Molecular characteristics of Rheb gene in Megalobrama amblycephala and its response to ammonia nitrogen stress

【Objective】This experiment was conducted to investigate the molecular characteristics of Rheb gene in Megalobrama amblycephala and its response to ammonia nitrogen stress to provide theoretical basis for the role of pathway mediated by Rheb protein in regulating fish growth and later drug targeted treatment under ammonia nitrogen stress.【Method】Juvenile fish of M. amblycephala were selected as research objects and cDNA sequence of Rheb gene in the brain of M. amblycephala was cloned using rapid amplification of cDNA ends(RACE). Then bioinformatics analysis was performed and a phylogenetic tree constructed. Real-time fluorescence quantitative PCR was applied to detect the expression pattern of Rheb gene in different tissues of M. amblycephala. Ammonia nitrogen stress group and control group were set.Then the 96 h half lethal concentration(96 h-LC₅₀)of M. amblycephala under ammonia nitrogen stress was studied via curve fitting, test of significance between groups on final weight and specific growth rate(SGR)was conducted. 【Result】The results showed that the full length of cDNA sequence of Rheb gene was 1488 bp, among which the non-coding region(UTR) of 5' and 3' ends were 245 bp and 688 bp, respectively, and the open reading frame(ORF)was 555 bp, encoding 184 amino acid residues.The mean hydrophilicity of Rheb protein was-0.196, indicating that Rheb was a hydrophilic protein. In the secondary structure of theprotein,α-helix accounted for 38.59%, extension chain 20.11%,β-turn 6.52%, and random coil 34.78%. According to the phylogenetic tree based on Rheb protein amino acid sequence similarity,M. amblycephala clustered with Danio rerio which belonged to the same family of M. amblycephala, but had a distant genetic relationship with mollusc(Mizuhopecten yessoensis and Crassostrea virginica)and arthropod(Scylla paramamosain and Portunus trituberculatus).The 96 h-LC₅₀ of M. amblycephala was 54.45 mg/L under ammonia nitrogen stress. And the final weight and SGR of M. amblycephala under ammonia nitrogen stress significantly decreased(P< 0.05, the same below)compared to that in the control group. Meanwhile, the relative expression level of Rheb gene in ammonia nitrogen stress group was significantly lower than that in the control group. 【Conclusion】Rheb protein is present in tissues of M. amblycephala,with the highest content in the brain. Long-term ammo nianitrogen stress reduces the growth indexes and Rheb gene expression levels of M. amblycephala, so it is speculated that Rheb protein may involve in the weight regulation of M. amblycephala through a certain signaling pathway under ammonia nitrogen stress.