Molecular Cloning and Expression Profiling of a Bax-Homologous Gene (EsBax) in the Chinese Mitten Crab (Eriocheir sinensis) Under Exogenous Stimulations
Mingqiao Ran | Chao Liu | Ying Deng | Wenbin Liu | Dingdong Zhang | Hengtong Liu | Cheng Chi
EsBax (bcl-2 Associated X protein), a member of the bcl-2 family involved in the mitochondrial apoptosis pathway, plays a crucial role in immune response and defense in invertebrates. In this study, we successfully cloned the full-length cDNA of EsBax from the Chinese mitten crab (Eriocheir sinensis) and investigated its immune-related functions. The EsBax cDNA is 3374 bp in length, including a 1563 bp open reading frame (ORF) encoding 521 amino acids, a 142 bp 5&prime: untranslated region (UTR), and a 1699 bp 3&prime: UTR. The predicted EsBax protein has a molecular weight of 58.0786 kD, a theoretical isoelectric point of 7.28, and contains three conserved BH domains (BH1-BH3), and a transmembrane domain (TM). Amino acid sequence analysis revealed the highest sequence identity (99.42%) with E. sinensis. For the expression analysis, three biological replicates were performed for each tissue and treatment group. Real-time quantitative PCR showed that EsBax mRNA was ubiquitously expressed in all examined tissues, with the highest expression in the hepatopancreas, followed by hemocytes, intestine, gill, and the lowest in muscle. Upon stimulation with lipopolysaccharide (LPS), Aeromonas hydrophila (AH), or cycloheximide (CHX), EsBax expression increased and peaked at 24 h (LPS and CHX) or 48 h (A. hydrophila), then decreased. These results suggest that EsBax expression is dynamically responsive to exogenous stimulants (LPS, A. hydrophila, and CHX) in E. sinensis, implying a potential role of EsBax in the molecular events associated with pathogen-induced apoptosis in this species.
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