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Molecular Biology Reports

, Volume 46, Issue 1, pp 309–315 | Cite as

Evaluation of the expression stability of β-actin under bacterial infection in Macrobrachium nipponense

  • Wen-Yi Geng
  • Feng-Jiao Yao
  • Ting TangEmail author
  • Shan-Shan ShiEmail author
Original Article
  • 63 Downloads

Abstract

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 qPCR Reference gene Prokaryotic expression 

Notes

Acknowledgements

This study was supported by Natural Science Foundation of Hebei Province (Grant No. C2015201013).

Author contribution

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.

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.School of DentistryJinan University Faculty of Medical ScienceGuangzhouChina
  2. 2.College of Life SciencesHebei UniversityBaodingChina
  3. 3.School of Basic MedicalJinan University Faculty of Medical ScienceGuangzhouChina

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