Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from Enteromorpha prolifera (Ulvophyceae, Chlorophyta)

Abstract

Heat shock protein 70 (Hsp70) is one of the important members of heat shock protein (Hsp) families and plays essential roles in folding nascent protein, translocation, refolding denatured protein, protein degradation, adverse stress resistance, and so on. In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone full-length cytosolic heat shock protein 70 of Enteromorpha prolifera (designed as EPHsp70). Bioinformatics was used to analyze structural feature, homologous relationship, and phylogenetic position of EPHsp70. The full length of EPHsp70 cDNA was 2,265 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 217 bp, and an open-reading frame of 1,983 bp encoding a polypeptide of 660 amino acids with an estimated molecular weight of 71.39 kDa and an estimated isoelectric point of 5.03. EPHsp70 had five degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The C-terminus amino acid sequence of cytosolic EPHsp70 was EEVD, and the conservation of EPHsp70 of N-terminus was higher than that of C-terminus. The homology between EPHsp70 and the cytosolic Hsp70s of other algae and land plants was more than 70%.Keywords Enteromorpha prolifera . Heat shock protein 70 . Molecular cloningUnder inherently stressful environment, intertidal seaweeds have to face different stress factors such as high temperature, low temperature, ultraviolet radiation, and desiccation with the turning tides and alterations caused by diurnal, seasonal, and meteorological variations. In the process of adaptation, seaweeds must make appropriately physiological and biochemical changes in order to deal with environmental stresses, at the same time, seaweeds upregulate expression of stress genes and downregulate expression of protein synthesis-related genes to resist abiotic stresses (Collén et al. 2007). Organisms respond to stress at the cellular level with the rapid synthesis of a number of so-called stress proteins (heat shock proteins (Hsps)). Investigations of stress response in nonmobile organisms such as seaweeds are especially interesting because they are unable to escape from unfavorable conditions. Therefore, it is meaningful to study the role of Hsps in adverse stress resistance mechanism of seaweeds. Stress responses have been studied in few species of seaweeds to date (Vayda and Yuan 1994;Lewis et al. 1998;Ireland et al. 2004;Li and Brawley 2004;Roeder et al. 2005;Collén et al. 2007; Henkel and Hofmann 2008a, b;Fu et al. 2009).Hsps are highly conserved throughout evolution and play an essential role in protecting cells, folding and translocating nascent proteins, refolding denatured proteins, disassembling already formed protein aggregates, and so on under both stress and nonstress conditions (Nelson et al

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