temperature, and salinity); the results showed significant changes in skin, intestine and gills. Recombinant SmLTL purified from Escherichia coli was able to haemagglutinate mouse erythrocytes and does not require calcium, and was inhibited in the presence of D-mannose. In addition, SmLTL display selectively binding against several kinds of bacteria, such as E. tarda and V. anguillarum. Results also indicated that Sm LTL were toxic to the Philasterides dicentrarchi. Even when the concentration of 200μgml-1 more than 80% mortality was observed after 24 h. We also found that the mRNA expression of SmLTL increased significantly in the skin, gill and intestine when turbot were infected with P. dicentrarchi. The results showed pronounced fluorescence on the surface of its body. All these results suggested that Lily type lectins, serving as the first line of defense against microbial, play pivotal roles in mucosal immune system.3 In the study, we researched environmental stress experiments, recombinant protein and agglutination test for studing features of Sm Lec1. As the theoretical basis for the guidance of Sm Lec1 used in the actual production and aquaculture. By analyzing the activity characteristics of Sm Lec1, the impact of environmental stress on expression and regulation, we explored its potential function and culture environment response during application in turbot innate immune system. In this study, the molecular mass of the SmLec1 with fusion protein was found to be 19 kDa as shown by SDS-PAGE, obtained the target protein after purification. SmLec1 showed agglutination activity against V. anguillarum and Edwardsiella in the six selected bacterias to test SmLec1 bacterial agglutination activity. On this basic, eight sugars were taken for the inhibitory agglutination studies. Among the carbohydrates tested, lactose, D-xylose, D-fructose, sucrose and D-mannose effectively inhibited the agglutination activity of Sm Lec1. In physical and chemical factors(PH and temperature),the activity of SmLec1 was inhibited after PH <7, T> 37 ℃. qPCR was performed to examine the relative expression of the Sm Lec1 in V.anguillarum challenged before and after, the expression level is relatively low before pathogenic stimulation, the highest at 6h, then gradually began to decline and restored to its original level at 32 h. Expression of SmLec1 in temperature stress was analyzed by qPCR, results show that the expression of liver mRNA starts to rise 19 ℃, 22 ℃ to 27 ℃ showed a decreasing trend, other organizations did not change expression significant. The impact of salinity stress to expression did not change significantly. The study will provide a scientific basis for further study of antibacterial mechanism and immune mechanism of SmLec1.Sm Lec1 and SmLTL are important immune factors, we analyzed the activity of Sm Lec1 and Sm LTL, first time to analysis these two lectins potential functions by environmental stress experiments.We investigated the potential function and application of innate immune response in culture environment of turbot of these two lectins.