Cloning And Expression Analysis Of Akirin2 Gene Regulatory Sequences And Related MiR-142 In Different Chicken Breeds

AKIRIN2,a highly conserved nuclear factor,regulates target genes and plays biological functions through forming complexes with other proteins.At present,the study on AKIRIN2 protein mainly focuses on the field of muscle development and immune regulation,but the transcriptional regulation mechanism of akirin2 gene has not been reported.Promoters and miRNAs play key roles in the pre-transcriptional and post-transcriptional regulation of target gene.In this study,chickens were selected as model animals.The 5'-terminal upstream 2832 bp sequence and 3'-terminal downstream 417 nt sequence of akirin2 gene from different chicken breeds were cloned.Bioinformatics analysis predicted that miR-142-3p might target the 3'-untranslated region(3'UTR)of akirin2 gene,and the expression and function of miR-142-3p and miR-142-5p were analyed comprehensively.The main findings are as follows:Firstly,the 2832 bp sequences of the akirin2 gene promoter region of Hi-Line Brown layer,AA~+broiler and Sanhuang broiler were cloned respectivly,and sequence alignment and bioinformatics analysis were performed.The results showed that there were 38 SNPs between layer(Hi-Line Brown layer)and broiler(AA~+broiler and Sanhuang broiler),while there were only 4 SNPs between AA~+broiler and Sanhuang broiler.It was predicted by transcription factors that 19 SNPs between layer and broiler caused the changes in transcription factor binding sites,which led to the differences in the type and number of transcription factors between layer and broiler.It was predicted by CpG islands that the CpG island sequence between layer and broiler was not much different,but there were more SNP loci distributed in CpG islands.We speculated that there are different regulatory mechanisms of the akirin2 gene promoters between different chicken breeds.Secondly,the 417 nt sequences of 3'UTR part of akirin2 stop codon downstream of Hi-Line Brown layer,AA~+broiler and Sanhuang broiler were cloned respectively,and sequence alignment and bioinformatics analysis were performed.The results showed that the sequences of 3'UTR were identical in the three breeds,which suggested that the post-transcriptional mechanisms of chicken akirin2 were similar in different breeds.Bioinformatics prediction found that miR-142-3p could target the akirin2 3'UTR.Finally,partial sequence analysis of pri-miR-142 showed that the sequences of the three chicken breeds were completely identical.The expression profile analysis of miR-142-3p/5p showed that miR-142-3p/5p were widely expressed in all tissues of three chicken breeds,and the expression activities of miR-142-3p/5p were related to tissue development and immune function,and miR-142-3p might have more functions than miR-142-5p.Tissues expression profile analysis of miR-142-3p/5p in inflammatory model and vaccine immunization model indicated that miR-142-3p/5p were involved in inflammatory response and immune response,and played different regulatory roles in different immune tissues.The differences of expression activity between miR-142-3p and miR-142-5p might be related to their functional characteristics,raits of different chicken breeds and different pathological processes.This study shows that the SNPs of akirin2 promoter sequences may be one of the key factors affecting the differential expression of akirin2 gene in different chicken breeds.Study on the expression and characteristics of akirin2 gene promoter and its related miR-142,it has important theoretical and practical significances for deeply understanding the regulation mechanism of akirin2 gene expression and the function and application of miR-142 in inflammation and immunity.