Molecular characterization, expression analysis of myostatin gene and its negative regulation by miR-29b-3p in Chinese concave-eared frogs (Odorrana tormota).

The molecular characteristics, expression patterns and functions of the amphibian myostatin (MSTN) gene are unknown. Here, we isolated a full-length Odorrana tormota MSTN cDNA sequence of 1701 bp (Ot-MSTN), containing a putative N-terminal signal peptide, a TGF-β propeptide domain and an active peptide. Ot-MSTN was expressed in 9 selected tissues examined, and the highest level of expression was in thigh muscle, followed by brain and female gonadal tissue. The expression of Ot-MSTN in multiple O. tormota tissues supported that the activities of MSTN may be not limited to skeletal muscle. Ot-MSTN expression was decreased from stage 31 to stage 40, while the growth rate was increased. The expression of Ot-MSTN in adult male frogs increased with age, indicating that adult male frogs may inhibit the continued hypertrophy of thigh muscle fibers and decrease the growth rate of thigh muscle to ensure muscles do not grow too large. Luciferase reporter assays showed that miR-29b-3p directly targeted the 3'-UTR of Ot-MSTN. miR-29b-3p expression in the thigh muscle of 2 yrs. females who grew faster was significantly lower than that of the slow-growing 2 yrs. male individuals, which showed an opposite trend with Ot-MSTN expression. In addition，miR-29b-3p expression reversed trends of Ot-MSTN expression at different developmental stages in thigh muscle. Therefore, these data indicate that miR-29-3p may negatively regulate the expression of MSTN and regulate thigh muscle growth and development in O. tormota.