巴什拜羊不同发育阶段骨骼肌中miR-486靶基因的研究

[目的]为了深入挖掘巴什拜羊不同发育阶段骨骼肌中miR-486的靶基因,为最终阐明巴什拜羊优良肉质性状形成的分子调控机制奠定基础,同时为巴什拜羊的持续选育提供理论依据.[方法]分别采集了巴什拜羊胎儿期40,50,60,80,100和120 d,以及出生当天,出生后30,60和90 d共计10个不同发育阶段的骨骼肌组织,...

Investigation of miR-486 Target Genes in Skeletal Muscle of Bashbay Sheep in Different Development Periods

【Objective】The aim of this study was to deeply reveal the target genes of miR-486 in skeletal muscle of Bashbay sheep during different development periods, and to get basic data for finally uncover the molecular regulation mechanism of excellent meat traits of this sheep breed, then finally to support to further breeding. 【Method】The skeletal muscle of Bashbay sheep were collected during 40 d, 50 d, 60 d, 80 d, 100 d and 120 d of fetal period, and 1, 2, and 3 months old of new born sheep. The total RNA was extracted, and two cDNA libraries of miR-486 target genes were constructed using mRNA of fetal and postnatal period, respectively, then the library was sequenced applied high-throughput sequencing technology. Based on the function analysis, 10 candidate target genes were selected, and their express patterns in Bashbay sheep skeletal muscle of 10 development periods mentioned above were detected by qRT-PCR. By analyzing the expression pattern of these 10 candidate target genes and miR-486 in skeletal muscle of Bashbay sheep, their target regulation relationship and functions were primarily verified. Finally, 4 candidate target genes were chosen to confirm their regulation relationship using the double luciferase reporter assay and target regulation assay in satellite cell of skeletal muscle of Bashbay sheep. 【Result】Total 123 and 118 target genes of fetal and postnatal period were obtained, respectively. Due to these target genes were not confirmed by further function assay, so they were called candidate target genes temporarily. Among of them, 96 genes were expressed in skeletal muscle of fetal and postnatal period, 27 and 22 genes were specially expressed in these two periods respectively. The result of GO and KEGG analysis showed that these candidate target genes regulated mass pathways related to muscle cell differentiation and development, just like PI3k-Akt, MAPK, Wnt, Adherens junction and Regulation of actin cytoskeleton, etc. All 10 candidate target genes were expressed in skeletal muscle of Bashbay sheep, but their expression patterns were different. Among of them,PTEN, Foxo1, Dock3, PAX7, IGF1R, PIK3I1 and FBN1 were expressed at a relatively high level in the fetal period skeletal muscle of Bashbay sheep, but were significantly down regulated during postnatal period, and OLFM4 showed an opposite expression pattern compared with above 7 genes. The expression of ARHGAP5 and PDCD4 gene did not found significantly change in all 10 development periods of Bashbay sheep. The result of double luciferase reporter assay showed that miR-486 could bind the target sites of PTEN, Foxo1, IGF1R and PIK3I1 efficiently, and significantly suppressed the activity of firefly luciferase. In skeletal muscle satellite cell, mir-486 also could down-regulate mRNA of these four genes significantly, and finally suppressed their biology functions. So it was ultimately confirmed that miR-486 could regulate these 4 target genes indeed in skeletal muscle of Bashbay sheep. 【Conclusion】The investigation deeply revealed the target genes of miR-486 in skeletal muscle of Bashbay sheep during different development periods for the first time, and comprehensively analyzed the biology process and signaling pathway they participated. The further function assay proved that the data of target genes were reliable. These data would help to uncover the molecular regulation mechanisms related to excellent meat traits of Bashbay sheep.