Journal of Applied Phycology

, Volume 24, Issue 5, pp 1303–1310 | Cite as

Isolation of the β-carotene ketolase gene promoter from Haematococcus pluvialis and expression of ble in transgenic Chlamydomonas

  • Chaogang Wang
  • Zhangli HuEmail author
  • Changnan Zhao
  • Xiaomo Mao


The β-carotene ketolase gene (bkt1) is a key enzyme in the biosynthesis of astaxanthin in the green alga Haematococcus pluvialis. We constructed a genomic library of H. pluvialis from which the upstream sequence of bkt1 was cloned. It was just 27% identical to the β-C-4-oxygenase gene (crto1) promoter. A TATA-box and a number of CAAT-boxes were found in the bkt1 promoter region. Analysis of the sequence revealed the presence of cis-acting elements associated with light and stress-related responses. Seven novel GTAC core sequences involved in copper response were also detected. The bkt1 promoter was transferred into Chlamydomonas reinhardtii CC-849 to drive the expression of ble. The antibiotic resistance and expression of ble in TranBCO transgenic lines confirmed the promoter activity of the cloned bkt1 promoter sequence. The results of this study confirm that the bkt1 promoter owned cis-acting elements involved in light and environmental stresses and the genetic transformation system of C. reinhardtii can be used to study the functions of bkt1promoters from H. pluvialis.


Haematococcus pluvialis β-Carotene ketolase gene (bkt1Chlamydomonas reinhardtii Transgenic alga Promoter 



We would like to thank the anonymous reviewers for their constructive suggestions. This work was supported by the National Natural Science Foundation of China (grant no. 31000162 and 31070323).


