The selection of a suitable reference gene is an important prerequisite for the precise analysis of target gene expression by real-time quantitative PCR (qPCR). The present study aims to explore the expression pattern of the Macrobrachium nipponense (M. nipponense) β-actin gene under Aeromonas hydrophila bacterial infection conditions. The complete sequence of the β-actin gene from M. nipponense was cloned by PCR. Identified and named β-actin genes were searched in the NCBI database, and the characteristics of the β-actin gene were analyzed using bioinformatics methods. The expression profiles of β-actin under stresses challenged by bacteria after 3, 6, 12, 24 and 48 h were investigated by measuring Ct values by qPCR. The prokaryotic expression vector pET-30a-actin was constructed by PCR and recombinant DNA techniques. Fused protein was induced by IPTG in the transformed Escherichia coli BL21 (DE3). Recombinant rActin was purified by nickel column. The bioinformatics analysis result revealed that the deduced protein encoded by the β-actin gene from M. nipponense had the highest homology with other prawns in the homologous assay (99%). The phylogenetic tree indicates that the β-actin from M. nipponense and other crustaceans have a single cluster. The qPCR results revealed that a stable expression of β-actin was observed in response to the A. hydrophila challenge for 3-48 h, and the Ct value was 22 ± 1.5. β-actin was ranked as a stable gene after the bacterial challenge, which was selected as the appropriate reference gene in M. nipponense.
Keywords: Macrobrachium nipponense; Prokaryotic expression; Reference gene; qPCR.