siRNA-mediated knockdown of two tyrosinase genes from Schistosoma japonicum cultured in vitro.

ABSTRACT

The cross-linking process of eggshell proteins in helminths is dependent on the activities of tyrosinases (TYRs), which can be inhibited by phenol oxidase inhibitors. Two genes encoding TYRs, SjTYR1 and SjTYR2, have been identified in Schistosoma japonicum. In this study, siRNA-mediated RNA interference (RNAi) was performed to silence these two SjTYR genes to evaluate their roles in eggshell formation. The effects of individual or double knockdown of the SjTYR genes were compared by determining SjTYR1/SjTYR2 transcript levels, enzyme activities, and by observing the morphology and amounts of intrauterine eggs. Results showed that SjTYR transcript levels were significantly reduced on the 3rd day post-RNAi. Significant reductions in TYR enzyme activities, as well as obvious changes in morphology and the number of intrauterine eggs followed the reductions in SjTYR transcript levels. On the 8th day after simultaneous knockdown of both SjTYR genes, which effected a 40% reduction in SjTYR1 transcript level and a 59% reduction in SjTYR2 transcript level, we observed an 80% reduction in diphenol oxidase (DPO) activity of TYRs, and a 74% reduction in the number of normal eggs in female uteri. Knockdown of both SjTYR genes has a greater effect than single knockdown of the SjTYR genes. These results demonstrate that both SjTYRs play an important role in eggshell sclerotization of S. japonicum, and that their enzyme activities depend on the transcript levels of two SjTYR genes.