Advertisement

Journal of Applied Phycology

, Volume 23, Issue 4, pp 681–690 | Cite as

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Ulva pertusa (Ulvophyceae, Chlorophyta)

  • Wandong Fu
  • Li Shuai
  • Jianting Yao
  • Bin Zheng
  • Mingjie Zhong
  • Delin Duan
Article

Abstract

In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone a full-length cytosolic heat shock protein 70 of Ulva pertusa (designated as UPHsp70). Bioinformatics was used to analyze structural features, homologous relationship, and phylogenetic position of UPHsp70. The full length of UPHsp70 cDNA was 2,283 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 247 bp, and an open reading frame of 1,971 bp encoding a polypeptide of 656 amino acids with an estimated molecular weight of 71.13 kDa and an estimated isoelectric point of 5.04. The UPHsp70 had four degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The specific C-terminus amino acid sequence of cytosolic UPHsp70 was EEVD, and the conservation of Hsp70s of N-terminus was higher than that of C-terminus. The homology between UPHsp70 and Hsp70s of other known algae and land plants was more than 70%. Under different stress conditions, mRNA expression levels of UPHsp70 were quantified by quantitative reverse transcriptase-polymerase chain reaction. When U. pertusa samples were kept in different temperatures (5–40°C) for 1 h, the expression level of UPHsp70 at 5°C, 35°C, or 40°C was over onefold higher than that at 25°C. When U. pertusa samples were kept at 30°C for different times (0–12 h), the mRNA expression level of UPHsp70 had a trend of rise first then fall. The expression level of UPHsp70 reached maximum level after 5 h. When U. pertusa samples were kept in different salt concentrations (0–45‰) for 2 h, the expression level of UPHsp70 at 0‰ or 5‰ salt concentration was twofold higher than that at 30‰ for 2 h. The expression levels of UPHsp70 at 30‰, 35‰, and 40‰ were low and had no difference (P < 0.05). When U. pertusa samples were kept at ultraviolet irradiation or desiccated for different times (0–4 h), the mRNA expression level of UPHsp70 reached maximum level after 3.0 h; after that, it was maintained at high level.

Keywords

Ulva pertusa Heat shock protein 70 Molecular cloning mRNA expression 

Notes

Acknowledgments

This work was funded by the Zhejiang Provincial Natural Science Foundation of China under grant no. Y3100437, the Ministry of Major Science and Technology of Zhejiang Province (2009C13SAA00053), and the Project of the Shandong Agriculture Seedstock Breeding. The authors thank the anonymous reviewers for the critical comments and suggestions for the manuscript.

