| Interleukin(Interleukin,IL)is a subset of cytokines that are involved in intercellular regulation of the immune system.Interleukin,expressed in various tissues and organs,plays an important role in individual immunity.Interleukin can be divided into several families according to the homology of its protein structure.The interleukin-12 family,which includes IL-12,IL-23,IL-27,IL-35,and IL-39,has attracted much attention due to its unique heterodimer structure,in which each member is composed of an α subunit(p19,p28,and p35)and a β subunit(p40 and Ebi3)in mammals.To investigate the roles of interleukin 12 family members in fish immunity,we performed phylogenetic analyses of the interleukin 12 family genes in vertebrates.We studied the expression changes of interleukin family 12 members in the immune organs of tilapia after bacterial and viral infection.Finally,we investigated the functions of il12ba(A member of the interleukin 12 family),which was dominantly expressed in ovary,in ovarian development in Nile tilapia(Oreochromis niloticus)using CRISPR/Cas9 gene editing technique.The main results of this study are as follows:1)Isolation and evolutionary analysis of interleukin-12 family membersBased on homology search and transcriptome data analysis,In this study,interleukin-12 family genes were isolated from the vertebrate genome database,and IL12 A,IL27A,IL23 A,IL12B and IL27 B genes were successfully isolated from tetrapods.Two il12a(il12aa and il12ab)and three il12b(il12ba,il12 bb and il12bc)were isolated from the third round of genomic replication in true bone fish such as zebrafish,fugu,medaka and tilapia.There were four il12a(il12aa1,il12aa2,il12ab1 and il12ab2)in the carp with the fourth round of genome replication,and five il12b(il12ba1,il12ba2,il12bb1,il12bb2 and il12bc)in the Atlantic salmon.A total of six IL-12 family members were isolated from the tilapia genome,namely il27 b,il12aa,il12 ab,il12ba,il12 bb and il12 bc.Since bony fish have undergone a genomic replication unique to evolution,we infer that the expansion of the interleukin-12 family in fish may be caused by genomic replication unique to bony fish.By constructing phylogenetic tree and analysis,we concluded that tilapia il12 aa is the homologous gene of mammalian IL12 A.Compared with il12 bc,il12ba and il12 bb in tilapia have higher homology with IL12 B in mammals.2)Transcriptome analysis and expression of interleukin-12 family members in tilapiaTranscriptome analysis of 10 adult tissues of tilapia showed that all members of the IL-12 family were expressed in the immune organs of tilapia.And il27 b,il12bb and il12 bc were highly expressed in the head kidney.In addition,il12 ba was highly expressed in the ovary,while il12 aa and il12 ab were highly expressed in the brain.These results suggest a possible role for these genes in the corresponding tissues.After 4 and 12 hours of LPS or poly(I:C)injection,the expression levels of il12 aa,il12ab,il12 ba,il12bb and il12 bc in liver,kidney and head kidney tissues of Nile tilapia were examined by real-time PCR.It was found that the m RNA levels of these genes were significantly upregulated suggesting that they might play a role in bacterial and viral infection in tilapia.3)Verification of il12 ba expression pattern and cellular localizationBased on transcriptome analysis,we found that he highest expression of il12 ba was detected in ovaries.To further verify the expression profile of il12 ba,real-time PCR was carried out.The results showed that the expression of il12 ba in ovary was significantly higher than that in other tissues,which was consistent with the results of transcriptome analysis.ISH(In situ hybridization)revleaed that il12 ba was expressed in the cytoplasm of phase I and II oocytes of the ovary at 2,3 and 6 months after hatching.4)Roles of IL12 BA on ovarian development of Nile tilapiaIt has never been reported that interleukin is highly expressed in fish gonads.Based on this new finding,we successfully established a homogous mutant line of il12 ba in tilapia by using CRISPR/Cas9 gene editing technology.At 5 months after hatching,ovarian development was abnormal in female with il12 ba homozygous mutation,and the link between ovary and genital pore became vary narrow.By H.E.staining,only phase I and II oocytes,but no phase III oocytes,were found in the ovaries of mutant female fish.These results indicate that IL12 BA may play a role in the development of oocytes and the maintenance of normal ovarian function.It also indicates the potential connection between the immune system and the reproductive system.In summary,six interleukin-12 family genes were isolated from the Nile tilapia genome,and il12 a and il12 b genes were replicated in the tilapia genome.Transcriptome analysis of 10 adult tissues of Nile tilapia indicated that these genes were expressed in the immune organs of tilapia.The role of these genes in the immune process of tilapia was verified by LPS and poly(I:C)treatment.Real-time PCR analysis demonstrated that il12 ba was localized in the cytoplasm of oocytes.Through the gene knockout model,we found that the deletion of il12 ba gene could lead to abnormal ovarian development in female fish,which indicated that il12 ba may also play an important role in the ovarian development of tilapia.This study provides a new perspective for further understanding the extension of interleukin-12 family in bony fish and the new function of il12 ba in fish reproduction. |
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