Characterization Of TLR Gene Family Based On Lampetra Japonica Liver Transcriptome And Molecular Cloning And Functional Study Of TLR3

In this study,the liver transcriptome of Lampetra japonica was sequenced by high-throughput RNA-seq.A total of 47 293 unigenes were obtained by de novo assembly with the N50 of 1 447 bp.Based on the blast to multiple databases,65.04% of unigenes were annotated.In the KEGG analysis,a lot of innate immune-related unigenes were identified,suggesting that the liver of the lamprey may play an important role in immune defense against pathogens invasion.After subseq uent identification,the genetic compositions of the complement and coagulation cascades were comprehensively characterized,which will accelerate the further studies of functional evolution of important genes.The assembled transcriptome was also screened of 25419 possible SSR molecular markers,providing useful candidates for future genetic diversity studies and germplasm conservation.The above results have greatly enriched the expressed genes database of L.japonica,and provided an important theoretical and practical basis for in-depth data-mining of important functional genes,exploring the species immunological adaption to the complex aquatic life and the protection of population genetic diversity.Based on Petromyrzon marinus genome and L.japonica liver transcriptome,a total of 16 TLR sequences of L.japonica were identified at the whole-transcriptome level.Using the SMART program,most of the lamprey TLR proteins contain the typical TLR structure,consisting of multiple LRRs,a transmembrane domain,and a TIR domain.Phylogenetic analyses indicated that the orthologs of vertebrate TLR1,4,6,9 and 10 were absent in lamprey.Except for TLR3 as a single copy,other TLRs were formed subfamilies with multiple duplicated copies in a lamprey-specific manner.Some of the multiple members may represent the ancestral genes of jawed vertebrates TLRs;some may represent the novel genes in lampary,which were expanded the recongnition of different PAMPs for host defense.In addition,RNA-seq data from various samples of L.japonica were used to calculate the FPKM values of all lamprey TLRs.The expression profiling revealed that the identified TLRs were positively expressed in L.japonica,and most of them exhibited tissue-specific expression patterns.The Q-PCR experiments validated the similar trends and showed the expression levels of many TLRs were highly up-regulated after Poly I:C and/or LPS stimulation but at different responsed time.In the higher vertebrates,TLR3-TICAM mediated signaling pathway is one of the important immune pathways against RNA virus infection.However,the sequence and expression of the lamprey TLR3(L-TLR3)is unclear.In this study,the full-length ORF of L-TLR3 gene was cloned with 2670 bp,encoding 889 amino acids.L-TLR3 contained the typical TLR domain structure and a BB-loop conserved in higher vertebrates.Real-Time PCR analysis showed that TLR3 was expressed in various tissues,highly in the liver and the leukocyte cells.The expression levels of L-TLR3,as well as its potential adaptors TIC AMa and TIC AMb,were up-regulated significantly in the marrow after Poly I:C stimulation,but were not up-regulated to LPS stimulation.The Western blot results showed the expression of L-TLR3 protein was similar to that of Q-PCR,suggesting that L-TLR3 protein was involved in the immune response process to Poly I:C infection.These results provide an important theoretical basis for exploring the TLR3-TICAM-mediated antiviral signaling pathway and understanding the functional evolution of TLR gene family involved in the innate immune system of vertebrates.