Cloning,Expression And Functional Identification Of Arih2 Gene And Its Related MiRNA In Different Breeds Of Chicken

ARIH2,an E3 ubiquitin-protein ligase,plays key roles in immune,inflammatory and developmental processes,but the specific mechanism is not clear.At present,there are no reports on Arih2 gene in birds.In this study,chicken was used as model animal and the full-length open reading frame(ORF)sequences and 3?-termainal 1169 bp untranslated region sequences of Arih2 gene were cloned from different breeds of chickens.The ARIH2 protein structure and target miRNA of Arih2 gene were predicted by bioinformatics methods.The temporal and spatial expression characteristics of Arih2gene and its target miRNA were analyzed by quantitative real-time PCR(qRT-PCR),and its expression and function in inflammation and immune responses were also preliminary explored.The main results are as follows:Firstly,the full-length ORF sequences of Arih2 gene were cloned from three breeds of AA~+broiler,Sanhuang broiler and Hy-line Brown layer,which nucleotide lengths were all 1473bp and encoding 490 amino acids.There were 11 SNP sites among the Arih2 gene of the three breeds,and ARIH2 protein was highly conserved between different species.Arih2 gene had transcriptional activities in all tissues of different breeds of chicken.In the same old chickens(14-day old)of different breeds,Arih2 had higher expression activities in blood and brain,and significantly high expression in liver of two breeds of broilers(P<0.05);In different development stages of Hy-line Brown layers,Arih2expression activities in heart and skeletal muscle increased gradually,and high expressed in large intestine of 10-month-old layers(P<0.05).It is suggested that Arih2 gene might play roles in intestinal mucosal immunity and tissue development.Secondly,the3?-termainal 1169nt sequences of Arih2 gene untranslated region(UTR)were cloned from three breeds of chicken,and bioinformatics predicted that miR-200b-3p might target 3?UTR sequence of Arih2 gene.So,203bp of partial pri-miR-200sequences of three breeds of chicken were cloned.Tissue expression profiles analysis showed that mi R-200b-3p was expressed in all tissues of the three breeds of chicken.MiR-200b-3p had significantly high expression activity in bursa of 14-day old Hy-line Brown layer(P<0.05),and its expression activities were all higher in epithelioid tissues(such as kidney,lung,skin and small intestine)of different development stages from different breeds of chicken(P<0.01).Through the comparison of expression activities,it was found that Arih2 and miR-200b-3p showed reverse differential expression among the several corresponding tissues.The experiment showed that miR-200b-3p might play an important role in the development of chicken epithelioid tissues and a part in the post transcriptional regulation of Arih2 gene.Finally,qRT-PCR was used to detect the expression of Arih2 gene and miR-200b-3p in inflammatory and immune model chickens.In the inflammatory model chickens prepared by LPS,the expression activities of Arih2 gene was significantly down regulated in blood(P<0.01)and thymus(P<0.05),but up-regulated in spleen(P<0.01)and bursa(P<0.05)of Hy-line Brown layer and AA~+broiler;In the immune model Hy-line Brown layer immunized with Newcastle attenuated vaccine and Avian influenza inactivated vaccine,the expression of Arih2 gene was down-regulated in liver and bursa(P<0.01),up-regulated in blood(P<0.01),and it showed reverse changes in spleen of the two immune models.The expressions of mi R-200b-3p in the blood and spleen of inflammatory model Hy-line Brown layer were significantly down regulated(P<0.05),and significantly up-regulated in the liver,thymus,bursa,small intestine and large intestine(P<0.05),but it was all significantly down regulated in the inflammation model AA~+broiler(P<0.05).The expression of miR-200b-3p in the blood of Newcastle and Avian influenza immune model layers was significantly down-regulated(P<0.05),while it was up-regulated in the liver,thymus and bursa(P<0.05).This experiment showed that miR-200b-3p and Arih2 gene were involved in inflammatory and vaccine immune response,and the differences of their expression activity might be related to the differences of their function and chicken breeds characteristics.In this study,the expression and functional characteristics of Arih2 gene and its related miRNA were explored.It was found that Arih2 and miR-200b-3p were important factors involving in the development and immune regulation,which laid a foundation for further exploring the functions and characteristics of Arih2 gene and its related miRNAs.