Regulation mechanism and functional verification of key functional genes regulating muscle development in black Tibetan sheep.

Black Tibetan sheep is a branch of Tibetan sheep on the Qinghai-Tibet Plateau (QTP). It is mainly distributed in Guinan County, Qinghai Province. In order to accurately identify the core regulatory genes in the process of muscle development of black Tibetan sheep, further explore the physiological processes of growth, development and myogenesis of black Tibetan sheep, and carry out molecular breeding of black Tibetan sheep, this experiment took the unique black Tibetan sheep on the Qinghai-Tibet Plateau as the experimental object, and selected three stages of 4-month-old embryo (embryonic stage, MF group), 10-month-old(breeding stage, ML group) and 36-month-old (adult, MA group). The longissimus dorsi tissues of 3 sheep were taken at each stage to quantify the expression of genes during muscle development at different developmental stages. Meanwhile, overexpression and interference techniques were used to detect the role of core genes in the proliferation of primary muscle cells of black Tibetan sheep. In the process from embryonic stage to mature stage and adulthood, more than 1000 genes were up-regulated and more than 4000 down-regulated in black Tibetan sheep, while from breeding to adulthood, there were only 51 up-regulated genes and 83 down-regulated genes. About 998 genes were newly identified in each group. During muscle development from embryonic stage to mature stage to adulthood, two significant differential trend gene sets of Profile1 and Profile 6 were screened and identified, in which there were 121 and 31 core regulatory genes identified, respectively. In the trend of first decreasing and then stable expression in the whole development stage, 121 genes are core regulatory transcripts, which are mainly related to axonal guidance, cell cycle and other functions. 31 genes are core regulatory transcripts in the first rising and then stable expression, which are mainly related to biological metabolic pathway, oxidative phosphorylation and other processes. In the MF-ML stage, 75 genes were selected as the core regulatory gene set, the core genes were PTEN, AKT3, etc., and there were 134 differentially expressed genes in the ML-MA stage, and the core regulatory genes were IL6, ABCA1 and so on. In the MF-ML stage, the core gene set widely plays a role in cell components, cell matrix and other biological processes, while in the ML-MA stage, the core gene set widely plays a role in cell migration, cell differentiation, tissue development and so on. Adenovirus vector overexpressed and interfered with the core gene PTEN in primary muscle satellite cells of black Tibetan sheep shown that, interference and overexpression of PTEN would correspondingly increase and decrease the expression of other core genes, like AKT3, CKD2, CCNB1, ERBB3, HDAC2, but the specific interaction mechanism of each gene still needs to be further explored.