Cloning of Bax gene in Macrobrachium nipponense and its role in hypoxia stress

Hypoxia phenomenon in pond water is a common and prominent stress factor in aquaculture, which will severely restrict the behavior, growth, reproduction and other aspects of aquaculture animals, and even lead to large-scale death. Bax is a pro-apoptotic protein in the Bcl-2 protein family, although most studies have investigated the function of Bax in many vertebrates, little information to date was observed in the crustaceans. The full-length cDNA sequence of pro-apoptotic Bax gene was obtained by RACE (rapid-amplification of cDNA ends) PCR technology to explore the role of Bax (B-cell lymphoma-2 associated X protein) in the oriental river prawn Macrobrachium nipponense under hypoxic stress. RT-PCR was used to analyze the distribution of Bax gene in different tissues of M. nipponense. Meanwhile, real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of Bax gene in different hypoxic stress stages. Bax recombinant protein and antiserum were prepared, and the localization of Bax protein was analyzed by immunohistochemistry. The present results showed that the full-length cDNA of Bax gene was 2 287 bp (NCBI accession no MZ823353), including 42 bp of 5' non-coding region (UTR), 814 bp of 3' UTR, 1 431 bp of open reading frame (ORF) encoding 476 amino acids. Sequence analysis showed that Bax was rich in highly conserved BH1, BH2 and BH3 domains. Phylogenetic tree analysis showed that Bax gene of M. nipponense was closely related to the Bax gene of Penaeus monodon. RT-PCR results showed that Bax mRNA expression was the highest in the hepatopancreas and the lowest in the brain. qPCR results indicated that Bax expression level in the gill and hepatopancreas tissues of M. nipponense was significantly higher than that of the control group under hypoxia 1-96 h. Western blot analysis also confirmed that Bax protein expression level was basically similar to gene transcription level. The recombinant Bax protein was obtained by constructing prokaryotic expression vector in vitro, which were immunized rabbits to obtain antiserum. Immunohistochemical results showed that the positive signal of Bax protein in the gill and hepatopancreas were mainly located in the gill epithelial cells and hepatocytes. Finally, flow cytometry analysis showed that the apoptosis rate of haemocytes in hypoxia 96 h group significantly higher than those in the control group, which was consistent with the expression pattern of Bax protein expression abundance and gene transcription level. These results suggest that Bax gene can promote apoptosis in different tissues of M. nipponense in response to hypoxic stress. The present study has provided theoretical reference for exploring the molecular mechanism of hypoxia sensitivity in M. nipponense.