Effect Of Toll-like Receptor On Immunity And Development Of Mytilus Coruscus

Mytilus coruscus is one of the important aquaculture species in China.In recent years,with the continuous deterioration of marine environment,microbial pollution of marine shellfish has become more and more common,which has seriously affected the healthy cultivation of mussels.In addition,shellfish diseases caused by microbial pathogens(such as marine Vibrio,etc.)often occur,resulting in huge economic losses.Starting from the immune system of M.coruscus,the necessary way to maintain the healthy development of mussels aquaculture industry is to deeply study the immune prevention mechanism of mussels and explore effective methods to improve the disease resistance of M.coruscus.Toll-like receptor(TLR)is one of the important pattern recognition receptors in invertebrates,which can recognize a variety of pathogenic microorganisms and non-self molecules.Myeloid differentiation factor 88(MyD88)is an adapter molecule directly acting with TLR in the TLR signaling pathway,and both of them play an important role in activating the subsequent series of immune responses.In this study,54 TLR genes were identified and preliminarily analyzed based on the data of mantle transcriptome of M.coruscus.The full-length of two TLR genes and one MyD88 gene were cloned.In addition,the roles of two TLR genes in the development of M.coruscus were explored.Main results are as follows: 1.TLR gene identification based on mantle transcriptome and its preliminary analysisA total of 54 TLR genes,named TLR1-54 respectively,were present in M.coruscus mantle transcriptome,and they all included LRR domain,transmembrane region and TIR domain.As predicted by SMART,among 54 TLRs,the number of LRR ranged from 1 to 19,and 32 TLRs had C-terminal LRR structure(LRRCT),16 TLRs had N-terminal LRR structure(LRRNT),and 9 TLRs had LRRCT,LRRNT and signal peptide at the same time.Phylogenetic tree results show that these 54 TLRs form two main clusters(cluster A and cluster B),which can be further subdivided into six subfamilies(A1,A2,B1,B2,B3,B4).TLR genes were randomly selected from each subfamily to detect their expression in different developmental stages and adult tissues.The expression trends of the six tested LTR genes were basically the same at different developmental stages,and all of them were significantly increased at the juvenile stage.In adult tissues,the LTR genes were expressed in all tissues except TLR31,which was not expressed in the foot.2.Cloning and structural analysis of the full-length sequences of two TLR genesThrough RACE cloning,we obtained the full-length sequences of two TLR genes(McTLR2 and McTLR3).The total length of the McTLR2 gene was 3968 bp,including a 71 bp of 5' untranslated region(UTR),1805 bp of 3' UTR and 2091 bp of the open reading frame(ORF),encoding 696 amino acids.The predicted molecular weight of the protein was 80.90 kDa,and the isoelectric point(pI)was 9.35.The amino acid sequence of McTLR2 contained a signal peptide,6 LRR domains,the LRR-CT transmembrane region and a TIR domain.While the total length of the McTLR3 gene was 4568 bp,including 258 bp of 5' UTR,1691 bp of 3' UTR and 2619 bp of ORF,encoding 872 amino acids.The predicted molecular weight of the protein was 98.97 kDa,and the pI was 9.20.The amino acid sequence of McTLR3 contained a signal peptide,13 LRR domains,a LRR-NT,4 LRR-TYP,1 LRR-CT,the transmembrane region and a TIR domain.3.Expression analysis of two TLR genes after bacterial infectionWe stimulated the mussels using bacteria Vibrio chagasii by injection into the adductor and exposure to water borne V.chagasii.The results showed that,when the mussels exposed to water borne V.chagasii,the mRNA expression levels of McTLR2 and McTLR3 in haemocytes and mantle were significantly increased,indicating that McTLR2 and McTLR3 could recognize Vibrios and participate in the immune response to remove bacteria.Moreover,the up-regulation time of these two genes in the mantle was significantly earlier than that of in haemocytes,indicating that the mantle was the first line of defense for shellfish immunity,and could immediately perceive external pathogens and make immune responses.However,the injection of bacteria only had a weak response to McTLR2,and even reduced the expression of McTLR3.But the activity of SOD,CAT and lysozyme in serum was significantly increased,indicating that immune-related enzymes in the body were involved in the reaction against bacteria.4.The full-length cloning and structural analysis of MyD88-4 geneThe full-length sequence of MyD88 gene(named McMyd88-4)was cloned through RACE.The gene has a total length of 3930 bp,including 188 bp of 5'UTR,2607 bp of ORF and 1135 bp of 3' UTR.It encodes 868 amino acids,including a dead domain(DD)and a TIR domain.The predicted molecular weight of the protein is 97.074 kDa and the isoelectric point is 5.32.Through RT-qPCR,it was found that McMyd88-4 gene was expressed in all tissues detected in the mussel M.coruscos,with the highest expression in the mantle and gill.After exposure to water borne V.chagasii,the expression of McMyd88-4 gene in the mantle,gill and digestive glands showed rapid and obvious upregulation,reaching a peak at 3 or 6 h.It was speculated that McMyd88-4 was involved in the immune response of mussel against Vibrio through TLR signaling pathway.The up-regulation level in the digestive glands was significantly higher than that in the mantle and gill,indicating that the digestive gland of M.coruscos may be more sensitive to the immune response of V.chagasii than the gill and mantle.5.The role of two TLR genes in the development of the mussel M.coruscosWe used biofilms of different induce activities to induce pediveliger larva to metamorphosis,and detected the expressions of McTLR2 and McTLR3 in larvae at different time points.The results showed that in the process of Shewanella loihica(high inducible activity)membrane induction,the expression of McTLR2 gene first decreased and then increased,while the expression of McTLR3 gene did not change significantly.During Pseudoalteromonas sp.4(low inducibility)membrane induction,McTLR2 gene increased significantly at 48 h,while McTLR3 gene expression was higher at 6 h,12 h and 48 h.The two genes showed different expression patterns after inducing by different biofilms,which was speculated to be related to the species of bacteria.While TLRs was up-regulated in the process of metamorphosis,suggesting that TLRs may regulate the disintegration,absorption or regeneration of the old tissues of larvae.