Molecular Biology Reports

, Volume 36, Issue 1, pp 207–214

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

  • Tianyun Wang
  • Lexun Xue
  • Xiang Ji
  • Jie Li
  • Yafeng Wang
  • Yingcai Feng
Article

Abstract

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca2+-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.

Keywords

14-3-3 Protein Dunaliella salina Molecular evolution 

Abbreviations

ORF

Open reading frame

RACE

Rapid amplification of cDNA ends

RT-PCR

Reverse transcriptase-polymerase chain reaction

References

  1. 1.
    Liebich I, Voigt J (1995) A Chlamydomonas homologue to the 14-3-3 proteins: cDNA and deduced amino acid sequence. Biochim Biophys Acta 1263:79–85PubMedGoogle Scholar
  2. 2.
    Voigt J, Frank R (2003) 14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall. Plant Cell 15:1399–1413PubMedCrossRefGoogle Scholar
  3. 3.
    Moore BW, Perez VJ (1967) Specific acid proteins in the nervous system. In: Carlson FD (ed) Physiological and biochemical aspects of nervous integration. Prentice-Hall, Englewood Cliffs Prentice Hall, New Jersey, pp 343–359Google Scholar
  4. 4.
    Aitken A, Colling DB, Van Heusden BPH, Isobe T, Roseboom PH, Rosenfeld G, Soll J (1992) 14–3-3 proteins: a highly conserved, widespread family of eukaryotic proteins. Trends Biochem Sci 17:498–501PubMedCrossRefGoogle Scholar
  5. 5.
    Ichimura T, Sugano H, Kuwano R, Sunaya T, Okuyama T, Isobe T (1991) Widespread distribution of the 14-3-3 protein in vertebrate brains and bovine tissues: correlation with the distributions of calcium-dependent protein kinases. J Neurochem 56:1449–1451PubMedCrossRefGoogle Scholar
  6. 6.
    Tang SJ, Suen TC, McInnes RR, Buchwald M (1998) Association of the TLX-2 homeodomain and 14-3-3 eta signaling proteins. J Biol Chem 273:25356–25363PubMedCrossRefGoogle Scholar
  7. 7.
    Morrison D (1994) 14-3-3: modulators of signaling protein. Science 266:56–57PubMedCrossRefGoogle Scholar
  8. 8.
    Wilker EW, van Vugt MA, Artim SA, Huang PH, Petersen CP, Reinhardt HC, Feng Y, Sharp PA, Sonenberg N, White FM, Yaffe MB (2007) 14-3-3 sigma controls mitotic translation to facilitate cytokinesis. Nature 446:329–332PubMedCrossRefGoogle Scholar
  9. 9.
    Yang X, Lee WH, Sobott F, Papagrigoriou E, Robinson CV, Grossmann JG, Sundstrom M, Doyle DA, Elkins JM (2006) Structural basis for protein-protein interactions in the 14-3-3 protein family. Proc Natl Acad Sci USA 103:17237–17242PubMedCrossRefGoogle Scholar
  10. 10.
    Aitken A, Howell S, Jones D, Madrazo J, Patel Y (1995) 14-3-3 alpha and delta are the phosphorylated forms of Raf-activating 14-3-3 beta and zeta. J Biol Chem 270:5706–5709PubMedCrossRefGoogle Scholar
  11. 11.
    Piotrowski M, Oecking C (1998) Five new 14-3-3 isoforms from Nicotiana tabacum L.: implications for the phylogeny of plant 14-3-3 proteins. Planta 204:127–130PubMedCrossRefGoogle Scholar
  12. 12.
    Wang W, Shakes DC (1996) Molecular evolution of the 14-3-3 protein family. J Mol Evol 43:384–398PubMedCrossRefGoogle Scholar
  13. 13.
    Vasara T, Keranen S, Penttila M, Saloheimo M (2002) Characterization of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components. Biochim Biophys Acta 1590:27–40PubMedCrossRefGoogle Scholar
  14. 14.
    Aitken A (2006) 14-3-3 proteins: a historic overview. Semin Cancer Biol 16:162–172PubMedCrossRefGoogle Scholar
  15. 15.
    Wu K, Rooney MF, Ferl RJ (1997) The Arabidopsis 14-3-3 multigene family. Plant Physiol 114:1421–1431PubMedCrossRefGoogle Scholar
  16. 16.
    Daugherty CJ, Ronney MF, Miller PW, Ferl RJ (1996) Molecular organization and tissue-specific expression of an Arabidopsis 14-3-3 gene. Plant Cell 8:1239–1248PubMedCrossRefGoogle Scholar
  17. 17.
    Voigt J, Stevanovic S, Schirle M, Fausel M, Maier J, Adam KH, Marquardt O (2004) A 14-3-3 protein of Chlamydomonas reinhardtii associated with the endoplasmic reticulum: nucleotide sequence of the cDNA and the corresponding gene and derived amino acid sequence. Biochim Biophys Acta 1679:180–194PubMedGoogle Scholar
  18. 18.
    Voigt J, Liebich I, Kiess M, Frank R (2001) Subcellular distribution of 14-3-3 proteins in the unicellular green alga Chlamydomonas reinhardtii. Eur J Biochem 268:6449–6457PubMedCrossRefGoogle Scholar
  19. 19.
    Wang T, Xue L, Hou W, Yang B, Cha Y, Ji X, Wang Y (2007) Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions. Appl Microbiol Biotechnol 76(3):651–657PubMedCrossRefGoogle Scholar
  20. 20.
    Silflow CD, Youngblom J (1986) Chlamydomonas reinhardtii tubulin gene structure. Ann NY Acad Sci 466:18–30PubMedCrossRefGoogle Scholar
  21. 21.
    Combet C, Blanchet C, Geourjon C, Deleage G (2000) Network protein sequence analysis. Trends Biochem Sci 25:147–150PubMedCrossRefGoogle Scholar
  22. 22.
    Voigt J, Liebich I, Wöstemeyer J, Adam KH, Marquardt O (2000) Nucleotide sequence, genomic organization and cell-cycle-dependent expression of a Chlamydomonas 14-3-3 gene. Biochim Biophys Acta 1492(2–3):395–405PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Tianyun Wang
    • 1
    • 2
  • Lexun Xue
    • 1
  • Xiang Ji
    • 1
  • Jie Li
    • 1
  • Yafeng Wang
    • 1
  • Yingcai Feng
    • 1
  1. 1.Laboratory for Cell BiologyZhengzhou UniversityZhengzhouP.R. China
  2. 2.Department of Biochemistry and Molecular BiologyXinxiang Medical UniversityXinxiangP.R. China

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