  1. Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K (1997) Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. Plant Cell 9:1859–1868PubMedGoogle Scholar
  2. Boussiba S (2000) Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol Plantarum 108:111–117CrossRefGoogle Scholar
  3. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538PubMedCrossRefGoogle Scholar
  4. Bustamante CA, Rosli HG, Añón MC, Civello PM, Martínez GA (2006) β-Xylosidase in Strawberry fruit: isolation of a full-length gene and analysis of its expression and enzymatic activity in cultivars with contrasting firmness. Plant Sci 171:497–504Google Scholar
  5. Ceron MC, Garcia-Malea MC, Rivas J, Acien FG, Fernandez JM, Rio ED, Guerrero MG, Molina E (2007) Antioxidant activity of Haematococcus pluvialis cells grown in continuous culture as a function of their carotenoid and fatty acid content. Appl Microbiol Biotechnol 74:1112–1119PubMedCrossRefGoogle Scholar
  6. Claudia AB, Pedro MC, Gustavo AM (2009) Cloning of the promoter region of β-xylosidase (FaXyl1) gene and effect of plant growth regulators on the expression of FaXyl1 in strawberry fruit. Plant Sci 177:49–56CrossRefGoogle Scholar
  7. Debuchy R, Rochaix JD, Purton S (1989) The argininosuccinate lyase gene of Chlamydomonas reinhardtii is an important tool for nuclear transformation and for correlating the genetic and molecular maps of the arg7 locus. EMBO J 8:2803–2809PubMedGoogle Scholar
  8. Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19:353–361PubMedCrossRefGoogle Scholar
  9. Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 54:1665–1669PubMedCrossRefGoogle Scholar
  10. Grünewald K, Eckert M, Hirschberg J, Hagen C (2000) Phytoene desaturase is localized exclusively in the chloroplast and upregulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, Chlorophyceae). Plant Physiol 122:1261–1268PubMedCrossRefGoogle Scholar
  11. Grünewald K, Hirschberg J, Hagen C (2001) Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis. J Biol Chem 276:6023–6029PubMedCrossRefGoogle Scholar
  12. Hagen C, Braune W, Greulich F (1993) Functional aspects of secondary carotenoids in Haematococcus lacustris (Girod) Rostafinski (Volvocales): IV. Protection from photodynamic damage. J Photochem Photobiol 20:153–160CrossRefGoogle Scholar
  13. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  14. Huang JC, Chen F, Sandmann G (2005) Stress-related differential expression of multiple β-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis. J Biotechnol 2:1–10Google Scholar
  15. Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N, Nagi S, Misawa N (1995) Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli. Plant Mol Biol 29:343–352PubMedCrossRefGoogle Scholar
  16. Kazuko YS, Kazuo S (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264Google Scholar
  17. Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87:1228–1232PubMedCrossRefGoogle Scholar
  18. Kindle KL, Schnell RA, Fernandez E, Lefebvre PA (1989) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 109:2589–2601PubMedCrossRefGoogle Scholar
  19. Kobayashi M, Kakizono T, Yamaguchi K, Nishio N, Nagai S (1992) Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic condition. J Ferment Bioeng 74:17–20CrossRefGoogle Scholar
  20. Kobayashi M, Kakizono T, Nishio N, Nagai S, Kurimura Y, Tsuji Y (1997) Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis. Appl Microbiol Biotechnol 48:351–356CrossRefGoogle Scholar
  21. Kropat J, Tottey S, Birkenbihl RP, Depège N, Huijser P, Merchant S (2005) A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element. Proc Natl Acad Sci USA 102:8730–8735CrossRefGoogle Scholar
  22. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de PY, Rouzé P, Rombauts S (2002) PlantCARE: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327PubMedCrossRefGoogle Scholar
  23. Lotan T, Hirschberg J (1995) Cloning and expression in Escherichia coli of the gene encoding ß-C-4-oxygnease that converts ß-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. FEBS Lett 364:125–128PubMedCrossRefGoogle Scholar
  24. Meng CX, Teng CY, Jiang P, Qin S, Tseng CK (2005) Cloning and characterization of β-carotene ketolase gene promoter in Haematococcus pluvialis. Acta Biochim Biophys Sinica 37(4):270–275CrossRefGoogle Scholar
  25. Nishiuchi T, Shinshi H, Suzuki K (2004) Rapid and transient activation of transcription of the ERF3 gene by wounding in tobacco leaves: possible involvement of NtWRKYs and autorepression. J Biol Chem 279:5355–5361CrossRefGoogle Scholar
  26. Quinn JM, Barraco P, Eriksson M, Merchant S (2000) Coordinate copper- and oxygen-responsive Cyc6 and Cpx1 expression in Chlamydomonas is mediated by the same element. J Biol Chem 275:6080–6089PubMedCrossRefGoogle Scholar
  27. Raman V, Lakshmanan V, Ravi S, Gokare AR (2008) Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions. J Exp Bot 59:1409–1418CrossRefGoogle Scholar
  28. Randolph-Anderson BL, Boynton JE, Gillham NW, Harris EH, Johnson AM, Dorthu MP, Matagne RF (1993) Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet 236:235–244PubMedCrossRefGoogle Scholar
  29. Schroda M, Vallon O, Wollman FA, Beck CF (1999) A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition. Plant Cell 11:1165–1178PubMedGoogle Scholar
  30. Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. Plant J 33:259–270PubMedCrossRefGoogle Scholar
  31. Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277:221–229PubMedCrossRefGoogle Scholar
  32. Stevens DR, Rochaix JD, Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 251:23–30PubMedGoogle Scholar
  33. Sun Z, Cunningham FX, Gantt E (1998) Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte. Proc Natl Acad Sci USA 95:1482–1488Google Scholar
  34. Tan S, Cunningham FX, Youmans M, Grabowski B, Sun ZR, Grantt E (1995) Cytochrome f loss in astaxanthin accumulation red-cells of Haematococcus pluvialis (Chlorophyceae) comparison of photosynthetic enzymes and thylakoid membrane polypeptides in red and green cells. J Phycol 31:897–905CrossRefGoogle Scholar
  35. Teng CY, Qin S, Liu JG, Yu DZ, Liang CW, Tseng CK (2002) Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis. J Appl Phycol 14:495–500CrossRefGoogle Scholar
  36. Wang CG, Hu ZL, Hu W, Lei AP (2004) Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii. Chin Sci Bull 49(16):1713–1717Google Scholar
  37. Yong YYR, Lee YK (1991) Do carotenoids play a photoprotective role in the cytoplasm of Haematococcus lacustris (Chlorophyta). J Phycol 30:257–261CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Chaogang Wang
    • 1
  • Zhangli Hu
    • 1
    • 2
    Email author
  • Changnan Zhao
    • 2
  • Xiaomo Mao
    • 2
  1. 1.College of Resources and Environmental ScienceWuhan UniversityWuhanPeople’s Republic of China
  2. 2.Institute of Eco-Environmental Science, College of Life SciencesShenzhen UniversityShenzhenPeople’s Republic of China

Personalised recommendations