TSC2 gene characterization and mechanism of ammonia nitrogen stress inhibiting growth through AMPK/mTOR pathway mediated by TSC2 in Megalobrama amblycephala

Abstract

This study was performed to evaluate the role of TSC2 (tuberous sclerosis complex 2) in the regulation of growth inhibition of fish exposed to chronic ammonia nitrogen. Firstly, the full-length cDNA of TSC2 in juvenile blunt snout bream, Megalobrama amblycephala was cloned using RACE technology (5’ and 3’ rapid amplification of cDNA ends). Then, TSC2 was analyzed by bioinformatics methods containing Seqman, BLAST, DNAMAN 8.0, EXPASY, and MEGA 6.0 program. Finally, the growth and gene expression level of AMKP-mTOR signaling pathway of blunt snout bream under ammonia nitrogen were investigated. The results showed that the full length of TSC2 was 6145 bp containing 5’- untranslated region of 179 bp, 3’-UTR of 992 bp and an open reading frame of 4974 bp encoding a peptide of 1657 amino acids. The estimated theoretical PI and MW (molecular weight) of TSC2 protein were 7.13 and 183016 Da, respectively. Meanwhile, TSC2 protein had three structural domains: DUF3384 domain (residues 5–303), Tuberin domain (residues 386–735) and Rap-GAP domain (residues 1409–1597). TSC2 from Megalobrama amblycephala gathered Pimephales promelas and Danio rerio clade belonging to Cyprinidae, and showed further distance to Ictalurus punctatus and Tachysurus fulvidraco. Final body weight and weight gain of fish under ammonia nitrogen stress significantly decreased (p < 0.05). Meanwhile, ammonia nitrogen stress significantly decreased Rheb, mTOR and S6K1 mRNA expression levels of blunt snout bream (p < 0.05). However, ammonia nitrogen stress significantly up-regulated the AMPKα1, AMPKα2, TSC2, 4E-BP2 mRNA expression levels (p < 0.05). Taken together, ammonia nitrogen stress inhibited protein synthesis of muscle via activating the AMKP-mTOR signaling pathway mediated by TSC2, which led to decreased body weight of blunt snout bream. The results obtained here will provide the theoretical foundation for completing the research of TSC2 functions involving protein synthesis in fish exposed to chronic ammonia nitrogen.