| Interleukin-17(IL-17)is a characteristic cytokine secreted by T helper cells(TH17)and plays an important role in host defense against extracellular bacteria,fungal infections,and various autoimmune diseases.Up to now,sequence characteristics,gene structure and locations of chromosomes of mammalian IL-17 family members and their receptor genes have been clearly understood.But research on fish IL-17 is not comprehensive enough.In this paper,13 IL-17 family members of carps were obtained by homology search and PCR;sequence characteristics,gene structure and chromosomal location of these genes were revealed.The phylogenetic tree of IL-17 family of fish was constructed.Real-time quantitative PCR was used to determine expression characteristics of IL-17 family members and some their receptor genes in embryonic development stage and tissues of different developmental stages of carps in order to further study expression regulation of IL-17 family members and lay the foundation of their functions under physiological and pathological conditions.13 IL-17 family member genes were extracted from the salmon genome using BLAST homology search;the sequence accuracy was verified by RT-PCR and they were named as CcIL-17A/F1a,CcIL-17A/F1b,CcIL-17A/F2a,CcIL-17A/F2b,CcIL-17A/F3,CcIL-17B1,CcIL*17B2,CcIL-17C1,CcIL-17C2,CcIL-17D1,CcIL-17D2,CcIL-17Na,CcIL-17Nb based on their homology with zebrafish IL-17 genes.Comparing cDNA sequence and DNA sequence of the carp IL-17 family,FancyGene was used to map gene structure.Results:Except for CcIL-17Ds having two coding exons,other IL-17 family members have three coding exons.They encode 136～207 amino acids;homology analysis showed that they all exist in the characteristic domain of IL-17 family and have 4 Cys residues,which can form two conserved disulfide bonds.Molecular weights of mature peptide were 12.86～20.83kD and isoelectric points were 5.47～10.11.A comparative analysis of linkage groups of IL-17 family members of carps and other fishes using Genomicus(v95.01)showed that genes near CcIL-17C1 and CcIL-17C2,near CcIL-17D1 and CcIL-17D2,near CcIL-17Na and CcIL-17Nb are identical,and two linkage groups of each pair are highly consistent;only one linkage group containing CcIL-17B was found during the analysis,and inconsistent genes were found on CcIL-17A/Fl and CcIL-17A/F2‘s both sides in two linkage groups containing both.In addition,amino acid sequences encoded by two paralogous gene sequences are highly similar(all similarities are more than 86.2%),supporting that these genes are derived from specific chromosome doubling events that occur during formation process of carp species.Compared with zebrafish,tilapia and medaka,the upstream gene of CcIL-17B became rspo2,which was inconsistent with the rest of bony fishes;Genes near IL-17A/F3 of carp,medaka,tilapia and Amazon moly are stmn4 and mcm3,but stmn4 of zebrafish became the downstream gene of IL-17A/F3 by replacement;upstream and downstream genes of other IL-17 family members are highly conserved.The identity/similarity between homologous genes of carps was between 10.6～33.3%/19.3～48.8%,and the similarity between CcIL-17A/F2s and CcIL-17Ds was the lowest(19.3%).The identity between CcIL-17Ns and CcIL-17Ds was the lowest(10.6%),and the identity/similarity of CcIL-17A/Fls and CcIL-17A/F3 were the highest(33.3%/48.8%).The identity/similarity between vertical homologous genes of carps and zebrafishes ranged from 62.3～85.4%/68.5～89.6%.The identity/similarity between IL-17A/F1 was 69.8%/77.4%,and the identity/similarity between IL-17A/F2 is 75%/88.6%;IL-17A/F3 had the lowest identity/similarity(62.3%/68.5%);the identity/similarity between IL-17C is 63.1%/69.6%;and IL-17D had the highest identity/similarity(85.4%/89.6%).The identity/similarity between vertical homologous genes of carps and humans ranged from 20.9～52.8%/28.2～64.2%.The identity/similarity between CcIL-17A/Fs and HumanIL-17A was similar to this between CcIL-17A/Fs and HumanIL-17F;the identity/similarity between CcIL-17A/Fs and HumanIL-17A was 24.5～26.8%/32.7～36.6%.The identity/similarity between CcIL-17A/Fs and HumanIL-17F was 20.9～25.5%/28.2～39.4%,and the identity/similarity between CcIL-17Bs and HumanIL-17B was 40.9%/56.6%.The identity/similarity between CcIL-17Cs and HumanIL-17C was 23.2%/32.3%,and the identity/similarity between CcIL-17Ds and HumanIL-17D was the highest(52.8%/64.2%).The NJ phylogenetic tree of fish IL-17 family shows that it can be divided into 6 branches,and IL-17N of fish and its ancestor gar is attributed to a 99%confidence value,IL-17A/F1 and IL-17A/F3 formed a branch with 76%confidence.IL-17A/F2,IL-17B and IL-17D each formed a branch with high confidence(greater than 80%),and the confidence of IL-17C clades was relatively low(50%).Real-time quantitative PCR was used to detect temporal and spatial expression levels of IL-17 family members and receptor genes in carps.RESULTS:Expression of CcIL-17A/F1a was low in embryonic stage and increased after 7 phd(7 posthatching days,7 phd);expression levels of 14 phd and 26 phd were significantly higher than those of 7 phd(P<0.05);expression level in fingerlings’ skin was the highest and significantly higher than that in other tissues except gill(P<0.01);expression levels in adult carps’ gill and skin were significantly higher than those in other tissues except heart,liver and spleen(P<0.05).The expression level of CcIL-17A/F1b was low in embryonic period and increased from 1 phd;the expression level of 14 phd was the