Target Genes Primary Identification And Functional Characterization Study Of MiR-1704 In Chicken

Micro RNAs are endogenous small noncoding RNAs that negatively regulate gene expression at post-transcriptional level by binding to the 3’ untranslated region of target gene m RNAs. Mi RNAs participate in the regulation of many important biological processes of boiology. Mi R-1704 was found in the chick embryo, but its function has not been reported until now. In this study, the mi R-1704 expression profiles were detected by quantitative real-time PCR in 9 tissues in 1 day, 6 weeks and 16 weeks old Gushi chickens. Target genes of mi R-1704 were predicted by Target Scan. Then, the target genes were further analysed and annotated by DAVID and GO Term Finder. In addition, dual-luciferase reporter system was utilized to identify the predicted targets. An F2 resource population of Gushi chicken and Anka broilers was employed to detect SNPs of mi R-1704 and elucidate their associations with chicken performance traits. Moreover, mi R-1704 expression vector of different alleles were constructed to investigate the function of polymorphisms. The main results are as follows:1. Mi R-1704 was widely and differently expressed in the different periods and tissues. The expression of mi R-1704 in 1 day and 6 weeks old chickens was low abundance, while its expression in 16 weeks old chickens was very high. In 1 day old chickens, mi R-1704 was highly expressed in the leg muscle, hypothalamus and liver, low expressed in the kidney and brain. In 6 weeks old chickens, the expression of mi R-1704 in all tissues was low. In 16 weeks old chickens, mi R-1704 was highly expressed in the spleen, leg muscle and hypothalamus, low expressed in the small intestine.2. The total 173 target genes of mi R-1704 were predited by Target Scan. GO and KEGG analysis showed that target genes were significantly enriched in the regulation of gene expression, biosynthesis, biological process and metabolism of substances, as well as the cell cycle, calcium signaling pathway and Hedgehog signaling pathway. Then, the relationship between mi R-1704 and predicted target genes were verified by dual-luciferase report system. We primarily confirmed that BMP2 and IRS2 were the targets of mi R-1704.3. The association analysis showed that rs14668705 C>G mutation was significant associated with chicken birth weight, 6, 8, 10 and 12 weeks body weight, 4, 8, and 12 weeks shank girth, carcass weight, semi eviscerated weight, water loss rate of leg muscle, chest muscle p H and aspartate aminotransferase(P<0.05). Furthermore, chickens carrying the CC genotype consistently showed higher value than GC and GG genotypes. As for the effect of different alleles, C>G mutation could significant affect mature mi R-1704 biogenesis.Conclutions:1. mi R-1704 was widely and differently expressed in the different periods andtissues of chicken.2. BMP2 and IRS2 were the target genes of mi R-1704.3. The rs14668705 C>G polymorphism within mi R-1704 precusor was significant associated chicken body weight, shank girth and carcass weight. Chickens carrying the CC genotype showed higher value than GG genotype. In addition, C>G mutation could significant affect mature mi R-1704 biogenesis.