Cloning Of IRAKs Gene Of Mytilus Coruscus And Their Related Immune Function

With the increase in human demand for seafood,the development of marine aquaculture continues to accelerate,and along with economic development,coastal industrialization has also become more rapid,resulting in the continuous deterioration of the marine aquaculture environment,and the ecological environment of the offshore waters has affected shellfish farming.Economic development is crucial.Therefore,studying the immunobiological mechanism of marine shellfish will provide an important theoretical basis for its healthy breeding.Mytilus coruscus as filter-feeding animals,are widely distributed and have strong growth ability,but poor mobility.They are widely exposed to various pathogenic microorganisms in the marine environment in daily environments,but the resulting disease infections are rare.It shows that there is a strong antibacterial infection mechanism in the body,so studying the natural immune defense mechanism of Mytilus coruscus will provide an important theoretical reference for its healthy breeding.Interleukin-1receptor-associated kinases(IRAKs)are important linker molecules in TLR(Toll-like receptor)-mediated signaling pathways,and play an important role in biological innate immune systems.Therefore,in this paper,the immune factor IRAKs in the TLR signaling pathway of Mytilus coruscus was identified,phylogenetic analysis,related expression mechanism and in vitro protein expression studies.The main results obtained are as follows:1.Based on the transcriptome data of Mytilus coruscus,interleukin 1receptor-associated kinase a(named McIRAK-a,GenBank accession number:MK592614)and b(named McIRAK-b,GenBank accession number: MK592615)were obtained)The full-length cDNA sequence of McIRAK-a is 3168 bp,its open reading frame(ORF)is 2937 bp,encoding 978 amino acids;McIRAK-b is 1815 bp,its ORF is 1599 bp,encoding 532 Amino acids.Multiple sequence alignment and phylogenetic tree analysis showed that the amino acid sequences of McIRAK-a and McIRAK-b have high homology with IRAK-1 and IRAK-4 of other mollusks,respectively.2.The expressions of McIRAK-a and McIRAK-b can be detected in five tissues including adductor muscle,gills,hepatopancreas,gonad and hemocytes,among which McIRAK-a is the highest expressed in gills,followed by hepatopancreas,hemocytes and gonads have the lowest transcription level in the adductor muscle,and the expression level in the gill tissue is 11.82 times that in the adductor muscle.McIRAK-b also has the highest gene expression in gill tissue,71.31 times higher than the hemocytes with the lowest expression,followed by gonads,adductor muscle,and hepatopancreas.After Vibrio infection,McIRAK-a and McIRAK-b were expressed in the gill tissue in a time-dependent manner,and the highest point of McIRAK-a gene expression appeared 12 h after infection with Vibrio alginolyticus(5.16 times higher than the control group),while After being attacked by Vibrio parahaemolyticus,it peaked at 8h(4.21 times higher than the control group).After injection with Vibrio alginolyticus,McIRAK-b was significantly expressed and peaked at 8 hours(9.47 times higher than the control group),but peaked 4 hours after being infected with Vibrio parahaemolyticus(12.02 times higher than the control group).The results show that IRAK-a and IRAK-b may play an important role in the antibacterial immune signaling pathway of Mytilus coruscus.3.In vitro construction of recombinant proteins containing active sites for protein interaction functions,that is,the plasmids of McIRAK-a and McIRAK-b kinase functional domains(S_TKc)are linked to pET28 a and pET32 a plasmids,respectively,and are in E.coli BL21(DE3)Expression,purified with Ni-NAT Superflow resin kit,the target protein band obtained conforms to the expected molecular weight,after ultrasonic fragmentation,SDS-PAGE detection showed that the expression was all inclusion body protein,consistent with the expected molecular weight,both the interaction relationship needs to be further verified.The above experimental results show that McIRAK-a and McIRAK-b are important transmission molecules on the Toll receptor-mediated signaling pathway ofmollusks such as Mytilus coruscus and play an important role in the body’s natural immune function.Antibacterial infections played an important role.Therefore,the results of this experiment will provide a reference for future research on the functional mechanism of natural immune molecules from the protein level.