References

  1. Bischof K, Gómez I, Molis M, Hanelt D, Karsten U, Lüder U, Roleda MY, Zacher K, Wiencke C (2006) Ultraviolet radiation shapes seaweed communities. Rev Environ Sci Biotechnol 5:141–166CrossRefGoogle Scholar
  2. Boorstein WR, Ziegelhoffer T, Craig EA (1994) Molecular evolution of the Hsp70 multigene family. J Mol Evol 38:1–17PubMedCrossRefGoogle Scholar
  3. Boutet I, Tanguy A, Rousseau S, Auffret M, Moraga D (2003) Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas. Cell Stress Chaperones 8:76–85PubMedCrossRefGoogle Scholar
  4. Bukau B, Deuerling E, Pfund C, Craig EA (2000) Getting newly synthesized proteins into shape. Cell 101:119–122PubMedCrossRefGoogle Scholar
  5. Cara JB, Aluru N, Moyano FJ, Vijayan MM (2005) Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout. Comp Biochem Physiol 142:426–431Google Scholar
  6. Collén J, Guisle-Marsollier I, Leger JJ, Boyen C (2007) Response of the transcriptome of the intertidal red seaweed Chondrus crispus to controlled and natural stresses. New Phytol 176:45–55PubMedCrossRefGoogle Scholar
  7. Davison IR, Pearson GA (1996) Stress tolerance in intertidal seaweeds. J Phycol 32:197–211CrossRefGoogle Scholar
  8. Feder ME, Hofmann GE (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol 61:243–282PubMedCrossRefGoogle Scholar
  9. Fu WD, Yao JT, Liu FL, Wang XL, Fu G, Duan DL (2009) Molecular cloning and expression analysis of a Hsp70 gene from Laminaria japonica (Laminariaceae, Phaeophyta). Mar Biotechnol 11:738–747PubMedCrossRefGoogle Scholar
  10. Guy CL, Li QB (1998) The organization and evolution of the spinach stress 70 molecular chaperone gene family. Plant Cell 10:539–556PubMedCrossRefGoogle Scholar
  11. Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–579PubMedCrossRefGoogle Scholar
  12. Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852–1858PubMedCrossRefGoogle Scholar
  13. Henkel SK, Hofmann GE (2008a) Differing patterns of hsp70 gene expression in invasive and native kelp species: evidence for acclimation-induced variation. J Appl Phycol 20:915–924CrossRefGoogle Scholar
  14. Henkel SK, Hofmann GE (2008b) Thermal ecophysiology of gametophytes cultured from invasive Undaria pinnatifida (Harvey) Suringar in coastal California harbors. J Exp Mar Biol Ecol 367:164–173CrossRefGoogle Scholar
  15. Ireland HE, Harding SJ, Bonwick GA, Jones M, Smith CJ, Williams JHH (2004) Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor. Biomarkers 9:139–155CrossRefGoogle Scholar
  16. Jolly C, Morimoto RI (1999) Stress and the cell nucleus: dynamics of gene expression and structural reorganization. Gene Expr 7:261–270PubMedGoogle Scholar
  17. Kakinuma M, Coury DA, Kuno Y, Itoh S, Kozawa Y, Inagaki E, Yoshiura Y, Amano H (2006) Physiological and biochemical responses to thermal and salinity stresses in a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta). Mar Biol 149:97–106CrossRefGoogle Scholar
  18. Kregel KC (2002) Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 92:2177–2186PubMedGoogle Scholar
  19. Kristensen TN, Dahlgaard J, Loeschcke V (2002) Inbreeding affects Hsp70 expression in two species of Drosophila even at benign temperatures. Evol Ecol Res 4:1209–1216Google Scholar
  20. Lewis S, May S, Donkin ME, Depledge MH (1998) The influence of copper and heat shock on the physiology and cellular stress response of Enteromorpha intestinalis. Mar Environ Res 46:421–424CrossRefGoogle Scholar
  21. Li R, Brawley SH (2004) Improved survival under heat stress in intertidal embryos (Fucus spp.) simultaneously exposed to hypersalinity and the effect of parental thermal history. Mar Biol 144:205–213CrossRefGoogle Scholar
  22. Lindquist SL (1986) The heat-shock responses. Annu Rev Biochem 55:1151–1191PubMedCrossRefGoogle Scholar
  23. Lindquist S, Craig EA (1988) The heat-shock proteins. Annu Rev Genet 22:631–677PubMedCrossRefGoogle Scholar
  24. Morimoto RI, Kline MP, Bimston DN, Cotto JJ (1997) The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem 32:17–29PubMedGoogle Scholar
  25. Neal F, Gordon NF, Clark B (2004) Heat shock proteins and immune response, the challenges of bringing autologous HSP–based vaccines to commercial reality. Methods 32(1):63–69CrossRefGoogle Scholar
  26. Nelson RJ, Ziegelhoffer T, Nicolet C, Werner-Washburne M, Craig EA (1992) The translation machinery and 70 kD heat shock protein cooperate in protein synthesis. Cell 71:97–105PubMedCrossRefGoogle Scholar
  27. Neupert W, Brunner M (2002) The protein import motor of mitochondria. Nat Rev Mol Cell Biol 3:555–565PubMedCrossRefGoogle Scholar
  28. Pratt WB, Toft DO (2003) Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med 228:111–133Google Scholar
  29. Renner T, Waters ER (2007) Comparative genomic analysis of the Hsp70s from five diverse photosynthetic eukaryotes. Cell Stress Chaperones 12:172–185PubMedCrossRefGoogle Scholar
  30. Roeder V, Collén J, Rousvoal S, Corre E, Leblanc C, Boyen C (2005) Identification of stress genes from Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis. J Phycol 41:1227–1235CrossRefGoogle Scholar
  31. Rubin DM, Mehta AD, Zhu J, Shoham S, Chen X, Wells QR, Palter KB (1993) Genomic structure and sequence analysis of Drosophila melanogaster HSC70 genes. Gene 128:155–163PubMedCrossRefGoogle Scholar
  32. Ryan MT, Pfanner N (2002) Hsp70 proteins in protein translocation. Adv Protein Chem 59:223–242CrossRefGoogle Scholar
  33. Schoembeck M, Norton TA (1978) Factors controlling the upper limits of fucoid algae on the shore. J Exp Mar Biol Ecol 31:303–313CrossRefGoogle Scholar
  34. Snyder MJ, Girvetz E, Mulder EP (2001) Induction of marine mollusk stress proteins by chemical or physical stress. Arch Environ Contam Toxicol 41:22–29PubMedCrossRefGoogle Scholar
  35. Sørensen JG, Loeschcke V (2001) Larval crowding in Drosophila melanogaster induces hsp70 expression, and leads to increased adult longevity and adult thermal stress resistance. J Insect Physiol 47:1301–1307PubMedCrossRefGoogle Scholar
  36. Sung DY, Kaplan F, Guy CL (2001) Plant Hsp70 molecular chaperones: protein structure, gene family, expression and function. Physiol Plant 113:443–451CrossRefGoogle Scholar
  37. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  38. Vayda ME, Yuan ML (1994) The heat shock response of an Antarctic alga is evident at 5°C. Plant Mol Biol 24:229–233PubMedCrossRefGoogle Scholar
  39. Yao JT, Fu WD, Wang XL, Duan DL (2009) Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta). J Appl Phycol l21:233–238CrossRefGoogle Scholar
  40. Young JC, Barral JM, Ulrich Hartl F (2003) More than folding: localized functions of cytosolic chaperones. Trends Biochem Sci 28:541–547PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Wandong Fu
    • 1
    • 2
  • Li Shuai
    • 3
  • Jianting Yao
    • 2
  • Bin Zheng
    • 1
  • Mingjie Zhong
    • 1
  • Delin Duan
    • 2
  1. 1.Zhejiang Marine Development Research InstituteZhoushanPeople’s Republic of China
  2. 2.Institute of OceanologyChinese Academy of SciencesQingdaoPeople’s Republic of China
  3. 3.College of Chemistry, Chemical Engineering and Environmental ScienceQingdao UniversityQingdaoPeople’s Republic of China

Personalised recommendations