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

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.