Skip to main content
SpringerLink
Log in

Functional analysis of the promoter region of amphioxus β-actin gene: a useful tool for driving gene expression in vivo

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Delsuc F, Brinkmann H, Chourrout D, Philippe H (2006) Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439:965–968

    Article  CAS  PubMed  Google Scholar 

  2. Putnam NH et al (2008) The amphioxus genome and the evolution of the chordate karyotype. Nature 453:1064–1071

    Article  CAS  PubMed  Google Scholar 

  3. Koop D, Holland LZ (2008) The basal chordate amphioxus as a simple model for elucidating developmental mechanisms in vertebrates. Birth Defects Res C Embryo Today 84:175–187

    Article  CAS  PubMed  Google Scholar 

  4. Bertrand S, Escriva H (2011) Evolutionary crossroads in developmental biology: amphioxus. Development 138:4819–4830

    Article  CAS  PubMed  Google Scholar 

  5. Stokes D, Holland ND (1991) Reproduction of the Florida lancelet (Branchiostoma floridae): spawning patterns and fluctuations in gonad indexes and nutritional reserves. Invertebr Biol 115:349–359

    Article  Google Scholar 

  6. Fuentes M, Schubert M, Dalfo D, Candiani S, Benito E et al (2004) Preliminary observations on the spawning conditions of the European amphioxus (Branchiostoma lanceolatum) in captivity. J Exp Zool B Mol Dev Evol 302:384–391

    Article  PubMed  Google Scholar 

  7. Zhang QJ, Sun Y, Zhong J, Li G, Lü XM, Wang YQ (2007) Continuous culture of two lancelets and production of the second filial generations in the laboratory. J Exp Zool B Mol Dev Evol 308:464–472

    Article  PubMed  Google Scholar 

  8. Edwin GC (1932) The embryology of amphioxus. J Morphol 54:69–151

    Article  Google Scholar 

  9. Tung TC, Wu SC, Tung YF (1958) The development of isolated blastomeres of amphioxus. Sci Sin 7:1280–1320

    CAS  PubMed  Google Scholar 

  10. Tung TC, Wu SC, Yeh YF (1961) Experimental studies on neural induction in Amphioxus. Sci Sin 11:805–820

    Google Scholar 

  11. Tung TC, Wu SC, Yeh YF (1961) The presumptive areas of the egg of amphioxus. Sci Sin 11:629–644

    Google Scholar 

  12. Holland LZ, Holland ND (1989) Fine structural study of the cortical reaction and formation of the egg coats in a lancelet (= Amphioxus), Branchiostoma floridae [Phylum Chordata: subphylum Cephalochordata = Acrania]. Bio Bull 176:111–122

    Article  Google Scholar 

  13. Fuentes M et al (2007) Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum). J Exp Zool B Mol Dev Evol 308:484–493

    Article  PubMed  Google Scholar 

  14. Li G, Yang X, Shu Z, Chen X, Wang YQ (2012) Consecutive spawnings of Chinese amphioxus, Branchiostoma belcheri, in captivity. PLoS One 7:e50838

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Wang Y, Zhao P, Wang J (2002) The comparison of the growth and development of the lancelets Branchiostoma belcheri tsingtaunese reared with natural and artificial seawater. Nat Sci J Hainan Univ 20:62–65

    CAS  Google Scholar 

  16. Theodosiou M, Colin A, Schulz J, Laudet V, Peyrieras N, Nicolas JF, Schubert M, Hirsinger E (2011) Amphioxus spawning behavior in an artificial seawater facility. J Exp Zool B Mol Dev Evol 316:263–275

    Article  PubMed  Google Scholar 

  17. Li G, Shu ZH, Wang YQ (2013) Year-Round Reproduction and Induced Spawning of Chinese Amphioxus, Branchiostoma belcheri, in Laboratory. PLoS ONE 8:e75461

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Yu JK, Holland ND, Holland LZ (2004) Tissue-specific expression of FoxD reporter constructs in amphioxus embryos. Dev Biol 274:452–461

    Article  CAS  PubMed  Google Scholar 

  19. Holland LZ, Onai T (2011) Analyses of gene function in amphioxus embryos by microinjection of mRNAs and morpholino oligonucleotides. Methods Mol Biol 770:348–423

    Google Scholar 

  20. Liu X, Li G, Feng J, Yang X, Wang YQ (2013) An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri. Dev Genes Evol 233:269–278

