Response Of Alternative Oxidase In Urechis Unicinctus To Sulfide Stress

Exogenous sulfide is harmful to organisms in several fields, such as damaging thestructure of protein and nucleic acid, restraining the cytochrome c oxidase activity,blocking the transport of electron and further resulting in the production andaccumulation of oxygen radicals in mitochondria respiratory chain. It is known thatsome marine invertebrates can detoxify by oxiding the exogenous sulfide. However ithas still not been revealed how these animals can avoid the damage of oxygen radicalsfrom electrons released by sulfide oxidation when exogenous sulfide restrains theelectronic transport in mitochondria. In plants such as tobacco, it is found thatalternative oxidase (AOX) plays key roles in harsh environment, such as the sulfideexposure, in which the electrons in mitochondria will be transported by AOX whenthe classic electron transport chain is affected or restrained in mitochondria. Toinvestigate the performance of alternative oxidase in the adaption of marineinvertebrates to sulfide, we choose sulfide-tolerant marine benthic invertebratesUrechis unicinctus as the studied animal in this study and obtained the recombinantproteins of AOX by prokaryotic expression system in vitro and made the polyclonalantibody using the purified AOX protein. Furthermore, we revealed the expressioncharacteristics and function of AOX in U. unicinctus organs (body wall, hindgut,mid-gut and anal sac) and analyzed adaptive response to sulfide in the4organs of U.unicinctus exposed in sulfide by indirect competitive ELISA, immunohistochemistry,and AOX enzyme activity analysis. The results are as follows:In this study, the prokaryotic expression vector pET28a-AOX had been constructedand the recombinatnt AOX was expressed successfully in E. coli as inclusion bodies.The AOX was detected in total tissued protein and mitochondria of U. unicinctus with Western blot and the polyclonal antibodies obtained from New Zealand rabbitsimmunized with the purified recombinant AOX, and AOX protein molecular mass isabout40kDa.The AOX was located mainly in epithelia of the4organs detected byimmunohistochemistry. Indirect competitive ELISA test results showed the AOX wasexpressed in body wall, mid-gut, hind-gut and anal sac, and the highest AOX contentwas in hind-gut, however no significant difference was presented among the fourorgans. Furthermore, the AOX activity in hind-gut was significantly higher than otherthree organs (p<0.05).When U. unicintus was exposed in50μmol·L-1and150μmol·L-1sulfide, the AOXprotein amount and enzyme activity in4organs increased with elevation of sulfideconcentration and delay of sulfide exposure time. AOX expression and activity in the4organs of U. unicinctus exposed in150μmol·L-1is almost higher than that of50μmol·L-1. Combining with changes of activity of cytochrome c oxidase which hasbeen reported in U. unicinctus exposed to sulfide, we discussed the role of AOX in4organs of U. unicinctus exposed to different concentrations of sulfide, and suggestedthat the AOX may play a role as the second electrons transportor when U. unicinctusstressed by sulfide and some important roles in stress adaption of echiura and evenmarine invertebrates.