| JNK, p38 and ERK are three main members of MAPK pathways and are involved in a series of cellular processes in organisms, such as cell proliferation, differentiation, adhesion, survival, death, metabolism, and responses to a variety of environmental stress, etc. Up to now, most of the studies about MAPK pathways have been focused on mammals and conducted in the cultured cells in vitro. Marine invertebrates present a great variety of species, physiological characteristics and habitats. Research in MAPK pathways of marine invertebrates will help people understand systematically their biological functions and expound their role in animal adapting to environment. Urechis unicinctus, a species of Echirua, inbitats a U-shape burrow in marine coast sediment and sulfide concentration in U. unicinctus burrow increases obviously during the low tide. Previous studies have indicated that U. unicinctus can tolerate the sulfide exposure, and the mechanism has been revealed in the cytological characteristics, analysis of metabolic products and roles of the key enzymes, etc. In this study, three key members of MAPK pathways, JNK, p38 and ERK were focused and investigated in the hindgut of U. unicinctus exposed to sulfide. Aims are to identify the key molecules, determine role of MAPK pathways during the sulfide exposure, and provide scientific data to reveal the molecular mechanism of U. unicinctus tolerating sulfide exposure. Main results are as follows:The cDNA sequences of three MAPK members, JNK, ERK and p38 were obtained by RACE (rapid-amplification of cDNA ends) in hindgut of U. unicinctus. The full-length cDNA of JNK is 2102 bp, encoding a protein of 403 amino acids; the full-length cDNA ofp38 is 2772 bp, encoding a protein of 363 amino acids; and a cDNA segment of ERK is 507 bp, encoding 169 amino acids, respectively. Two full-length ORF (open reading frame) sequences of JNK, p38 were connected with pET28a to construct recombinant expression plasmid, and the recombinant proteins were obtained as inclusion body with a prokaryotic expression system in vitro. These proteins were purified by Ni-His and their purities are all higher than 85%. Polyclonal antibodies against New Zealand rats were prepared with the purified proteins, respectively.Furthermore, the content changes of JNK, p38 and ERK at total-protein level and activated protein level were analyzed with the JNK, p38 polyclonal antibody and commercial purchased ERK polyclonal antibody and three monoclonal antibody of phospho-JNK (p-JNK), phospho-ERK (p-ERK) and phospho-p38 (p-p38) for total-protein by western blot in the hindgut of U. unicinctus exposed to sulfide. Results showed:① when the worms were exposed to 50 μM and 150 μM sulfide, contents of active p-JNK increased constantly in U. unicinctus hindgut, and an initial significant increase (p<0.05) occurred at 0.5 h post-exposure, and then the contents of p-JNK continued to increase and reached the highest in 150 μM group at 48 h which was 10.2-fold higherthan that of control;② a transitory activation (p<0.05) of the ERK was induced at early stage (0.25 h～1 h) of sulfide exposure, and then the difference disappeared among the different group;③ no significant change of the p-p38 content occurred throughout the experiment. Moreover, no significant difference in total protein content of the JNK and p38 was detected by western blotting during the sulfide exposure, except p38 whose content increased significantly (p<0.05) at 0.25 h post-exposure of sulfide; Total-ERK level decreased before restored the original level after first decreases in 150 μM group, but continuously decreased to 35% folds as the level of the original state.Our findings suggested that the JNK pathway is a key pathway, and the ERK pathways may play role at early stage of sulfide exposure, effect of p38 is not obvious in the hindgut of U. unicinctusexposed to sulfide. |
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