Genetic Diversity Of Intron 1 Of Chicken IL18 Gene And Their Effects On Gene Expression Regulation

As a member of Interlukin family,IL18 plays important roles in both regulating host immunity and energy homeostasis.During chicken artificial selection or domestication,its genome has been adaptively shaped to high-production performance,along which its host immunity seems reduced.In a previous study of our lab,Luo et al(Unpublished),through whole-genome resequencing of a number of chicken breeds,found IL18 was in close linkage with chicken skin color locus BCO2,and the Intron 1 of IL18 could be finely grouped into two haplotypes,i.e.,Wild type(White skin)and Mutant type(Yellow skin),according to the skin color tag SNP.Further transcription factor binding site prediction analysis indicated that these two haplotypes of IL18 Intron probably had functionally differentiated.In this study,we firstly amplified the complete 5’-UTR of IL18 with 5’-RACE and identified an intron between 5’-UTR and Exon 1,named 5’-UTR Intron.We further analyzed the single nucleotide polymorphisms(SNPs)from 5’-flanking region,5’-UTR,5’-UTR Intron and Intron 1 of IL18 in different populations,and found the SNPs from above regions could be truly clustered into two haplotypes,consistent with the results obtained by previous high-sequencing.To investigate whether the IL18 had functionally differentiated with respect to these two haplotypes,we measured the IL18 m RNA expression of different tissues in Yao breed(4-week old),and found the Wild-type IL18 in liver harbored a higher expression compared with that of the Mutant-type,suggesting the differentiation of these two types in terms of transcription activity.Based on p GL3-promoter and p GL3-basic vector systems,we identified the transcriptional differentiation in 5’-flanking region,5’-UTR Intron and Intron 1,respectively.In detail,the Wild-type 5’-flanking region and 5’-UTR Intron both harbored a higher transcriptionally regulatory ability than that of the Mutant-type,while the Wild-type Intron 1 harbored a weaker one than that of the Mutant-type;Co-transfection of 5’-UTR Intron and Intron 1 showed that the Wild-type harbored a higher transcriptionally regulatory ability than the Mutant-type.These results suggest the differentiation of 5’-flanking region and 5’-UTR Intron probably underlies the two types of IL18 transcriptional differentiation.Additionally,to investigate the roles of IL18 in chicken host immune response,normally-expressed,over-and down-regulated IL18 of chicken DF-1 cell lines separately used as hosts,we performed the challenge experiments with Eimeria tenella(E.tenella).After a 3-h infection,we observed the cell line with over-regulated IL18 was significantly(P<0.01)resistant to E.tenella infection compared with control.Intriguingly,the cell line with down-regulated IL18 wasn’t predictably more susceptible to E.tenella infection compared with control.Collectively,we herein identified in chicken,the Wild-type IL18 are stronger in transcriptional activity compared with the Mutant-type,and its expression level is probably closely associated with chicken host immune response.More importantly,the results presented here is conducive to shedding light on reduction in immunity during chicken artificial selection or domestication.