Molecular Biology Reports

, 37:1143 | Cite as

A structurally novel salt-regulated promoter of duplicated carbonic anhydrase gene 1 from Dunaliella salina

Article

Abstract

It has been demonstrated that the duplicated carbonic anhydrase is induced by salt in the Dunaliella salina (D. salina) and duplicated carbonic anhydrase 1 (DCA1) is a member of carbonic anhydrase family. The purpose of this study was to identify whether both the DCA1 gene and its promoter from D. salina are salt-inducible. In this study, the results of real time RT–PCR showed that the transcripts of DCA1 were induced by gradient concentration of sodium chloride. Subsequently, a structurally novel promoter containing highly repeated GT/AC sequences of the DCA1 gene was isolated, which was able to drive a stable expression of the foreign bar gene in transformed cells of D. salina, and the gradient concentrations of sodium chloride in media paralleled regulations in the levels of both proteins and mRNA of the bar gene driven by the DCA1 promoter. Furthermore, analysis of GUS activities revealed that the salt-inducible expression of the external gus gene was regulated by the promoter fragments containing highly repeated GT sequences, but not by the promoter fragments deleting highly repeated GT sequences. The findings above-mentioned suggest that the highly repeated GT sequence in the DCA1 promoter is involved in the salt-inducible regulation in D. salina and may be a novel salt-inducible element.

Keywords

bar gene Dunaliella salina Duplicated carbonic anhydrase 1 Promoter 

Abbreviations

DCA1

Duplicated carbonic anhydrase 1

PAT

Phosphinothricin acetyl transferase

PPT

Phosphinothricin

References

  1. 1.
    Ben-Amotz A, Avron M (1990) The biotechnology of cultivating the halotolerant alga Dunaliella. Trends Biotech 8:121–126CrossRefGoogle Scholar
  2. 2.
    Geng DG, Wang YQ, Li WB, Sun YR (2002) Transient expression of GUS gene in Dunaliella salina. High Tech Lett 12:35–39Google Scholar
  3. 3.
    Sun Y, Yang ZY, Gao XS, Li QY, Zhang QQ, Xu ZK (2005) Expression of foreign in Dunaliella by electroporation. Mol Biotechnol 30:185–192CrossRefPubMedGoogle Scholar
  4. 4.
    Tan CP, Qin SP, Zhang QP, Jiang PP, Zhao FQ (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiol 43:361–365PubMedGoogle Scholar
  5. 5.
    Geng DG, Han Y, Wang YQ, Wang P, Zhang LM, Li WB, Sun YR (2004) Construction of a system for the stable expression of foreign genes in Dunaliella salina. Acta Bot Sin 46:342–346Google Scholar
  6. 6.
    Li J, Qu DQ, Liu LL, Feng SY, Xue LX (2007) Comparison of stable expressions of foreign genes driven by different promoters in transgenic Dunaliella salina. China Biotech 27:47–53Google Scholar
  7. 7.
    Jiang GZ, Lu YM, Niu XL, Xue LX (2005) The actin gene promoter-driven bar as a dominant selectable marker for nuclear transformation of Dunaliella Salina. Acta Gen Sin 32:424–433Google Scholar
  8. 8.
    Li J, Xue LX, Yan HX, Wang LL, Liu LL, Lu YM, Xie H (2007) The nitrate reductase gene-switch: a system for regulated expression in transformed cells of Dunaliella salina. Gene 403:132–142CrossRefPubMedGoogle Scholar
  9. 9.
    Li J, Xue LX, Yan HX, Wang LL, Liu HT, Liang JY (2008) Inducible EGFP expression under the control of the nitrate reductase gene promoter in transgenic Dunaliella salina. J Appl Phycol 20:137–145CrossRefGoogle Scholar
  10. 10.
    Fisher M, Pick U, Zamir A (1994) A salt-induced 60-kilodalton plasma membrane protein plays a potential role in the extreme halotolerance of the alga Dunaliella. Plant Physiol 106:1359–1365PubMedGoogle Scholar
  11. 11.
    Fisher M, Gokhman I, Pick U, Zamir A (1996) A salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina. J Biol Chem 271:17718–17723CrossRefPubMedGoogle Scholar
  12. 12.
    Premkumar L, Bageshwa UK, Gokhman I, Zamir A, Sussman JL (2003) An unusual halotolerant a-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli. Protein Exp Purif 28:151–157CrossRefGoogle Scholar
  13. 13.
    Bageshwar UK, Premkumar L, Gokhman I, Savchenko T, Sussman JL, Zamir A (2004) Natural protein engineering: a uniquely salt-tolerant, but not halophilic, a-type carbonic anhydrase from algae proliferating in low- to hyper-saline environments. Protein Eng Des Select 17:191–200CrossRefGoogle Scholar
  14. 14.
    Xie H, Xu PY, Jia YL, Li J, Lu YM, Xue LX (2007) Cloning and heterologous expression of nitrate reductase genes from Dunaliella salina. J Appl Phycol 19:497–504CrossRefGoogle Scholar
  15. 15.
    Overbergh L, Giulietti A, Valckx D, Decallonne R, Bouillon R, Mathieu C (2003) The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech 14(1):33–43PubMedGoogle Scholar
  16. 16.
    Chai YR, Hou WH, Wang TY, Wang N, Yuan BM, Wang JM, Xue LX (2005) Identification of transcription start site of Dunaliella salina salt inducible carbonic anhydrase gene and construction of its expression vector. Mar Sci 29:26–30Google Scholar
  17. 17.
    Jin ES, Potle JE, Melis A (2001) Involvement of zeaxanthin and of the CBR protein in the repair of photosystem II from photoinhibition in the green alga Dunaliella salina. Biochim Biophys Acta 1506:244–259CrossRefPubMedGoogle Scholar
  18. 18.
    Premkumar L, Greenblatt HM, Bageshwar UK, Savchenko T, Gokhman I, Sussman JL, Zamir A (2005) Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog. Proc Natl Acad Sci USA 102(21):7493–7498CrossRefPubMedGoogle Scholar
  19. 19.
    Davies JP, Weeks DP, Grossman AR (1992) Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucl Acids Res 20:2959–2965CrossRefPubMedGoogle Scholar
  20. 20.
    Wang JF, Li YK, He HQ, Chen ZJ, Liu LS (2000) Promoter function of a rice repetitive DNA sequence RRD3 in transgenic plants. Acta Bot Sin 42:1057–1061Google Scholar
  21. 21.
    Cannons AC, Shiflett SD (2001) Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: identification of regulatory elements controlling expression. Curr Genet 40(2):128–135CrossRefPubMedGoogle Scholar
  22. 22.
    Butler JE, Kadonaga JT (2002) The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev 16:2583–2592CrossRefPubMedGoogle Scholar
  23. 23.
    Carey M, Smale ST (2000) Transcriptional regulation in eukaryotes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  24. 24.
    Ott RW, Hansen LK (1996) Repeated sequences from the Arabidopsis thaliana genome function as enhancers in transgenic tobacco. Mol Gen Genet 252:563–571PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Laboratory for Cell Biology, Department of BiologyZhengzhou UniversityZhengzhouPeople’s Republic of China
  2. 2.Shanghai Institute of Materia MedicaChinese Academy of SciencesShanghaiChina

Personalised recommendations