The Relation Of Expression Patterns Betwen TLR-i Gene With Genes From NF-?B Pathway In Mussel Mytilus Coruscus Under The Stress Of Bacteria

Nowadays,along with the industrialization of coastal cities and the pollution of daily life wastes,most marine economic species are facing damage.As a kind of economic shellfish along Chinese coast,the mussel Mytilus coruscus is one of the important shellfish of the southeast coastal provinces.Due to the benthic fixation and filter-feeding lifestyle of adults,M.coruscus is an ideal reservoir for many Marine pathogens,among which Marine Vibrio are kinds of the main pathogens.Innate immunity plays a important role in the immune system of mussels like M.coruscus,while as we all know toll-like receptors play a part in innate immunity.Therefore,in this study,the McTLR-i cDNA was cloned as a major research object,furthermore,several cDNA fragment of TLR/NF-?B pathway proteins for RT-qPCR and a lysozyme cDNA sequence called as McLYZ were cloned.With RT-qPCR,the result whether there is a connection among the there genes was be showed.The main experimental results were as follows:?1?The full length sequence of McTLR-i is 2821bp including a 2022bp open reading frame.An amino acid sequence with 673 residues is encoded,which is similar to the mccTLR of mollusks using the SMART domain analysis.NCBI Blast showed that there are90.64%identity between McTLR-i and MgTLR-i of the mussel M.galloprovincialis.According to the result of multiple sequences alignment,McTLR-i and toll-like receptors of other shellfish have less conservation in the extracellular region.Reversely,the transmembrane region and TIR domains are more conservative.The conserved motifs BOX1 and BOX3 contained were successfully identified in the MgTLR-i TIR domain by this study,however the BOX2 is deficient.The phylogenetic tree shows that McTLR-i is located in the cluster of mccTLRs,and in the same branch as MgTLR-i.?2?The results of tissues differential expression showed that McTLR-i was only significantly expressed in gill?P<0.05?.In the gill,after infection with V.parahaemolyticus,the McTLR-i expression peak appeared at 2h,which was about 10.27 times compared to the control group.However,under the stimulation of V.alginolyticus,the expression peak appeared at 8h,which was about 7.09 times compared to the control.It can be inferred that McTLR-i plays a role in the immune response of M.coruscus with challenge of the both Vibrio.Almost all temporal expression of TLR/NF-?B signal pathway gene presented inconsistent results with the McTLR-i expression profile.It was speculated that the missing BOX2 in the TIR domain was a possible key reason.?3?A full length cDNA of McLYZ is 691bp with an open reading frame length of564bp and encode a putative protein of 187 amino acids.Domain prediction and multiple sequence alignment analysis revealed that McLYZ has a highly conserved destabilase domain.There are three conserved amino acid identified including two catalytic sites Glu⁸³,Asp⁹⁵ of destabilase activity and one His¹⁵⁹,which is considered to have isopeptidase activity.Multiple sequence comparison showed that the similarity between McLYZ and M.edulis LYZ was 87%.The predicted molecular properties suggest that McLYZ may play a part in the immune system and digestive system of M.coruscus.The results of modeling Three dimensional model of the protein showed that the three catalytic sites were located in the predicted activity center of McLYZ and had the possibility of functional correlation.?4?The results of tissues differential expression showed that the most tissues showed significant expression?P<0.05?,except the adductor and gonad.After the stimulation of V.parahaemolyticus,McLYZ reached a highest expression at 24h.After A.hydrophila injection,McLYZ peaked at 48h.In gill,under the stress of V.parahaemolyticus,McLYZ was only significantly expressed at 24h.However,under the stimulation of A.hydrophila,the relative expression of McLYZ had a first rising trend at 2h after stress,and reached the peak at 4h.The temporal expression of McLYZ in gill was also not similar to the expression profile of McTLR-i.The results can not support that McTLR-i transduce the immune signaling through the possible TLR/NF-?B pathway proteins which were selected in this experiment to promote the formation of McLYZ.