Evaluation of the expression stability of β-actin under bacterial infection in Macrobrachium nipponense
- 63 Downloads
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.
KeywordsMacrobrachium nipponense qPCR Reference gene Prokaryotic expression
This study was supported by Natural Science Foundation of Hebei Province (Grant No. C2015201013).
All authors have contributed significantly to the manuscript
Compliance with ethical standards
Conflict of interest
None of the authors have any financial disclosure or conflict of interest.
- 1.Yang P, Chen LQ, Wang W, Yu N, Song DX, Liu ZJ (2010) Genetic diversity of oriental river prawn (M. nipponense De Haan) revealed by ISSR markers. J Fish Sci China 17:913–921. [Chinese Journal, Article in English]Google Scholar
- 5.Jin S, Fu H, Sun S, Jiang S, Xiong Y, Gong Y, Qiao H, Zhang W, Wu Y (2018) iTRAQ-based quantitative proteomic analysis of the androgenic glands of the oriental river prawn, M. nipponense, during nonreproductive and reproductive seasons. Comp Biochem Physiol D 26:50–57Google Scholar
- 19.Yuan M, Lu Y, Zhu X, Wan H, Shakeel M, Zhan S, Jin BR, Li J (2014) Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR. PloS ONE 9:e86503CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Shi CH, Hu JR, Li CR, Wang WK (2017) Research progress of reference gene for real-time quantitative reverse transcription PCR (qRT-PCR) of insects. Jiangsu Agric Sci 45:1–7Google Scholar