    Article  Google Scholar 

  21. Li G, Feng J, Lei Y, Wang J, Wang H, Shang LK, Liu DT, Zhao H, Zhu Y, Wang YQ (2014) Efficient TALEN-Mediated Genome Editing in a Promising Model Animal Amphioxus. J Genet Genomics (in press)

  22. Beaster-Jones L, Schubert M, Holland LZ (2007) Cis-regulation of the amphioxus engrailed gene: insights into evolution of a muscle-specific enhancer. Mech Dev 124:532–542

    Article  CAS  PubMed  Google Scholar 

  23. Holland LZ et al (2008) The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res 18:1100–1111

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Li DL, Li G, Wang KR, Liu X, Li WY, Chen XH, Wang YQ (2012) Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene. Gene 510:39–46

    Article  CAS  PubMed  Google Scholar 

  25. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  26. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Huang S, Wang X, Yan QY, Guo L, Yuan SJ, Huang GR, Huang HQ, Li J, Dong ML, Chen SW, Xu AL (2011) The evolution and regulation of the mucosal immune complexity in the basal chordate amphioxus. J Immunol 186:2042–2055

    Article  CAS  PubMed  Google Scholar 

  28. Yu JK, and Holland LZ (2009) Amphioxus whole-mount in situ hybridization. Cold Spring Harb Protoc, 9,pdb-prot5286

  29. Corbo JC, Levine M, Zeller RW (1997) Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589–602

    CAS  PubMed  Google Scholar 

  30. Wei YH, Zhang YJ, Chen Y, Mao BY (2009) Expansion of the actin gene family in amphioxus. Zool Res 30:473–479

    Article  CAS  Google Scholar 

  31. Kakunaga R, Kakunaga T, Satoh N, Holland ND, Holland LZ (1997) Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev Genes Evol 207:203–215

    Article  Google Scholar 

  32. Kost TA, Theodorakis N, Hughes SH (1983) The nucleotide sequence of the chick cytoplasmic beta-actin gene. Nucleic Acids Res 11:8287–8301

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Nudel U, Zakut R, Shani M, Neuman S, Levy Z, Yaffe D (1983) The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res 11:1759–1771

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Liu ZJ, Moav B, Faras AJ, Guise KS, Kapuscinski AR, Hackett PB (1990) Functional analysis of elements affecting expression of the beta-actin gene of carp. Mol Cell Biol 10:3432–3440

    PubMed Central  CAS  PubMed  Google Scholar 

  35. Hwang GL, Azizur Rahman M, Abdul Razak S, Sohm F, Farahmand H, Smith A, Brooks C, Maclean N (2003) Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter. Biochim Biophys Acta 1625:11–18

    Article  CAS  PubMed  Google Scholar 

  36. Feng H, Cheng J, Luo J, Liu SJ, Liu Y (2006) Cloning of black carp β-actin gene and primarily detecting the function of its promoter region. Yi Chuan Xue Bao 33:133–140

    CAS  PubMed  Google Scholar 

  37. Quitschke WW, Lin ZY, DePonti-Zilli L, Paterson BM (1989) The beta actin promoter high levels of transcription depend upon a CCAAT binding factor. J Biol Chem 264:9539–9546

    CAS  PubMed  Google Scholar 

  38. Liu ZJ, Moav B, Faras AJ, Guise KS, Kapuscinski AR, Hackett PB (1991) Importance of the CArG box in regulation of beta-actin-encoding genes. Gene 108:211–217

    Article  CAS  PubMed  Google Scholar 

  39. Wang W, Zhong J, Wang YQ (2010) Comparative genomic analysis reveals the evolutionary conservation of Pax gene family. Genes Genet Syst 85:193–206

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of the Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, 361102, Fujian, China

    Jun Feng, Guang Li, Xin Liu, Jing Wang & Yi-Quan Wang

  2. Shenzhen Research Institute of Xiamen University, Shenzhen, 518058, China

    Yi-Quan Wang

Authors
  1. Jun Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yi-Quan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yi-Quan Wang.

Additional information

Jun Feng and Guang Li were equally contributed to this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Feng, J., Li, G., Liu, X. et al. Functional analysis of the promoter region of amphioxus β-actin gene: a useful tool for driving gene expression in vivo. Mol Biol Rep 41, 6817–6826 (2014). https://doi.org/10.1007/s11033-014-3567-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-014-3567-x

Keywords

Search

Navigation