| Heat shock protein 70 is one of important members of heat shock protein families and plays essential roles in nascent protein folding, translocation, denatured protein refolding, protein degradation, adverse stress resistance and so on. Laminaria japonica and Undaria pinnatifida, representative species of brown algae, inhabit subtidal zone; Ulva pertusa and Enteromorpha prolifera, representative species of green algae, inhabit intertidal zone. The four kinds of macroalgae have important economical and ecological values. These macroalgae confront serious environmental stresses with the turning tides and rapid alterations of other physical and chemical factors. Therefore, it is meaningful to study adverse stress resistance mechanism of macroalgae.In this study, homologous cloning coupled with the rapid amplification of cDNA ends (RACE) was used to clone full length cytosolic heat shock protein 70s of L. japonica, U. pertusa, U. pinnatifida and E. prolifera (designed as LJHSP70, UPHSP70, QDHSP70 and EPHSP70, respectively). Bioinformatics was used to analyze structural features, homologous relationship and phylogenetic position of HSP70s. The full length of LJHSP70 cDNA was 2918 bp, with a 5'untranslated region (5UTR) of 248 bp, a 3'untranslated region (3UTR) of 696 bp, and an open reading frame (ORF) of 1974 bp encoding a polypeptide of 657 amino acids with an estimated molecular weight (Mw) of 72.03 kDa and an estimated isoelectric point (PI) of 4.97. The full length of QDHSP70 cDNA was 3243 bp, with a 5UTR of 248 bp, a 3UTR of 1021 bp, and the ORF of 1974 bp encoding a polypeptide of 657 amino acids with an estimated Mw of 72.03 kDa and an estimated PI of 4.96. The full length of UPHSP70 cDNA was 2283 bp, with a 5UTR of 65 bp, a 3UTR of 247 bp, and the ORF of 1971 bp encoding a polypeptide of 656 amino acids with an estimated Mw of 71.13 kDa and an estimated PI of 5.04. The full length of EPHSP70 cDNA was 2265 bp, with a 5UTR of 65 bp, a 3UTR of 217 bp, and the ORF of 1983 bp encoding a polypeptide of 660 amino acids with an estimated Mw of 71.39 kDa and an estimated PI of 5.03. The HSP70s of four kinds of seaweeds had degenerate repeats of tetrapeptide GGMP and three typical HSP70 signature motifs. The C-terminus amino acid sequences of cytosolic HSP70s were EEID or EEVD and the conservation of HSP70s of N-terminus was higher than that of C-terminus. The homology between LJHSP70 and QDHSP70 was 98%, the homology between UPHSP70 and EPHSP70 was 96%, the homology between the four HSP70s and HSP70s of other algae and land plants was 70-80%.Under different stress conditions, mRNA expression levels of LJHSP70 and UPHSP70 were quantified by quantitative RT-PCR (qRT-PCR). When L. japonica samples were kept in different temperatures (5-40℃) for 1 h, the expression level of LJHSP70 at 30℃was highest and two-fold higher than that at 10℃. The expression level of LJHSP70 at 35℃or 40℃was lower than that at 25℃or 30℃. When L. japonica samples were kept at 25℃for different times (0-12 h), the mRNA expression level of LJHSP70 had a trend of rise first then fall. The expression level of LJHSP70 reached maximum level after 7 h, which was three-fold higher than that of control group. When L. japonica samples were kept in different salt concentrations (0‰-45‰) for 2 h, the expression level of LJHSP70 at 0‰or 5‰salt concentration was two-fold higher than that at 30‰for 2 h. The expression levels of LJHSP70 at 30‰, 35‰and 40‰were low and had no difference (P<0.05).When U. pertusa samples were kept in different temperatures (5-40℃) for 1 h, the expression level of UPHSP70 at 20℃or 25℃was low. The expression level of UPHSP70 at 5℃, 35℃, or 40℃was over one fold higher than that at 25℃. When U. pertusa samples were kept at 30℃for different times (0-12 h), the mRNA expression level of UPHSP70 also had a trend of rise first then fall. The expression level of UPHSP70 reached maximum level after 5 h, which was 2.5-fold higher than that of control group. 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 two-fold 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 UV radiation or desiccated for different times (0-4 h), the mRNA expression level of UPHSP70 reached maximum level after 3.0 h, after that maintained at high level.To further study the biological function of seaweed HSP70, the ORF of LJHSP70 gene was subcloned into expression vector of pEASY-E2. The recombinant plasmid was transformed to E.coli BL21(DE3)pLysS. The positive recombinant was cultured in LB media with 100 U/mL ampicillin, induced by IPTG and determined by SDS-PAGE. The expression level of LJHSP70 reached high level after 5 h of induction, after that the expression level increased very slowly. The expression level of LJHSP70 at 5 mM IPTG was higher than that at 1 mM.
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