highest;the expression level of fingerlings’ liver was highest and significantly higher than that of heart,spleen and kidney(P<0.05);it was the highest in adult carps’ liver,which was significantly higher than other tissues(P<0.01).In embryonic period,the expression of CcIL-17A/F2a at 26 phd was significantly higher than other time points(P<0.01);the expression was the highest in fingerlings’ skin,which was significantly higher than that of heart.liver,spleen and kidney(P<0.05);in adult stage,the expression in body kidney was highest,significantly higher than heart,spleen,intestine and muscle(P<0.05).The expression level of CcIL-17B2 was low in embryonic period and increased from 14 phd,which was highest at 26 phd;the expression level in fingerlings’gill was highest and significantly higher than that of other tissues except muscle(P<0.05);it was expressed in adult carps’ liver,gill and muscle,and the expression in liver was highest and significantly higher than other tissues except skin,gill and muscle(P<0.01).The expression level of CcIL-17C1 was high in early embryos;the expression level at 25 postfertilizing hours(25 pfh)was significantly higher than those at 35 pfh,60 pfh and 120 pth(P<0.05);the expression level at 14 phd was the highest and significantly higher than previous time points except for 26 phd(P<0.01);the expression level in fingerlings’ gill was significantly higher than that in other tissues except liver,kidney,skin and intestine(P<0.05);the expression level in adult carps’ gill was extremely significant higher than other tissues(P<0.01).The expression level of CcIL-17D2 was low in embryonic period and increased at 7 phd;the expression level at 14 phd was the highest and significantly higher than other time points except 7 phd and 26 phd(P<0.01);the expression of CcIL-17D2 in fingerlings’ kidney was significantly higher than that in other tissues(P<0.01);the expression in adult carps’ skin was the highest and higher than other tissues except body kidney(P<0.01).CcIL-17Ns had the highest expression at 0.5 pfh and 12 pfh,which was significantly higher than other time points(P<0.01);expression of CcIL-17Ns in fingerlings’ brain was significantly higher than that in other tissues except gill(P<0.01);at adult carp stage,the expression level of CcIL-17Ns in brain was significantly higher than that in other tissues(P<0.01).The expression of CcIL-17RA1 was low in embryonic stage and began to increase at 14 phd.The expression level at 26 phd was significantly higher than other time points(P<0.01).The expression level of 14 phd was significantly higher than those at other tissues except 26 phd(P<0.05);at fingerlings stage,the expression level of CcIL-17RA1 in skin was the highest and higher than other tissues except liver,kidney,gill and intestine(P<0.05).it was expressed slightly in fingerlings’ liver,kidney,intestine and gill;CcIL-17RA1 was significantly higher in adult carps’ gill and skin than other tissues(P<0.05).CcIL-17RB was lowly expressed in early stage of embryo,but it was in an upward trend.The expression level at 26 phd was significantly higher than other time points(P<0.01);the expression in fingerlings’ liver was significantly higher than other tissues(P<0.01),and the expression of intestine was significantly higher than other tissues except skin and gill(P<0.05);at adult carps stage,the expression level of CcIL-17RB in gill was significantly higher than that in other tissues except liver and brain(P<0.01).CcIL-17RC1 was expressed in early embryos,and the expression level decreased to the lowest at 25 pfh;the expression level began to increase at 14 phd and was highest at 26 phd,which was significantly higher than other time points except 14 phd(P<0.05);the expression of CcIL-17RCl in fingerlings’ intestine was the highest;the expression of liver and intestine was significantly higher than those in other tissues(P<0.01);the expression level in adult carps’ body kidney was the highest,which was significantly higher than other tissues except liver and skin(P<0.01),followed by liver significantly higher than other tissues except skin,gill and intestine(P<0.05).The expression level of CcIL-17RD was high in early stage of embryo and began to decrease at 12 pfh;the expression at 0.5 pfh was the highest,which was significantly higher than other time points except 12 pfh and 25 pfh(P<0.05);there was no significant difference between expression at other time points;the expression of CcIL-17RD in fingerlings’ brain was significantly higher than that in other tissues(P<0.01);the expression of CcIL-17RD in adult carps’ brain was highest and significantly higher than that in other tissues except heart,skin and gill(P<0.05).CcIL-17RE had the highest expression in fingerlings’ gill,which was significantly higher than other tissues except spleen and skin(P<0.01),followed by skin;the expression in adult carps’ gill was significantly higher than other tissues(P<0.01).In this experiment,13 gene sequences of IL-17 family members were excavated and identified,which are characteristic sequences of IL-17 family.Homology analysis and linear analysis showed that IL-17A/F,IL-17D and IL-17N of fish has high conversation,IL-17C is more variable,and IL-17B was not excatated from some model fish,such as zebrafish,during this experiment.Results of real-time PCR showed that there were significant differences in temporal and spatial expression patterns of IL-17 family members. |
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