Mechanisms Of The IKK?/TBK1/IRF Signaling Pathway In Antiviral Innate Immunity Of Black Carp

Fish possess innate and acquired immune system,but they more rely on the innate immune system to resist the invasion of pathogenic microorganisms(such as bacteria and viruses).Black carp is one of the traditional " Four Major Chinese Carps" and an important freshwater economic fish in China.Diseases caused by bacteria,viruses or parasites in natural and farmed waters pose a serious threat to the healthy breeding of black carp.In order to elucidate the innate immune mechanism of black carp,we sequenced the full-length transcriptome of the black carp tissue under the condition of pathogen microorganism stimulation,and interpreted the innate immunology-related IKK?/TBK1/IRF signaling pathway.One of the contents of this paper is using the third-generation sequencing technology to sequence the full-length transcriptome of black carp and analyzing the sequencing results by bioinformatics method.The main research results are as follows:With the help of Pacbio Sequel sequencing system,RNA from spleen,liver and kidney of black carp stimulated by GCRV and Edwardsiella tarda were mixed and sequenced in equal amounts.The total number of reads was 5,663,655,and the total data was 10.9Gb.Quality control through transcripts resulted in 7,183 high quality Isform and 49,942 low quality Isform.Based on the previous results of the second-generation transcriptome of black carp,35,608 Unigene were selected from the whole isform.The BUSCO database was used for data integrity assessment,and the number of matching genes was 221,accounting for 73%.The transcripts were annotated by common databases,such as NR,COG/KOG,GO,Swissprot,eggNOG,KEGG and Pfam.In the NR annotation,29068 Unigene were annotated,accounting for 81.63% of the total number of Unigene.Homology analysis revealed that 6,392 genes were homologous to Sinocyclocheilus rhinocerous,accounting for 22.05%.Among the KEGG annotations,most transcripts were involved in signal transduction,followed by transport and catabolism,and molecular folding,arrangement and degradation.In KOG annotations,transcripts were focused in signal transduction mechanisms and post-translational modifications,protein transformation,and molecular chaperones.In the GO annotation,the transcripts were focused in constitute cells,cell components and organelles in the cytological components.Among the molecular functions,most transcripts perform binding function,followed by catalytic activity.In biological pathways,the largest number of transcripts annotate cellular processes,followed by metabolism and biological regulation.In addition to the common database annotation,transcription factor,lncRNA and transposon analysis were performed on the obtained transcripts in this study.The analysis of this experiment showed that there were 3,956 transcription factor sequences,2,274 lncRNA and 1,338 transposon.The second part of this study is the functional mechanism of the related genes in the IKK?/TBK1/IRF signaling pathway.The main research results are as follows:qPCR experiments showed that LPS,poly(I:C),SVCV and GCRV could activate the expressions of bcIRF3 and bcIRF7 in MPK and MPF cells.EPC cells overexpressed bcIRF3 and bcIRF7 showed enhanced resistance to SVCV and GCRV viruses.Immunofluorescence experiments showed that bcIRF3 and bcIRF7 were transferred from cytoplasm to nucleus due to viral infection.The co-immunoprecipitation experiment proved that bcIRF3 could bind to each other,suggesting that they can form homodimer.The co-immunoprecipitation experiment proved that bcIRF3 and bcIRF7 could also bind to each other,suggesting that they can form heterodimer to stimulate the expression of interferon.We constructed structural mutants of bcIRF3 and bcIRF7 to explore the functions of different structural domains on bcIRF3 and bcIRF7.The results showed that DBD domain was necessary for bcIRF3 and bcIRF7.The missing of SRD partially affected the function of bcIRF3 and bcIRF7.The IAD domain of bcIRF3 was essential for the full function of bcIRF3,but the IAD domain of bcIRF7 could inhibit the function of bcIRF7.In addition,immunofluorescence staining showed that the deletions of bcIRF3 and bcIRF7 in the IAD domain of black carp were mostly concentrated in the nucleus.The relationship between upstream kinases TBK1/IKK? and IRF3/IRF7 was also investigated: It was showed that the ability of TBK1 to regulate interferon was dependent on bcIRF3 and bcIRF7;both TBK1 and IKK? shown the ability to enhance bcIRF3 and bcIRF7 to stimulating interferon;both TBK1 and IKK? could improve the antiviral ability of bcIRF7.In conclusion,bcIRF3 and bcIRF7 are both activated by TBK1 and IKK? respectively,which made them to modulated the expression of interferon.The third part of this study is the cloning and functional study of SIKE.The main research results are as follows:With the full-length transcriptome information of black carp,the full length sequence of SIKE gene was screened from immune-related genes.Full-length of bcSIKE was composed of 1,144 nucleotides,with 5-UTR of 210 bp,3-UTR of 292 bp,ORF of 642 bp,and expected molecular weight of 25 kDa.Homology analysis showed that the amino acid similarity of SIKE with common carp and zebrafish was 93.0% and 94.4%,respectively.qPCR results showed that bcSIKE was involved in the host innate immune response initiated by SVCV and GCRV.Immunofluorescence data showed that bcSIKE was mainly distributed in the cytoplasm.The dual-luciferase data showed that bcSIKE itself did not have the ability to regulate interferon,but it played an obvious inhibitory role in the RLR/IFN signaling pathway of black carp.Immunofluorescence data showed that bcSIKE and bcTBK1 showed the same subcellular distribution in MPF cells,and Co-IP data showed the direct interaction between these two proteins.Virus titer assay showed that bcSIKE could inhibit bcTBK1-mediated antiviral activity in the process of host innate immune activation.In addition,bcSIKE was able to bind to IKK? and inhibit its ability to regulate IRF7.The results above indicated that SIKE had an inhibitory effect in the RLR signaling pathway,and its target proteins were TBK1 and IKK?.In conclusion,we sequenced and analyzed the transcriptome of the innate immune response,investigated the function and regulatory mechanism of the IKK?/TBK1/IRF signaling pathway on the antiviral innate immunity of black carp.It provides new data for the systematic elucidation of the antivirus mechanism of the innate immunity of black carp and provides a new reference for the healthy breeding of black carp.