Identification And Functional Study Of A Fusion Gene NLRC3-NLRP12 In Miiuy Croaker(Miichthys Miiuy)

The fusion gene is a novel gene formed by all or part of the sequences of two genes,which is caused by chromosome translocation of two chromosomes,chromosome deletion or chromosome inversion.This chimeric gene will form abnormal transcripts or proteins in the subsequent biological process,which will lead to or promote the occurrence of tumors.A great quantity of research in the past few years have shown that fusion genes are closely related to the initiation and development of various diseases,especially the tumor.Many fusion genes have been detected in various human diseases.However,few fusion genes have been found in fish when studying the disease and immune response of fish.The study of fusion genes in fish has a certain new value and significance for studying the regulation mode and role of immune response in fish against various diseases.In this study,the existence of a new NLRC3-NLRP12 fusion gene was verified in miiuy croaker(Miichthys miiuy)by PCR,quantitative real-time PCR(q RT-PCR)and Sanger sequencing.A new NLRC3-NLRP12 fusion gene was verified in Miichthys miiuy.The fusion gene NLRC3-NLRP12 is fused by two genes related to nucleotidebinding domain and oligomerization domain-like receptors(NLRs).They are NLR Family CARD Domain Containment 3(NLRC3)and NOD Family Pyrin Domain Containment 12(NLRP12),respectively.Based on the whole genome and transcriptome data of miiuy croaker,the NLRC3-like gene and NLRP12-like gene in miiuy croaker genome were identified and analyzed by means of biological informatics such as Blast search,multiple sequence alignment,phylogenetic analysis and domain analysis.As a pattern recognition receptor,NLRs play an important role in regulating the host’s natural immune response.In addition,mutations in some members of the NLRs family are also related to a variety of inflammatory diseases.These molecules play an important role in host-pathogen interactions and inflammatory reactions.This study analyzed the expression level of fusion gene NLRC3-NLRP12 in the liver and kidney of immune-related tissues in fish after miiuy croaker was infected by Vibrio anguillarum and stimulated by lipopolysaccharide(LPS).This study also found that the fusion gene NLRC3-NLRP12 can inhibit nuclear transcription factors κB(nuclear factor κB,NF-κB)activation of the pathway.Specific research conclusions are as follows:1.Based on miiuy croaker genome and transcriptome database,this study analyzed the NLRC3-like gene and NLRP12-like gene that comprise the NLRC3-NLRP12 fusion gene.Through sequence alignment and evolutionary analysis,it is found that the NLRC3-like gene of miiuy croaker is closest to the NLRC3-like gene of yellow croaker and yellow croaker,and is close to the NLRC3 gene of zebrafish,human and mouse,the NLRP12-like gene of miiuy croaker is the closest to the NLRP12-like gene of grouper and large yellow croaker,and is close to the NLRP12 gene of zebrafish,human and mouse.SMART domain analysis shows that miiuy croaker NLRC3-like both of the NLRP12-like gene and the NLRP12-like gene have the centrally located nucleotidebinding(NACHT)domain unique to the NLR family and the FISNA(Fish-specific NACHT associated domain)domain unique to fish.The difference is that NLRC3-like gene has an effect domain PYRIN,while NLRP12-like gene contains a leucine-rich repeats domain(LRR).2.PCR amplification,q RT-PCR and Sanger method sequencing were used to identify the existence of a new NLRC3-NLRP12 fusion gene,and q RT-PCR also detected that the expression level of NLRC3-NLRP12 fusion gene in the liver,spleen and kidney of miiuy croaker was high,and Sanger sequencing was used to detect the fusion breakpoint of the fusion gene.Through SMART domain analysis,the NLRC3-NLRP12 fusion transcript encodes a protein with 529 amino acids.The N-terminal of NLRC3-NLRP12 fusion protein is composed of 79 amino acid PYRIN domains encoded by NLRC3-like gene.The C-terminal is composed of a part of FISNA domain,NACHT domain and LRR domain of 450 amino acids encoded by NLRP12-like gene.It is speculated that the NLRC3-NLRP12 gene fusion of miiuy croaker is caused by the translocation of chromosome 11 containing NLRC3-like gene and chromosome 12 containing NLRP12-like gene.3.The results showed that the expression of NLRC3-NLRP12 was significantly up-regulated after the stimulated of LPS and the infected by Vibrio anguillarum in miiuy croaker.In addition,when LPS stimulated and Vibrio anguillarum infected miiuy croaker at different time gradients,the results showed that the expression of NLRC3-NLRP12 in liver and kidney increased significantly after the 12 th hour of infection.Based on the above results,NLRC3-NLRP12 may play a potential regulatory role in the immune response of miiuy croaker.4.In order to further understand the role of NLRC3-NLRP12 in fish immunity,it is necessary to understand whether the NLRC3-NLRP12 fusion gene is expressed and its position in cells.The flag-labeled NLRC3-NLRP12 fusion gene plasmid was transfected into the carp epithelioma cell line(EPC).Western blot showed that the molecular weight of NLRC3-NLRP12 fusion gene was 78.51 KDa,which was the same as the predicted value.Then,the NLRC3-NLRP12 plasmid was transfected into EPC cells,and the cells were located by immunofluorescence analysis.The results showed that NLRC3-NLRP12 existed in the nucleus and cytoplasm of EPC cells.5.In order to explore the immune regulation of NLRC3-NLRP12,the fusion gene NLRC3-NLRP12 can inhibit the activation of NF-κB pathway through double luciferase reporter gene analysis.In order to further verify the inhibition effect,concentration gradient and time gradient experiments were carried out.The research has shown that NLRC3-NLRP12 could remarkably inhibit the activation of NF-κB signal pathway and lessen the amount of interleukin-1β(IL-1β)and interleukin-8(IL-8),which are all backward inflammatory cytokine.This study not only identified and verified a new fusion gene related to fish immunity: NLRC3-NLRP12,but also verified its effect on NF-κB Negative regulation of pathway.This provides a certain reference value for the discovery of new fusion genes in fish and even mammals in the future,and also provides a new idea and direction for the study of fish immune regulation.