关键词: 绵羊, 胚胎, 全转录组, 骨骼肌, 生长发育, circRNA

Abstract:

【Objective】The meat production of livestock, which is closely related to the development of skeletal muscle, is an important economic trait to measure the quality of livestock. For mammals, the skeletal muscle development depends on the growth and differentiation of embryonic myocyte, which has a significant impact on the subsequent growing potential. In this study, the developmental mode of skeletal muscle, the important transformation nodes, the formation of muscle fibers and the molecular regulation mechanism of transformation were mainly explored. 【Method】 Based on the previous research, the important nodes D85, D105 and D135 related to the myotube development were used in the experiment, and the longissimus dorsi muscles were sequenced by whole transcriptome sequencing. The differentially expressed (DE) circRNAs were screened by bioinformatics analysis and verified by quantitative real-time PCR (qRT-PCR). 【Result】 1 126 DE circRNAs were obtained by conditional screening (|log2| ≥1 and P≤0.05). The 3 groups were compared and many specific expressions of circRNA were found at each stage, but in the D85 vs D135 group, the amount was the most. 374 DE circRNAs were obtained, which contained 201 up-regulated and 173 down-regulated, and 44.7% of the DE genes were differentially expressed with a difference of more than 4 times. These DE circRNAs were subjected to run GO and KEGG functional analysis and targeted prediction, and they were enriched into some pathways, such as energy metabolism and signal transduction, which involved in muscle differentiation and muscle fiber development, including MAPK, PI3K-Akt, Ras, regulation of actin cytoskeleton and other signal transduction pathways. According to the results, it was confirmed that the DE circRNAs enriched during D85 to D105 were mostly associated with cell proliferation and survival, regulation of myocyte development and cell cycle, while D105 to D135 were mainly related to energy conversion, material transport, RNA transport, and DNA repair. By drawing co-expression visualization network with the targeted prediction results used by Cytoscape, the core regulatory transcripts, such as circRNA8239, circRNA19073, circRNA2765 and circRNA1616, were identified. In the D105 period, a key factor circRNA7527 that regulated the conversion of fast and slow muscle types was found, which targets the bta-miR-135a, bta-miR-615, and chi-miR-133a-5p to regulate the MEF2C gene. According to the differential expression and functional prediction in three comparison groups, 4 circRNAs related to muscle development and 4 target miRNA were selected for qRT-PCR, and the results showed that the gene expression trend was consistent with the sequencing data. 【Conclusion】 It was verified that the stabilization of the number of muscle fibers occurred between sheep embryos at D85 and D105, and muscle fiber hypertrophy happened during the D105 to D135 period, which lead to the conclusion that D105 was probably a key time point. In this study, we firstly constructed a circRNA map in sheep embryonic skeletal muscle development based on the whole transcriptome sequencing. The transcriptome differences at key stages were revealed, and multiple circRNAs and miRNAs targeting MEF2C that involved in the MAPK signaling pathway were found, which provided reference for livestock myofiber development research and other research on non-coding RNA.

Key words: sheep, embryo, whole transcriptome, skeletal muscle, growth and development, circRNA