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


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.


Macrobrachium nipponense qPCR Reference gene Prokaryotic expression 



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.


  1. 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
  2. 2.
    Wang Y, Tang T, Gu J, Li X, Yang X, Gao X, Liu F, Wang J (2015) Identification of five anti-lipopolysaccharide factors in oriental river prawn, M. nipponense. Fish Shellfish Immunol 46:252–260CrossRefGoogle Scholar
  3. 3.
    Tang T, Li X, Liu X, Wang Y, Ji C, Wang Y, Xie S, Liu F, Wang J (2017) A single-domain rhodanese homologue MnRDH1 helps to maintain redox balance in M. nipponense. Dev Comp Immunol 78:160–168CrossRefGoogle Scholar
  4. 4.
    Tang T, Ji C, Yang Z, Liu F, Xie S (2017) Involvement of the M. nipponense rhodanese homologue 2, MnRDH2 in innate immunity and antioxidant defense. Fish Shellfish Immunol 70:327–334CrossRefGoogle Scholar
  5. 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
  6. 6.
    Li F, Qiao H, Fu H, Sun S, Zhang W, Jin S, Jiang S, Gong Y, Xiong Y, Wu Y, Hu Y, Shan D (2018) Identification and characterization of opsin gene and its role in ovarian maturation in the oriental river prawn M. nipponense. Comp Biochem Physiol B 218:1–12CrossRefGoogle Scholar
  7. 7.
    Sun S, Guo Z, Fu H, Ge X, Zhu J, Gu Z (2018) Based on the metabolomic approach the energy metabolism responses of oriental river prawn M. nipponense hepatopancreas to acute hypoxia and reoxygenation. Front Physiol 9:76CrossRefGoogle Scholar
  8. 8.
    Uddowla MH, Salma U, Kim H-W (2013) Molecular characterization of four actin cDNAs and effects of 20-hydroxyecdysone on their expression in swimming crab, Portunus trituberculatus (Miers, 1876). Anim Cell Syst 17:203–212CrossRefGoogle Scholar
  9. 9.
    Hooper SL, Thuma JB (2005) Invertebrate muscles: muscle specific genes and proteins. Physiol Rev 85:1001–1060CrossRefGoogle Scholar
  10. 10.
    Razzaq A, Schmitz S, Veigel C, Molloy JE, Geeves MA, Sparrow JC (1999) Actin residue glu(93) is identified as an amino acid affecting myosin binding. J Biol Chem 274:28321–28328CrossRefGoogle Scholar
  11. 11.
    Sutoh K (1983) Mapping of actin-binding sites on the heavy chain of myosin subfragment 1. Biochemistry 22:1579–1585CrossRefGoogle Scholar
  12. 12.
    Moens PD, dos Remedios CG (1997) A conformational change in F-actin when myosin binds: fluorescence resonance energy transfer detects an increase in the radial coordinate of Cys-374. Biochemistry 36:7353–7360CrossRefGoogle Scholar
  13. 13.
    Perler F, Efstratiadis A, Lomedico P, Gilbert W, Kolodner R, Dodgson J (1980) The evolution of genes: the chicken preproinsulin gene. Cell 20:555–566CrossRefGoogle Scholar
  14. 14.
    Weeds A (1982) Actin-binding proteins–regulators of cell architecture and motility. Nature 296:811–816CrossRefGoogle Scholar
  15. 15.
    Yeap WC, Loo JM, Wong YC, Kulaveerasingam H (2014) Evaluation of suitable reference genes for qRT-PCR gene expression normalization in reproductive, vegetative tissues and during fruit development in oil palm. Plant Cell Tissue Organ Cult 116:55–66CrossRefGoogle Scholar
  16. 16.
    Saray P, Roytrakul S, Pangeson T, Phetrungnapha A (2018) Comparative proteomic analysis of hepatopancreas in Macrobrachium rosenbergii responded to poly(I:C). Fish Shellfish Immunol 75:164–171CrossRefGoogle Scholar
  17. 17.
    Sun Y, Zhang J, Xiang J (2018) Molecular characterization and function of β-N-acetylglucosaminidase from ridgetail white prawn Exopalaemon carinicauda. Gene 648:12–20CrossRefGoogle Scholar
  18. 18.
    Ning M, Xiu Y, Yuan M, Bi J, Liu M, Wei P, Yan Y, Gu W, Wang W, Meng Q (2017) Identification and function analysis of ras-related nuclear protein from Macrobrachium rosenbergii involved in Spiroplasma eriocheiris infection. Fish Shellfish Immunol 70:583–592CrossRefGoogle Scholar
  19. 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:e86503CrossRefGoogle Scholar
  20. 20.
    Provenzano M, Mocellin S (2007) Complementary techniques: validation of gene expression data by quantitative real time PCR. Adv Exp Med Biol 593:66–73CrossRefGoogle Scholar
  21. 21.
    Lourenço AP, Mackert A, Cristino AS, Simões ZLP (2008) Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie 39:372–385CrossRefGoogle Scholar
  22. 22.
    Gao Y, Tang T, Gu J, Sun L, Gao X, Ma X, Wang X, Liu F, Wang J (2015) Downregulation of the Musca domestica peptidoglycan recognition protein SC (PGRP-SC) leads to overexpression of antimicrobial peptides and tardy pupation. Mol Immunol 67:465–474CrossRefGoogle Scholar
  23. 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

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

Personalised recommendations